# Patent

## Title
Methods of treating metabolic disease

## Abstract

The invention provides methods for treating metabolic disease in a subject and methods for increasing the expression of ILDR2 in a subject. The invention further provides a method for identifying an agent which modulates expression of an Ildr2 RNA comprising contacting a cell with an agent; determining expression of the Ildr2 RNA in the presence and the absence of the agent; and comparing expression of the Ildr2 RNA in the presence and the absence of the agent, wherein a change in the expression of the Ildr2 RNA in the presence of the agent is indicative of an agent which modulates the level of expression of the RNA.

## Background
This application claims priority to U.S. Provisional Patent Application No. 61/919,367, filed on Dec. 20, 2013, the contents of which is hereby incorporated by reference in its entirety.


GOVERNMENT INTERESTS
This invention was made with government support under N.I.H. Grant Number DK66518-08. As such the United States government has certain rights in this invention.


This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 17, 2015, is named 19240.1030US2_SL.txt and is 288,360 bytes in size.
BACKGROUND
Metabolic risk factors can lead to heart disease, diabetes and stroke. Metabolic syndrome is becoming more common due to a rise in obesity rates in adults.
Type 2 diabetes (T2DM) afflicts 246 million people worldwide, including 21 million in the United States. Another 54 million Americans have pre-diabetes. If the incidence of T2DM continues to increase at the present rate, one in three Americans, and one in two minorities born in 2000 will develop diabetes in their lifetime (Cowie C, MMWR 52: 833-837, 2003). In addition to the human cost, direct medical costs associated with diabetes in the United States currently exceed $132 billion a year and consume  ̃10% of health care costs in industrialized nations (Saltiel A R Cell 104: 517-529, 2001). Diabetes is the leading cause of both end stage renal disease and blindness (in people aged 20-74 years), and its association with cardiovascular disease increases mortality rates two-fold.
Although intensive genetic analyses of human populations have confirmed contributory roles for some specific genes, these cannot account—even in the aggregate—for powerful genetic predisposition T2DM. The link between obesity and diabetes is the result of obesity-related insulin resistance stress on the insulin-producing cells of the pancreas. Genetic differences and differences in numbers of insulin producing beta cells can cause differential susceptibility among individuals to T2DM. Therefore, there is a need to identify relevant genes associated with susceptibility to diabetes. This invention addresses this need and provides treatment strategies for manipulating beta cells and treating T2DM.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of treating a metabolic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein.
In one embodiment, the metabolic disease is a fatty liver disease, dyslipidemia, metabolic syndrome, a cardiovascular disease, obesity, a leptin disorder, or any combination thereof. In one embodiment, the fatty liver disease is hepatic steatosis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, elevated liver cholesterol level, elevated liver triglyceride level, elevated liver fatty acid level, elevated liver LDL-cholesterol level, elevated liver VLDL cholesterol level, or elevated liver non-HDL cholesterol level, or any combination thereof. In one embodiment, the dyslipidemia is hyperlipidemia, mixed dyslipidemia, hypercholesterolemia, polygenic hypercholesterolemia, hypertriglyceridemia, hyperfattyacidemia, elevated ApoB, elevated cholesterol, elevated LDL-cholesterol, elevated VLDL-cholesterol, or elevated non-HDL cholesterol, or any combination thereof. In one embodiment, the cardiovascular disease is coronary heart disease, acute coronary syndrome, early onset coronary heart disease, or atherosclerosis, or any combination thereof. In one embodiment, the leptin disorder is hyperleptinemia, or tissue leptin resistance, or any combination thereof. In one embodiment, the treatment improves cardiovascular outcome. In one embodiment, the treatment results in slowed progression and/or amelioration of the metabolic disease. In one embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in liver tissue. In one embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in hepatocytes. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor. In one embodiment, the subject is administered an additional therapy. In one embodiment, the additional therapy is a lipid lowering therapy. In one embodiment, the lipid lowering therapy is a therapeutic lifestyle change, a HMG-CoA reductase inhibitor, niacin, a fibrate, a cholesterol absorption inhibitor, a MTP inhibitor, or any combination thereof. In one embodiment, the subject has type II diabetes or insulin resistance. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof. In one embodiment, the subject has a reduced level of expression of Ildr2 mRNA or ILDR2 protein compared to the level of expression of Ildr2 mRNA or ILDR2 protein in a subject without a metabolic disease. In one embodiment, the level of expression is determined before administrating to the subject the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof.
In another aspect, the invention provides a method of decreasing lipid levels in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein.
In one embodiment, the lipid level is a cholesterol level, triglyceride level, ApoB level, LDL-cholesterol level, VLDL-cholesterol level, small LDL-particle level, small VLDL-particle level, non-HDL-cholesterol level, phospholipid level, or fatty acid level, or any combination thereof. In one embodiment, the level is the concentration in blood plasma. In one embodiment, the level is the concentration in liver. In one embodiment, the lipid level is decreased relative to a lipid level in the subject before administrating the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in liver tissue. In one embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in hepatocytes. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor. In one embodiment, the subject is administered an additional therapy. In one embodiment, the additional therapy is a lipid lowering therapy. In one embodiment, the lipid lowering therapy is a therapeutic lifestyle change, a HMG-CoA reductase inhibitor, niacin, a fibrate, a cholesterol absorption inhibitor, a MTP inhibitor, or any combination thereof. In one embodiment, the subject has type II diabetes or insulin resistance. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof. In one embodiment, the subject has a reduced level of expression of Ildr2 mRNA or ILDR2 protein compared to the level of expression of Ildr2 mRNA or ILDR2 protein in a subject without a metabolic disease. In one embodiment, the level of expression is determined before administrating to the subject the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof.
In another aspect, the invention provides a method of increasing expression of Ildr2 mRNA or ILDR2 protein in a hepatocyte, the method comprising contacting the cell with an agent which increases expression of the Ildr2 mRNA or ILDR2 protein.
In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor. In one embodiment, the subject is administered an additional therapy. In one embodiment, the additional therapy is a lipid lowering therapy. In one embodiment, the lipid lowering therapy is a therapeutic lifestyle change, a HMG-CoA reductase inhibitor, niacin, a fibrate, a cholesterol absorption inhibitor, a MTP inhibitor, or any combination thereof. In one embodiment, the subject has type II diabetes or insulin resistance. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof. In one embodiment, the subject has a reduced level of expression of Ildr2 mRNA or ILDR2 protein compared to the level of expression of Ildr2 mRNA or ILDR2 protein in a subject without a metabolic disease. In one embodiment, the level of expression is determined before administrating to the subject the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In one embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or any fragment thereof.
In another aspect, the invention provide a method of treating fatty liver disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor.
In another aspect, the invention provides a method of decreasing liver fat deposits in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor.
In another aspect, the invention provides a method of modifying liver lipid metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein.
In another aspect, the invention provides a method of modifying liver lipid metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which decreases expression of Ildr2 mRNA or ILDR2 protein.
In another aspect, the invention provides a method of modifying ER stress responses in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein.
In another aspect, the invention provides a method of modifying ER stress responses in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which decreases expression of Ildr2 mRNA or ILDR2 protein.



BRIEF DESCRIPTION OF THE FIGURES
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 shows the predicted structure of major ILDR2 isoforms. Isoform 1 (GENBANK: FJ024495.1) is full-length. There are 10 predicted exons. Exon 1 is an amino terminal signal peptide; exons 2 and 3 code for an IgV-like immunoglobulin domain; exon 4 is amino proximal to the trans-membrane domain of exon 5; exons 6-10 comprise a randomly-coiled, carboxy-terminal tail (simplified in this depiction as rod-like). Based on results shown in FIGS. 2A-C, exons 1-4 are lumenal and exons 6-10 are cytosolic. Isoform 2 (GENBANK: FJ024496.1) lacks cytosolic exon 6. Isoform 4 (GENBANK: FJ024498.1) lacks lumenal exon 4. Isoform 3 (GENBANK: FJ024497.1) lacks exons 4, 5, and 6 and, therefore has no transmembrane domain, and is depicted as entirely cytosolic.
FIGS. 2A-C shows fluorescence microscopy of ILDR2 localization under basal conditions. ILDR2 fused on its C-terminus to mYFP (green) was transiently co-transduced into cell lines with DsRed-probes specific to either the ER (red) or the PM (red). The ER-specific probe is DsRed fluorescent protein attached to the ER-retention sequence KDEL (SEQ ID NO: 140). The PM-specific probe is DsRED attached to a farnesyl group that targets the protein to the inner leaflet of the PM. Cells were fixed without any further treatment 24 hr after transfection. Bar: 100 uM. Confocal images recorded at 636 magnification. (FIG. 2A) GT1-7 cells. ILDR2-isoform 2-YFP merges with DsRed-ER probe to produce a yellow signal over the ER, but does not merge with the red DsRed-PM probe. (FIG. 2B) Hepa1c1c7 cells. The green ILDR2-isoform 4-YFP probe merges with the red DsRed-ER probe to produce an orange signal over the ER; expression levels of labeled proteins are less uniform than in GT1-7 cells. The red DsRed-PM and green ILDR2-YFP signals do not merge in the PM. (FIG. 2C) Hepa1c1c7 cells. N-terminal fusion of ILDR2-isoform 1 with 3×FLAG epitope co-transduced with DsRed-probes to ER. Tag geometry does not interfere with subcellular localization.
FIGS. 3A-F shows liver morphology and histology in ADKD and ADOX WT and OB mice. Chow-fed, 10-week-old B6 males were sacrificed after 24-hr fast (Fasted) or following a 24-hr fast and 12-hr refeeding (Refed). Liver morphology is shown in the upper panels and hematoxylin and eosin staining of representative sections is shown in the lower panels at 200× magnification (scale is 100 mm). Asterisk (*) identifies large droplet, macrovesicular lipid vacuoles, particularly evident in Ob sections; large open arrows (M-D) denote intra-hepatocellular Mallory-Denk-like eosinophilic material; open yellow arrows (mF) denote small droplet, microvesicular fat within hepatocytes; short double black arrows (iMO) indicate mononuclear inflammatory cells, consistent with lymphocytes; large blue arrows (ap) indicate apoptotic hepatocytes; (glyc) identifies a “clear”-appearing hepatocyte with increased glycogen content (e.g., ADOX WT 10d Refed); Portal Tract (or PT) is above the hatched line in ADKD WT 10d Fasted); (CV) is Central Vein; (PV) is Portal Vein; (BD) is Bile Duct. (FIG. 3A) Wild-type mice, 3 days p.t. with adenovirus knockdown vectors expressing RNAi for lacZ or Ildr2 (FIG. 3B) Wild-type mice, 10 days p.t. with adenovirus knockdown vectors expressing RNAi for lacZ or Ildr2 (FIG. 3C) ob/ob mice, 10 days p.t. with adenovirus knockdown vectors expressing RNAi for lacZ or Ildr2 (FIG. 3D) Wild-type mice, 3 days p.t. with adenovirus vector over-expressing GFP or Ildr2; there is no significant steatosis or inflammation (FIG. 3E) Wild-type mice, 10 days p.t. with adenovirus vector over-expressing GFP or Ildr2 (FIG. 3F) ob/ob mice, 10 days p.t. with adenovirus vector over-expressing GFP or Ildr2. As described in the text, increased apoptosis without inflammation is consistent with a primary role for ILDR2 in ER stress responses.
FIGS. 4A-B shows TG secretion analysis in ADKD and ADOX WT mice. Chow-fed, 10-week-old B6 (WT) males were intravenously injected with ADKD or ADOX vectors expressing RNAi for lacZ or Ildr2. At 7 days p.t., following a 16 hr fast, mice were intravenously injected with 15% Triton WR1339 at a dose of 500 mg/kg. Plasma (from 100 ul of blood) was collected hourly for 4 hr and TG measured. (FIG. 4A) Wild-type mice, 7 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 4B) Wild-type mice, 7 days post-transduction with adenovirus vector over-expressing GFP or Ildr2. AUC: area under the curve. Insignificant differences by AUC analysis show that hepatic lipoprotein secretion is unaffected by Triton WR1339 administration in ADKD and ADOX mice.
FIGS. 5A-D shows FPLC analysis of plasma lipoprotein fractions in ADKD and ADOX WT mice. At 7 days p.t. with either ADKD or ADOX vectors, plasma from 6 wild-type mice was collected, pooled and TCH and TG profiles were analyzed by FPLC using Sepharose 6 Fast Flow columns. HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein. (FIG. 5A) TCH profile in wild-type mice, 7 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 5B) TCH profile in wild-type mice, 7 days p.t. with adenovirus vector over-expressing GFP or Ildr2; (FIG. 5C) TG profile in wild-type mice, 7 days after adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 5D) TG profile in wild-type mice, 7 days p.t. with adenovirus vector over-expressing GFP or Ildr2. These experiments show an increase in plasma TG (as VLDL) in ADKD mice but not in ADOX mice. TCH shifts in ADKD mice from HDL to LDL and VLDL, while in ADOX mice the decrease in HDL is accompanied by an increase in VLDL only.
FIGS. 6A-F shows relative expression of selected genes in ADKD and ADOX WT and OB mice. 10-week-old B6 male mice were chow-fed, intravenously injected with ADKD and ADOX vectors and sacrificed at 3 days p.t, following a 12-hr fast. Expression levels were determined by qPCR normalized to expression levels of the 36B4 housekeeping gene. Fold changes are relative to the GFP control in the same state as the Ildr2 (either fasted or refed compared to fasted or refed). * indicates p,0.05; ** indicates p,0.01 (2 tailed t-test). (FIG. 6A) Expression in wild-type mice, 3 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 6B) Expression in wild-type mice, 10 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 6C) Expression in wild-type mice, 10 days p.t. with adenovirus vector over-expressing GFP or Ildr2; (FIG. 6D) Expression in wild-type mice, 3 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2: (FIG. 6E) Expression in ob/ob mice, 10 days p.t. with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 6F) Expression in ob/ob mice, 10 days p.t. with adenovirus vector over-expressing GFP or Ildr2. Changes in transcriptional profiles appear to be secondary to changes in lipid content.
FIGS. 7A-B shows relative expression of selected genes in ADKD and ADOX primary hepatocytes. To identify short-term effects of changes in Ildr2 expression, hepatocytes from five 10-week-old B6 mice were extracted, pooled and plated into individual wells and exposed, in triplicate, for 24 hr to ADOX or ADKD viral vectors. RNA was extracted, transcribed into cDNA, and expression was determined by qPCR. (FIG. 7A) Expression in hepatocytes transduced with adenovirus knockdown vector expressing RNAi for lacZ or Ildr2; (FIG. 7B) Expression in hepatocytes transduced with adenovirus vector over-expressing GFP or Ildr2. These results recapitulate those seen the in vivo studies.
FIGS. 8A-B shows expression of Ildr2 in liver is increased by adiposity through high-fat diet or leptin deficiency. Expression of Ildr2 was determined by qPCR, normalized to 36B4 in mice sacrificed after either fasting for 24 hr or after fasting for 24 hr and followed by a 12-hr refeeding period. (FIG. 8A) Wild type B6 mice at 6 weeks of age were fed ad libitum either chow or a high fat diet (60% of kcal from fat) for 3 additional months. (FIG. 8B) Chow-fed wild type B6 and leptin-deficient OB mice (B6.Cg-Lepob/J) were purchased at 9 weeks and sacrificed at 10 weeks of age. Wild-type mice fed a high fat diet and genetically obese mice showed a similar (3.6 to 3.7-fold) increase in Ildr2 liver expression compared to age-matched wild-type mice (p value, 0.01) regardless of feeding status.
FIGS. 9A-D shows Respiratory Exchange Ratio (RER) in ADKD and ADOX WT and OB mice. Mice were chow-fed, 10-week-old B6 (WT) or B6.V-Lepob/J (OB) males, at 4 to 5 days p.t. with adenovirus knockdown vectors expressing RNAi for lacZ or Ildr2 or with adenovirus vectors over-expressing GFP or Ildr2. Data shown are mean 6 SEM (8 mice per group) and run in a TSE systems indirect calorimeter for 48 hr. (FIG. 9A) WT ADKD; (FIG. 9B) OB ADKD; (FIG. 9C) WT ADOX; (FIG. 9D) OB ADOX. ADKD mice show decreased RER at night, whereas ADOX mice show no differences, day or night. AUC calculations are shown in Table 9.
FIGS. 10A-D shows ipGTT in WT and OB mice 7 days p.t. At 7 days p.t. with adenovirus knockdown vectors expressing RNAi for lacZ or Ildr2 (left) or with adenovirus vectors over-expressing GFP or Ildr2 (right), the 10-week-old chow-fed male B6 mice that were used in the 10 day experiments were injected intraperitoneally after 12 hr fast with 2 g/kg glucose. The mice used in this experiment are the same mice that on which indirect calorimetry was conducted on day 5 p.t. (FIG. 10A) WT ADKD; (FIG. 10B) OB ADKD; (FIG. 10C) WT ADOX; (FIG. 10D) OB ADOX. In both ADKD and ADOX animals, IPGTT was unaffected.
FIG. 11 shows a schematic of diabetes susceptible and diabetes-resistant mouse strains.
FIG. 12 shows BMI versus glucose for ob crosses.
FIG. 13 shows genes in the minimal congenic interval on Chr1:168.1-170.3 Mb. Gray background corresponds to the minimal DBA “variable” interval from 168.1 Mb-169.9 Mb, between markers rs33860076 and rs30708865. Yellow background corresponds to the centromeric end of the DBA vs. B6 “conserved” interval (i.e. nominally invariant). Genes in blue are from RefSeq; genes in black are predicted and locally confirmed. The N-scan predicted gene chr1:1224.1 is designated here as “Lisch-like” (also known as ILDR2). Amino acid variants are shown in red to the right of the corresponding gene. Nucleotide substitutions were confirmed by bidirectional sequencing in both C57BL/6J and DBA/2J DNA.
FIG. 14 shows shows sub-congenic lines (1jc, 1jcdc, 1jcd, 1jcdt) in the interval Chr1:164-194 Mb that display hypoinsulinemic hyperglycemia in association with histological evidence of a relative reduction in β-cell mass in the first 21-28 days of life due to reduced β-cell proliferation. An expanded view of the L1 gene (chr1.1224.1) is shown at bottom. Above the map scale, in black type, are microsatellite markers that were used to genotype B6 and DBA alleles to establish general boundaries of these congenic intervals. D1mit110 is the peak of the F2/F3 QTL linkage map (see Mapping T2D-related Phenotypes in B6×DBA F2/F3 Progeny). Below map scale, RefSNP (rs) and D-markers in red type identify DBA sequence limits of the respective congenic lines. Markers in blue type identify the closest, confirmed non-DBA (B6) sequence. Sequences in intervals between markers in red and blue type are DBA vs. B6 invariant. Gray bars are DBA-derived sequences. Yellow box corresponds to a 3.2 Mb interval, conserved between DBA and B6. The red box identifies the N-scan predicted gene, chr1.1224.1, subsequently identified as Lisch-like (L1) or ILDR2, extending centromerically from line 1jcdt. In the expanded view of L1, the B6 boundary (rs31968429) for lines 1jcdc, 1jcd, 1jcdt is 333 bp centromeric of exon 7; the DBA boundary, (rs33860076) is 2,700 bp telomeric of exon 7. 5330438I03Rik is an anti-sense transcript described in detail in the text. Marker positions are from the mouse genome annotation (NCBI Build 36, February 2006). Variants in ILDR2=T587A; A647V.
FIGS. 15A-D show the phenotypes of those obese B6.DBA congenic mice were reduced β-cell mass (FIG. 15A), reduced plasma insulin/glucose ratio (FIG. 15B), reduced glucose tolerance (FIG. 15C), and persistent mild hypoinsulinemic hyperglycemia (FIG. 15D).
FIGS. 16A-B show plasma insulin/glucose ratios and hyperglycemic clamps in age-grouped 1jc congenic males. FIG. 16A: Fasting plasma insulin/glucose ratios in 30- and 62-day old 1jc Lepob/ob B/B and D/D male mice, chow-fed since weaning Asterisk (*) indicates significant difference between B/B and D/D animals; p-value <0.05 for 2-tailed t-test. FIG. 16B: Hyperglycemic clamping in 100-day old 1jc males on Surwit Diet for 18 weeks. 1jc DD male mice fed a Surwit diet for 18 wks were clamped at a blood glucose concentration of 250 mg/dl for 1 hr and serum insulin concentrations measured at 1 hr. Asterisk (*) indicates p-value <0.05 for 2-tailed t-test.
FIGS. 17A-B show the relationship between islet histology and insulin secretion. FIG. 17A: Islet histology in 21-day old ljcd Lepob/ob B/B and D/D male mice. 4 μm pancreatic sections from 21-day old ljcd Lepob/ob B/B and D/D male mice were insulin-stained with anti-guinea pig IgG and visualized by light microscopy at 10× magnification. In D/D animals, islets were smaller and less numerous. By histomorphometry, the proportion of small islets (250-2000 μm2) in 21-day old Lepob/ob males was greater in D/D (1jc and ljcd) mice (73%) than in B/B (60%); whereas the proportion of large islets (10,000-50,000 μm2) was lower (9% in D/D and 14% in B/B). FIG. 17B: In vitro glucose-stimulated insulin secretion in pancreatic islets in 28-day old 1jc Lepob/ob B/B and D/D males. Each congenic genotype group consisted of 3 male animals. Negative control consisted of 3 4-week old diabetes-prone Leprdb/db KsJ male animals that are hypo-responsive to glucose stimulation; positive control was 3 4-week old insulin-resistant animals segregating for a diabetes-susceptibility QTL on Chr5@78cM, characterized by hyperglycemia and hyperinsulinemia. B/B and D/D show dose response, but no B/B vs. D/D difference at any concentration of glucose. Response to 10 mM arginine in the same animals confirms that the β-cells of the B/B and D/D congenics are comparable with regard to insulin release to a non-glucose stimulus. The 0 mM arginine control in B/B is shown to establish baseline insulin levels.
FIGS. 18A-B show B-cell mass and replication rates in ljcd Lepob/ob males. FIG. 18A: Relative β-cell area in 20-, 60, and 150-day old Lepob/ob males segregating for B and D ljcd congenic intervals. In 60 and 150-day old males segregating for the D/D sub-congenic interval, relative β-cell mass was approximately half that of B/B littermates; B/D animals were intermediate at 150 days. N=10 for each of the 3 groups of animals. Mean+/−SEM. The asterisk (*) indicates that p<0.05 for D/D vs. B/B at 60 days, and D/D and B/D vs B/B at 150 days. These findings are consistent with in vivo data showing onset of elevated blood glucose at rest and during ipGTT by 60 days. FIG. 18B: β-cell replication rates (Ki67) in 1- and 21-day old Lepob/ob B/B and D/D ljcd males. To estimate the proportion of dividing cells, the number of Ki67-positive β-cells was normalized to the total number of insulin-positive cells. Replication of β-cells in 1-day old D/D males was  ̃1⁄3 that of B/B littermates (p=0.017). This difference, not present in 21-day old animals, was probably due to normally reduced β-cell replication by the time of weaning. Mean+/−SEM. The asterisk (*) indicates that p<0.05 for D/D vs. B/B in 1-day old animals.
FIG. 19 shows the mouse ILDR2 gene map. Isoform 2 is absent exon 6; isoform 3 is absent exons 4, 5, 6; isoform 4 is absent exon 4.
FIGS. 20A-B shows expression analysis of candidate genes and liver expression of ILDR2. FIG. 20A: Tissue-specific expression analysis of genes in the “variable” portion of the minimum congenic interval. Data for relative expression (B/B to D/D) for hypothalamus, islets, liver and EDL-muscle are displayed graphically and numerically below the graph. 21-day old DD and BB Lepob/ob 1jc congenic animals males were analyzed using Affymetrix #430A microarrays. FIG. 20B: Liver expression of ILDR2 in 1jc B/B and D/D males from 21-120 days. Samples from Lepob/ob 1jc males were analyzed by qPCR.
FIGS. 21A-B shows the expression patterns and morpholino knockdown in Zebrafish embryos. FIG. 21A: Developmental expression of zebra fish ILDR2 (Lisch-like) and Lsr-like orthologs. ILDR2 RNA was hybridized in situ to whole-mount zebra fish embryos at 48 hours post-fertilization (hpf), dorsal view with anterior towards the top; and 72 hpf, lateral view with anterior towards the top, ventral towards the right and yolk removed. Lsr-like RNA was hybridized at 48 hpf and 34 hpf. Ildr2 (Lisch-like) panels show ventral views of embryos with yolks removed and anterior towards the top. Lsr-like panels show the same image captured in the focal plane of the anterior (ap) and posterior (pp) pancreatic buds, respectively. i, intestine; ph, pharynx; pn, pronephric ducts; 1, liver; ap, anterior pancreatic bud; pp, posterior pancreatic bud; p, pancreas (after anterior and posterior bud fusion); b, brain; o, otic vesicle. FIG. 21B: Morpholino knockdown of ILDR2 (Lisch-like) and Lsr-like orthologs at 48 hpf. Two dimensional ventral views (anterior towards top) of confocal stacks of 48 hpf embryos, uninjected or injected with 15 ng morpholino: control, Lsr-like sp1, and ILDR2 (Lisch-like) ATG. Gut-GFP transgene expression (green); insulin immunolabelling (red).
FIG. 22 shows ENU mutations in ILDR2 in C3HeB/FeJ mice. The sequence (SEQ ID NO: 1) shows the positions and changes from wild-type of the five variants available (SEQ ID NO: 151). W87* can be introgressed on the diabetes-susceptible KsJ strain.
FIGS. 23A-B shows the body composition and blood chemistry in chow-fed Ildr2 W87* animals and in +1+(WT) littermates. FIG. 23A: Fat mass measured at 3 and 6 months; body weight and lean mass measured at 6 months. FIG. 23B: BG, blood glucose, measured in W87* and WT males following 24 hr fast and postprandially 12 hrs after fast. (*=p<0.05; T test; n=10 WT and 11 W87*).
FIGS. 24A-B shows ENU-mutagenized Ildr2-null mice that encode a transcript containing a stop codon at threonine-87 (W87*) in the second exon (W87*mice) have increased hepatic triglycerides and cholesterol ester stores (FIG. 24A). The phenotype of these mice (3 months old; diet: normal chow, 4 hr fasting) were consistent with B6. DBA congenic mice. These mice were also reduced beta cell mass and persistent mild hypoinsulinemic hyperglycemia. In addition, hepatic TG and CE levels were increased in 3 month old W87 mice. (*=p<0.05; n=10 WT and 11 W87*). CE levels were measured in fasted (24 hours) and refed (12 hours) mice (FIG. 24B).
FIGS. 25A-B shows the effects of W87* and Ildr2 RNAi on LDL cholesterol uptake in isolated hepatocytes. Primary hepatocytes show reduced LDL uptake when Ildr2 is functionally reduced. FIG. 25A: Isolated primary heptocytes were extracted from 2 month old wild type B6 mice. The hepatocytes were either not transfected, transfected with scrambled siRNA, siRNA targeting Ldlr (a positive control showing the assay was working), or siRNA targeting Ildr2. The cells were incubated for 2 hours in the presence of DiI-LDL. The red indicates the LDL that is taken up by the cell (nuclei are stained with Dapi-blue). FIG. 25B: Primary hepatocytes were isolated from both wildtype and W87* age matched male mice. Cells were incubated at 4° C. and 37° C. in the presence of bodipy-labled LDL to determine binding and internalization of LDL, respectively. The cells were then analyzed by facs and an average of X thousand cells per mouse per condition were measured for fluorescence intensity. The W87* isolated primary hepatocytes showed decreases in both binding and internalization of LDL compared to the wildtype mice.
FIG. 26 shows the relative B-cell area in 200-day old WT (+1+) and W87* mutants (−/−). At 200 days, the beta cell mass of the ENU −/− is reduced by about 2⁄3, but the animals remain euglycemic
FIG. 27 shows a schematic of ILDR2. ILDR2 is a type I transmembrane protein with an immunoglobulin-like domain facing the lumen of the ER and C-terminal cytoplasmic tail. ILDR2 is highly expressed in liver, hypothalamus, fat, and islets.
FIG. 28 shows the phenotype after Ildr2 knockdown or overexpression for 10 days in 10 week-old WT mice. 24 hrs fasted or 12 hrs refed. Adenovirus were made by invitrogen's adenovirus expression system using pAd/CMV-V5 DEST vector for OE and BLOCK-iT Adenoviral RNAi Expression System for KD. 10 days after Ildr2 knockdown or overexpression by adenovirus infection, the fasting-refeeding experiment was performed. P value's color indicate that values are significantly increased in red and significantly decreased in blue. ILDR2 knockdown liver were enlarged and had increased lipid droplet. Plasma TG and TC were increased in knockdown mice. Hepatic TG and TC were also increased. Regarding overexpression mice, ILDR2 liver were enlarged compared to control, but HE stain liver sections were not different. Plasma TG and TC levels were increased but hepatic TG and TC were markedly decreased. This results suggested that Ildr2 is involved in positively regulate lipid metabolism.
FIG. 29 shows relative gene expression in the liver from qRT-PCR data in WT mice. srebp1c and its target genes FAS and ACCC were decreased. TG synthesis genes were also decreased in knockdown mice. Regarding overexpression mice, these lipogenic genes expression were opposite to knockdown mice.
FIG. 30 shows Western Blots of 10 week knockdown C57BL/6J males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to lipid metabolism and insulin signaling pathway, western blots were performed. Quantification of each band is shown in the right. Phspho-Akt are decreased in refed knockdown mice. Lipogenic protein Fas and ACC were decreased. Regarding nuclear proteins, Phospho FoxO1 and FoxO1 were decreased in knockdown mice, consistent with decreased Fas and ACC, SREBP1 in refed state. In KD mice, decreased lipogenic proteins were caused by impairment of hepatic insulin signaling, suggesting that ILDR2 knockdown might induce hepatic insulin resistance.
FIG. 31 shows Western Blots of 10 week overexpression C57BL/6J males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to lipid metabolism and insulin signaling pathway, western blots were performed. Quantification of each band is shown in the right. In overexpression mice, phospho-Akt was slightly lower than control. Fas and Acc were increased. Phospho-Foxo and Foxo1 were increased. SREBP1 was also increased. These protein levels of OE mice livers were mostly opposite to KD mice (FIG. 30).
FIG. 32 shows the phenotype after Ildr2 knockdown or overexpression for 10 days in 10 week-old ob/ob mice. 24 hrs fasted or 12 hrs refed. To determine whether consistent with metabolic effects of wild-type mice, we infected ob/ob mice with knockdown and overexpression adenovirus. Knockdown liver were markedly enlarged and had increased lipid droplet compared to control. Both plasma fasted and refed TG levels were higher than control mice. Plasma TC levels were slightly higher in refed state. Consistent with WT knockdown mice, hepatic TG and TC were increased in knockdown mice. Regarding overexpression mice, overexpressing ILDR2 improve hepatic steatosis. Plasma TG and TC levels were no difference but hepatic TG and TC were markedly decreased. These results of hepatic lipids levels were totally consistent with both knockdown and overexpression WT mice.
FIG. 33 shows relative gene expression in the liver from qRT-PCR data in ob/ob mice. Regarding gene expression, consistent with knockdown WT mice, srebp1c and its target genes and TG synthesis genes were decreased in knockdown ob mice. In overexpression mice, these lipogenic genes expression were opposite to knockdown mice. Genes expression levels were also totally consistent with WT mice experiment.
FIG. 34 shows Western Blots of 10 week knockdown ob/ob males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to lipid metabolism and insulin signaling pathway, western blots were performed. Quantification of each band is shown in the right. Regarding protein levels, phspho-Akt/Akt ratio in KD mice are lower than control. Fas and ACC were decreased. Phospho-FoxO1 and FoxO1 were decreased. Consistent with Fas and ACC, SREBP1 decreased in both fasted and refed state.
FIG. 35 shows Western Blots of 10 week overexpression ob/ob males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to lipid metabolism and insulin signaling pathway, western blots were performed. Quantification of each band is shown in the right. In overexpression mice, phospho-Akt was significantly increased. Fas, Acc were increased. Phospho-Foxo and Foxo1 were increased. SREBP1 was also increased. These results were mostly consistent with WT mice, suggesting that ILDR2 knockdown in ob mice induced the worse hepatic insulin resistance, and that ILDR2 overexpression in ob mice might improve hepatic insulin resistance.
FIG. 36—Prior Art—shows a schematic of the unfolded protein response pathways (See Zhang, K and R. J. Kaufmann. Nature 454 2008).
FIG. 37 shows Western Blots of 10 week knockdown C57BL/6J males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to ER stress response proteins, western blots were performed. Quantification of each band is shown in the right. IRE1a, phospho-eIF2a, and Bip were reduced in knockout mice. Nuclear proteins, XBP1, ATF6, and ATF4 were decreased in knockout mice.
FIG. 38 shows Western Blots of 10 week overexpression C57BL/6J males, chow fed (*P<0.05, ** P<0.01). To examine levels of proteins related to ER stress response proteins, western blots were performed. Quantification of each band is shown in the right. IRE1a, phospho-PERK, phospho-eIF2a, and Bip were increased. Nuclear proteins, XBP1, ATF6, and ATF4 were also increased.
FIG. 39 shows global gene expression analysis on Affymetrix Gene Mouse 1.0 Expression Arrays. Cells were transfected (GT1-7 and BTC6) or transduced (primary hepatocytes) with RNAi for ildr2 mRNA Lower panels indicate the levels of knock down for ildr2 mRNA for the three cells types.
FIG. 40 shows mRNA level changes representative of the individual UPR branches (IRE1a pathway). Represented are the intensity values from the expression array data. First row: results from GT1-7 cells. Second row: results form BTC6 cells. Third row: results form primary hepatocytes.
FIGS. 41A-H shows the sequences of the mouse peptides used to make antibodies to the LL protein. FIG. 41A shows the amino acid sequence of the ILDR2 α-intracellular domain antigen (amino acid #298-401) (SEQ ID NO: 2). FIG. 41B shows the amino acid sequence of the ILDR2 α-extracellular domain antigen (amino acid #22-186) (SEQ ID NO: 3). FIG. 41C shows the amino acid sequence of the human (ILDR2) cytoplasmic domain corresponding to amino acid 298-401 of Mouse ILDR2 (SEQ ID NO: 4). FIG. 41D shows the amino acid sequence of the human (ILDR2) intracellular domain corresponding to amino acid 22-186 of Mouse ILDR2 (SEQ ID NO: 5). FIG. 41E shows the ILDR2 α-intracellular domain antigen (amino acid #354-363) for the anti-intracellular-ILDR2 antibodies of the invention (SEQ ID NO: 6). FIG. 41F shows the ILDR2 α-extracellular domain antigen (amino acid #124-136) for the anti-extracellular-ILDR2 antibodies of the invention (SEQ ID NO: 7). FIG. 41G shows the amino acid sequence of the human (ILDR2) cytoplasmic domain corresponding to amino acid 354-363 of Mouse ILDR2 (SEQ ID NO: 8). FIG. 41H shows the amino acid sequence of the human (ILDR2) extracellular domain corresponding to amino acid 124-136 of Mouse ILDR2 (SEQ ID NO: 9).
FIG. 42 shows the DBA mouse ILDR2 gene 5′UTR, transcript and 3′UTR (SEQ ID NO:10). Shown are the DBA sequence of the 5′UTR, coding exons and 3′UTR of the mouse Ildr2 gene. The positions corresponding to B6 variants are shown in uppercase and highlighted clear. The 5′UTR is underlined, and each exon is alternately highlighted in gray; the 3′UTR is underlined.
FIG. 43 shows variant positions in the mouse ILDR2 anti-sense Transcript in DBA and B6 mice (SEQ ID NO: 11). Shown is the genomic DBA sequence corresponding to the anti-sense transcript, 5330438103RiK. The sequences of the intron preceding exon 8 are underlined. Exon 8 is highlighted gray. The intron between exons 8 and 9 is italicized. Exon 9 is highlighted gray and underlined. The intronic sequences telomeric to exon 9 and underlying the anti-sense transcript are shown in bold.
FIG. 44 shows SNP variants and positions in the mouse ILDR2 anti-sense Transcript in DBA (SEQ ID NO: 12) and B6 mice (SEQ ID NO: 13). Shown is a display generated by a BLAT analysis of the anti-sense transcript of the Ildr2 gene in mouse strain DBA/2J on the reference c57BL/6j genomic sequence. Exons 8 and 9 are underlined.
FIG. 45 shows ClustalW analysis of Ildr2(Lisch-like) homologs and the LSR protein. ClustalW analysis was performed on the EMBL-EBI server using their default settings. Display was modified to emphasize exonic alignments. Positions of non-synonymous variants in exon 9 of Ildr2 are identified by blue background. Non-homologous extension of mouse Lsr exon 6 (green background) is drawn beneath line. Abbreviations: B6, strain C57BL/6J; DBA, strain DBA/2J; ECD, extra-cellular domain; hpf, hours post-fertilization; Ig-like, immunoglobulin-like; ICD; intra-cellular domain; QTL, quantitative trait locus; TM, trans-membrane domain; T2DM, type 2 diabetes; UTR, untranslated region. Mm_Lisch-like (mouse Ildr2) (SEQ ID NO: 14); Hs_clorf32 (humans ILDR2) (SEQ ID NO: 15); Dr_Lisch-like (SEQ ID NO: 16); Mm_LSR (SEQ ID NO: 17).
FIG. 46 shows an alignment of comparative amino acid sequences for ILDR2 (LL) and related proteins. LL_Musmus (SEQ ID NO: 18); LL_Ratnor (SEQ ID NO: 19); LL_Bostau (SEQ ID NO: 20); LL_Canfam (SEQ ID NO: 21); LL_Homsap (SEQ ID NO: 22); LL_Pantro (SEQ ID NO: 23); LL_Macmul (SEQ ID NO: 24); LL_Feldom (SEQ ID NO: 25); LL_Mondom (SEQ ID NO: 26); LL_Galgal (SEQ ID NO: 27); LL_Xentro (SEQ ID NO:28); LL_Danrer (SEQ ID NO: 29); LSR_Homsap (SEQ ID NO: 30); LSR_Pantro (SEQ ID NO: 31); LSR_Macmul (SEQ ID NO: 32); LSR_Bostau (SEQ ID NO: 33); LSR_Canfam (SEQ ID NO: 34); LSR_Musmus (SEQ ID NO: 35); LSR_Ratnor (SEQ ID NO: 36); LSR_Mondom (SEQ ID NO: 37); LSR_Danrer (SEQ ID NO: 38); ILDR1_Homsap (SEQ ID NO: 39); ILDR1_Pantro (SEQ ID NO: 40); ILDR1_Ponpy (SEQ ID NO: 41); ILDR1_Musmus (SEQ ID NO: 42); ILDR1_Ratnor (SEQ ID NO: 43); ILDR1_Canfam (SEQ ID NO: 44); ILDR1_Xenla (SEQ ID NO: 45); ILDR1_Galgal (SEQ ID NO: 46); and ILDR1_Danrer (SEQ ID NO: 47).
FIGS. 47A-C shows spliced and unspliced sequences of the human ILDR2 (C1Orf32) Antisense RNA transcript. FIG. 47A shows the sequence of the unspliced human ILDR2 (C1Orf32) Antisense RNA transcript (SEQ ID NO: 48). FIG. 47B shows DA322725, a spliced anti-sense transcript of human ILDR2 (C1Orf32) corresponding to chr1:165156961-165228581 (SEQ ID NO: 49). FIG. 47C shows DA565656, a spliced anti-sense transcript of human ILDR2 (C1Orf32) corresponding to chr1:165156982-165225636 (SEQ ID NO 50).
FIG. 48 shows a schematic of Ildr2 conditional knockout construct.
FIGS. 49A-E show a schematic of the proposed effects of ER-stress on ILDR2 and downstream VLDL secretion and uptake in the liver. ER stress decreases ILDR2 (1)(FIG. 49A), causing increased VLDLR(2) (FIG. 49B). This causes increased uptake of VLDR particles in the liver (3) (FIG. 49C). Meanwhile, reduced ILDR2 also reduced VLDL secretion (4) (FIG. 49D) and suppress VLDL uptake (5) (FIG. 49E), possibly due to reduced incorporation of apoE into VLDL particles.
FIG. 50 shows ILDR2 interacts with ER stress-related proteins. Flag-tagged-ILDR2 vector was co-transfected with V5-tagged vectors as indicated into HEK 293 cells. Immunoprecipitation was carried out with anti-V5 antibody. Western blots were immunoblotted with either anti-Flag or anti-V5 antibody as shown.
FIG. 51A shows PERK cleavage of ILDR2. Flag-tagged-ILDR2 vector was co-transfected with V5-tagged PERK vector into HEK 293 cells. Western blots were immunoblotted with either anti-Flag or anti-V5 antibody. FIG. 51B shows PERK dominant negative did not cleave ILDR2. Flag-tagged-ILDR2 vector was co-transfected with V5-tagged PERK vector into HEK 293 cells. Western blots were immunoblotted with either anti-Flag or anti-V5 antibody.
FIG. 52 shows reduction of ildr2 protein by IRE1a co-transfection (flag-tagged ILDR2; v5 tagged IRE1a). The basis for this reduction appears to be reduced levels of ildr2 mRNA.
FIG. 53 shows real time PCR of ILDR2. Flag-tagged-ILDR2 were co-transfected with either V5-tagged IRE1a-WT or IRE1a-K599A expression vector into HEK 293 cells. Mouse and human ILDR2 transcript levels were markedly reduced (real time PCR) by co-transfection with IRE1a (by not the dominant-negative IRE1a isoform).
FIG. 54 shows real time PCR of endogenous ILDR2. Hepa1c1c7 cells were transfected with either IRE1a-WT or IRE1a-K599A expression vector. After overnight incubation, cells were cultured in the presence of actinomycin D (1 μg/m1) for different lengths of time. Actinomycin D blocks mRNA transcription.
FIG. 55 shows an Ildr2 mRNA degradation assay, Actinomycin D was added in Hepa1c1c7 cells. Xbp1 was spliced by IRE1a in time dependent and spliced Xbp1 increased. Ildr2 mRNA transcription was decreased by IRE1a but not dominant negative K599A IRE1a.
FIGS. 56A-B shows ILDR2 and ER stress protein expression levels in response to ER stress induced using tunicamycin, FIG. 56A: Hepa1c1c7 cells were treated with various concentrations of tunicamycin, then harvested at 12 hrs. FIG. 56B: Hepa1c1c7 cells were treated with 10 mg/mL tunicamycin, then harvested at various time points
FIG. 57 shows the human ILDR2 promoter and luciferase assay. The human ILDR2 promoter (SEQ ID NO: 152) has an ERSE response element. Hepa1c1c7 cells were co-transfected with pGL4 human Ildr2 promoter vector and either the active form of ATF6 or spliced XBP1 expression vectors.
FIG. 58 shows Hepa1c1c7 cells transfected with either the active form ATF6 or spliced Xbp1 vectors of various concentrations, then harvested at 48 hrs.
FIG. 59 shows a summary of the ER stress responses related to ILDR2 (SEQ ID NO: 152). ILDR2 binds PERK which cleaves ILDR2 (1). Transcription factors, ATF6 and XBP1 are increased by ER stress and downregulate ILDR2 transcripts by binding to its promoter region (2). IRE1a activated by ER stress degrades Ildr2 mRNA (3).
FIGS. 60A-D show the in vivo effects of tunicamycin on liver ILDR2 (lowering), and the protective effect of overexpressing ILDR2 in liver exposed to tunicamycin. Liver morphology and histology and Ildr2 expression in tunicamycin-treated mice is shown. Chow-fed, 10-week-old B6 males were administered tunicamycin for 72 h. FIG. 60A: Liver morphology at 72 hours post-injection in untreated and tunicamycin-treated samples. Lower panel: HE staining of representative sections. FIG. 60B: Ildr2 expression level determined by qPCR. FIG. 60C: Hepatic triglyceride increased from 16.4±5.4 to 45.0±6.7 mg/g liver (n=4). FIG. 60D. Hepatic Cholesterol increased from 7.1±1.2 to 10.6±0.9 mg/g liver. * indicates p<0.05; ** indicates p<0.01 (2 tailed t-test).
FIGS. 61A-D Liver phenotypes in tunicamycin-treated mice in which Ildr2 was overexpressed in liver. Chow-fed, 10-week-old B6 males overexpressing Ildr2 (GFP as control) in liver (3 days) were administered tunicamycin for 72 h. FIG. 61A: Liver gross morphology at 72 hours post-injection in untreated and tunicamycin-treated samples in hepatic ILDR2 overexpression mice. Lower panel: HE staining of representative sections. FIG. 61B: Ildr2 expression level determined by qPCR. FIGS. 61C and D: Measurements of hepatic triglyceride and cholesterol.



DETAILED DESCRIPTION
The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present disclosed subject matter pertains.
As various changes can be made in the methods and compositions described herein without departing from the scope and spirit of the disclosed subject matter as described, it is intended that all subject matter contained in this application and claims, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative, and not in a limiting sense.
DEFINITIONS
The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.
As used herein the term “ILDR2” or “Ildr2” refers to immunoglobulin-like domain containing receptor 2. ILDR2 is also known as, “Lisch-like” (“D” or “LL”) and “C1ORF32”. “ILDR2” refers to all ILDR2 orthologs, including, but not limited to, those found in mice and humans.
As used herein the term “Ildr2 RNA” includes any RNA, for example but not limited to unprocessed RNA, any mRNA of any splice variant (isoform), which encodes a full length Ildr2 protein (ILDR2), any fragment, any protein isoform, or any Ildr2 protein variant thereof. The term Ildr2 RNA also includes an antisense RNA to any Ildr2 mRNA, including but not limited to an antisense RNA to a full length mRNA, any portion of the full length mRNA, or any splice variant.
As used herein the terms “ILDR2” and “Ildr2” which are used interchangeably, include a full length ILDR2 protein, any ILDR2 protein fragment, ILDR2 isoform, or ILDR2 protein variant thereof.
As used herein, a “ILDR2 molecule” refers to an ILDR2 protein, or a fragment thereof. A “ILDR2 molecule” can also refer to a nucleic acid (including, for example, Ildr2 RNA, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to an ILDR2 protein, or fragment thereof.
As used herein the term “variant” covers nucleotide or amino acid sequence variants which have about 95%, about 90%, about 85%, about 80%, about 85%, about 80%, about 75%, about 70%, or about 65% nucleotide identity, or about 95%, about 90%, about 85%, about 80%, about 85%, about 80%, about 75%, or about 70% amino acid identity, including but not limited to variants comprising conservative, or non-conservative substitutions, deletions, insertions, duplications, or any other modification. The term variant as used herein includes functional and non-functional variants, and variants with reduced or altered activity.
As used herein, the term “agent” include, but are not limited to, biological or chemical agents, such as peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e. including heteroorganic and organometallic compounds), and salts, esters, and other pharmaceutically acceptable forms of such compounds. Salts, esters, and other pharmaceutically acceptable forms of such compounds are also encompassed.
Type 2 Diabetes and ILDR2
The identification of susceptibility genes in humans is complicated by the polygenic nature of the phenotype (Cox et al, 1992, Diabetes 41:401-407). This is refected in convergent yet distinct metabolic processes producing identical phenotypes (phenocopies) in a background of gene/gene and gene/environment (e.g., obesity) interactions that characterize the disease. Clear genetic influences on the endophenotypes (intermediate phenotypes) of β cell mass/function and insulin resistance vary among ethnic groups (Pimenta et al, 1995, Jama 273:1855-1861; Gelding et al, 1995, Clin Endocrinol (Oxf) 42:255-264; Knowler et al, 1993, Care 16:216-227; Hanley et al, 2003, Diabetes 52:463-469). Although more than 20 genome scans in ethnic and racial groups have detected numerous diabetes-susceptibility intervals of modest statistical significance, many of these results have not been replicated in other populations. Despite some successes (e.g. PPARG, CAPN10, TCF7L2), the number of genes conveying diabetes can vary by race/environment (Permutt et al, 2005, J Clin Invest 115:1431-1439).
Like humans, mouse strains differ widely in susceptibility to diabetes when made obese. As described herein, the differential diabetes susceptibilities of the B6 and DBA strains segregating for the obesity mutation Lepob (Clee S M, Attie A D (2007) The genetic landscape of type 2 diabetes in mice. Endocr Rev 28: 48-83) were used to identify a diabetes susceptibility QTL in B6×DBA progeny and then used congenic lines derived from the implicated interval to clone a candidate gene accounting for the QTL. Similar strategies have been used to identify QTLs (and responsible genes) for other complex phenotypes in mice (Flint J, Valdar W, Shifman S, Mott R (2005) Strategies for mapping and cloning quantitative trait genes in rodents. Nat Rev Genet 6: 271-286) such as type 1 diabetes (Todd J A (1999) From genome to aetiology in a multifactorial disease, type 1 diabetes. Bioessays 21: 164-174), diet-induced obesity (York B, Lei K, West D B (1996) Sensitivity to dietary obesity linked to a locus on chromosome 15 in a CAST/Ei×C57BL/6J F2 intercross. Mamm Genome 7: 677-681), tuberculosis susceptibility (Mitsos L M, Cardon L R, Fortin A, Ryan L, LaCourse R, et al. (2000) Genetic control of susceptibility to infection with Mycobacterium tuberculosis in mice. Genes Immun 1: 467-477), atherosclerosis (Welch C L, Bretschger S, Latib N, Bezouevski M, Guo Y, et al. (2001) Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the Ldlr knockout mouse model. Proc Natl Acad Sci USA 98: 7946-7951), epilepsy (Legare M E, Bartlett F S, 2nd, Frankel W N (2000) A major effect QTL determined by multiple genes in epileptic EL mice. Genome Res 10: 42-48), schizophrenia (Joober R, Zarate J M, Rouleau G A, Skamene E, Boksa P (2002) Provisional mapping of quantitative trait loci modulating the acoustic startle response and prepulse inhibition of acoustic startle. Neuropsychopharmacology 27: 765-781) and, also, T2DM (Clee S M, Yandell B S, Schueler K M, Rabaglia M E, Richards O C, et al. (2006) Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus. Nat Genet 38: 688-693; Goodarzi M O, Lehman D M, Taylor K D, Guo X, Cui J, et al. (2007) SORCS1: a novel human type 2 diabetes susceptibility gene suggested by the mouse. Diabetes 56: 1922-1929; Freeman H, Shimomura K, Horner E, Cox R D, Ashcroft F M (2006) Nicotinamide nucleotide transhydrogenase: a key role in insulin secretion. Cell Metab 3: 35-45; Freeman H C, Hugill A, Dear N T, Ashcroft F M, Cox R D (2006) Deletion of nicotinamide nucleotide transhydrogenase: a new quantitative trait locus accounting for glucose intolerance in C57BL/6J mice. Diabetes 55: 2153-2156).
In one aspect of this invention, these differential diabetes susceptibilities were exploited to map diabetes-susceptibility regions of the mouse genome using genetic crosses between a diabetes-susceptible (DBA) and a resistant strain (B6). In another aspect, the invention provides the identification of the genes responsible for the diabetes-related phenotypes of B6.DBA Lepob/ob F2 and F3 mice segregating for a QTL in the distal portion of Chr1. As described in the Examples of section herein, molecular genetic methods were used to identify to immunoglobulin-like domain containing receptor 2 (Ildr2), as a gene that accounts for diabetes susceptibility conveyed by the DBA interval in the intercross, and in B6.DBA N12-15 congenic progeny. The gene affects the early development and replication of beta cells and a reduced beta cell mass resulting in a predisposition to diabetes. In certain aspects, the invention provides methods to increase Ildr2 activity to reverse these effects. The gene encodes multiple, tissue-specific transcripts in brain, liver and islets. The functional consequences of the hypomorphic DBA allele (diabetes-prone) in Lepob/ob mice appear to be late embryonic to early postnatal reductions in β-cell mass due to diminished rates of β-cell replication, some “catch-up” of β-cell mass by 2-3 months, followed by mild glucose intolerance at >6 months of age. These phenotypes are recapitulated in mice with an ENU-induced null allele of Ildr2.
Ildr2 is a gene that produces multiple tissue-specific transcripts and is most highly expressed in brain, liver, and islets. Encoding a 10-exon 646 amino acid protein with significant homology to Lsr on Chr1qB1 and to Lldr1 on Chr16B3, Ildr2 spans 62.7 kb on Chr1qH2. The largest ILDR2 isoform is a predicted single-pass trans-membrane molecule with a signal sequence, an immunoglobulin-like extra-cellular domain and a serine/threonine rich intra-cellular domain that also contains a 14-3-3 binding domain and a terminal PDZ-binding motif.
The amounts of L1 transcripts are reduced 2-10 fold in these organs in mice segregating for DBA (v. B6) congenic intervals containing Ildr2. A recombination event between exons 8 and 9 of the 10 exon Ildr2 gene, has allowed characterization of the phenotypes of lines segregating for the complete DBA allele of Ildr2 versus B6. DBA lines containing only the distal portions (exons 9, 10 and 3′UTR) of the gene. The latter lines display phenotypes and organ-specific rates of Ildr2 expression comparable to the line containing the entire DBA allele of Ildr2, implicating 3′ UTR-mediated effects on message stability as a potential primary mechanism for the DBA allele's affects on diabetes-related phenotypes. There is also a 2845 bp in-frame antisense transcript running centromeric from exon 9 of Ildr2. In one embodiment, this antisense sequence can be used to squelch message in DBA v. B6 alleles of Ildr2. In another embodiment, this antisense sequence can be used to protect message in DBA v. B6 alleles of Ildr2. (Lapidot and Pilpel 2006, EMBO Rep 7:1216-1222; Costa 2005, Gene 357:83-94.).
The amino acid sequence of Ildr2 is highly homologous to the so-called “Lipolysis-stimulated receptor” (Lsr) (Yen et al, 1999, J Biol Chem 274:13390-13398). “Knockdown” of embryonic Zebrafish (D. rerio) paralogs of Ildr2 and Lsr results in disruption of endodermal organization and the integrity of the single large pancreatic islet in these animals. The physiological role(s) of Lsr—an apparent plasma membrane receptor—are unclear. The molecule is expressed in different tissues, including brain and liver. Homozygosity for a null allele of Lsr is embryonic lethal at E12.5-15.5 and associated with hepatic hypoplasticity, whereas the heterozygotes appear normal (Mesli et al, 2004, Eur J Biochem 271:3103-3114). LSR binds to apoliproteins B/E in the presence of free fatty acids, and can assist in the clearance of triglyceride-rich lipoproteins (Yen et al, 1999, J Biol Chem 274:13390-13398; Yen et al, 1994, Biochemistry 33:1172-1180). While LSR and ILDR2 are structurally homologous and may have overlapping functions, they are distinct enough so that they may also have non-overlapping functions and that reagents designed to be specific to either protein would not be predicted to cross-react.
LSR protein domains are described in U.S. Pat. No. 7,291,709. The table 11 below and description that follows show the sequence of several LSR domains compared to the corresponding aligned sequence in mouse ILDR2. Start and end amino acid residues refer to SEQ ID NO:17 (mouse LSR) and SEQ ID NO:14 (mouse ILDR2).







TABLE 11







Sequence of several LSR domains compared to


the corresponding aligned sequence in mouse ILDR2








Domain in LSR
Amino acid sequence (LSR and ILDR2)





Potential fatty
LSR 23-41:


acid binding
CLFLIIYCPDRASAIQVTV ((SEQ ID NO: 51)


site
ILDR2 7-25:



GWTAVFWLTAMVEGLQVTV (SEQ ID NO: 52)


 


Transmembrane
LSR 204-213:


domain
LEDWLFVVVV (SEQ ID NO: 53)



ILDR2 184-193:



MPEWVFVGLV (SEQ ID NO: 54)


 


Potential
LSR 214-249:


cytokine
CLASLLFFLLLGICWCQCCPHTCCCYVRCPCCPDKC (SEQ ID NO: 55)


receptor
ILDR2 194-229:


site
ILGIFLFFVLVGICWCQCCPHSCCCYVRCPCCPDSC (SEQ ID NO: 56)


 


Potential
LSR 544-558:


lipoprotein
ERR--------------------------------RVYREEEEEEEE (SEQ ID NO: 57)


ligand binding
ILDR2 540-586:


site
ESSSRGGSLETPSKLGAQLGPRSASYYAWSPPTTYKAGASEGEDEDD (SEQ ID NO: 58)









There are other structural similarities between LSR and ILDR2. For example, the NPGY sequence (SEQ ID NO: 141) in LSR (104-107), referred to as a putative clathrin-binding sequence on LSR, is a phosphotyrosine binding ligand of the class NPXY, that is contained in β-amyloid precursor proteins. The sequence NPDY (SEQ ID NO: 142) is found between residues 370-373 in ILDR2. Additionally, the RSRS motif (SEQ ID NO: 143) is within a proline-rich domain in LSR (470-473); a similar motif RSRASY (SEQ ID NO: 144) (561-565 of ILDR2) was identified by Motif Scan as a putative 14-3-3 Mode 1 binding motif. The ILDR2 sequence RAGSRF (SEQ ID NO: 145) (451-456 of ILDR2) was identified by the ELM Server as a potential 14-3-3 ligand.
ILDR2 may participate in a variety of processes. Like LSR, ILDR2 may be involved in the transport of fatty acids and and/or cholesterol. ILDR2 is expressed in liver, islets and the hypothalamus, and, based upon developmental and physiological studies, has effects on beta cell development and, possibly, function. These effects could be conveyed directly on the beta cell, or could be secondary to changes in the liver and/or hypothalamus. The high specific expression of ILDR2 transcripts in the hypothalamus and the relatively high specific concentration of ILDR2 polypeptide in the hypothalamus are consistent with a role for ILDR2 in control of hepatic glucose homeostasis and/or beta cell function by autonomic efferents from the hypothalamus. These have not yet been directly tested.
Non-limiting examples include for islet cell ontogenesis, cellular lipid homeostasis, hepatic and muscle insulin responsiveness and islet β cell function and survival. Identification of such functions can be important for understanding aspects of the pathogenesis of T2DM. In certain aspects, the invention provides methods to characterize the molecular physiology of ILDR2 in mice.
The human ortholog of mouse ILDR2, is 90% identical to Ildr2 at the amino acid level, maps to a region of Chr1q23 that has been repeatedly implicated in T2DM in seven ethnically diverse populations including Caucasians (Northern Europeans in Utah) (Elbein et al, 1999, Diabetes 48:1175-1182), Amish Family Study (Hsueh et al, 2003, Diabetes 52:550-557, St. Jean 2000, American Journal of Human Genetics 67), United Kingdom Warren 2 study (Wiltshire et al, 2001 Am J Hum Genet 69:553-569), French families (Vionnet et al, 2000, Am J Hum Genet 67:1470-1480), and Framingham Offspring study (Meigs et al, 2002, Diabetes 51:833-840), Pima Indians (Hanson et al, 1998, J Hum Genet 63:1130-1138), and Chinese (Xiang, et al, 2004, Diabetes 53:228-234) with LOD scores as high as 4.3. There is evidence of association of alleles of Ildr2 with T2D in several of these populations. The mouse congenic interval examined as described herein is in the middle of, and physically  ̃10× smaller than, the 30 Mb human interval. Recent analysis of the broad interval ascertained in Utah identified two peaks, one of which, at D152762 (@163.6 Mb), is just 12 kb telomeric to the 5′ end of the Ildr2 gene (Das S K, Elbein S C (2007) The search for type 2 diabetes susceptibility Loci: the chromosome 1q story. Curr Diab Rep 7: 154-164). The genes, and gene order, are generally conserved between mouse and human in the region syntenic to the congenic interval. The metabolic phenotypes documented in human subjects with T2DM linked to 1q23 resemble diabetic phenotypes observed in congenic mice segregating for the DBA interval in B6.DBA congenics examined here (McCarthy M, Shuldiner, A. R., Bogardus, C., Hanson, R. L., Elbein, S., (2004) Positional Cloning of a Type 2 Diabetes Susceptibility Gene on Chromosome 1q: A collaborative effort by the Chromosome 1q Diabetes Positional Cloning Consortium. 1-39), suggesting that the diabetes-susceptibility gene in congenic mice and human subjects may be the same gene, or among the genes, acting in the same genetic pathway(s). The syntenic interval in the GK rat also correlates with diabetes-susceptibility (Chung W K, Zheng M, Chua M, Kershaw E, Power-Kehoe L, et al. (1997) Genetic modifiers of Leprfa associated with variability in insulin production and susceptibility to NIDDM. Genomics 41: 332-344).
Data described herein identify two non-synonymous amino acid variants in ILDR2 of DD mice: T587A and A647V (both found in exon 9 in Ildr2). These positions correspond to Glycine-572 and Alanine-625 in human Ildr2, respectively. In certain aspects, the invention provides methods to determine whether these amino acid variants: (a) decrease protein stability and (b) change protein function in any way. To determine the effect of these amino acids changes, these mutation can be engineered in expression vectors for mammalian transfections, and functional characterization experiments as described herein can be carried out for the mutant Ildr2 variants. The T587A mutation abolishes a potential phosphorylation site. Methods for investigating the role of phosphorylation are well known to those skilled in the art.
Insight into the function(s) of the mouse Ildr2 protein may be gained from similarities in structure, expression, and cellular location with the human paralog and with genes encoding related trans-membrane receptors, Ildr1 (Hauge H, Patzke S, Delabie J, Aasheim H C (2004) Characterization of a novel immunoglobulin-like domain containing receptor. Biochem Biophys Res Commun 323: 970-978) and Lsr (Yen F T, Masson M, Clossais-Besnard N, Andre P, Grosset J M, et al. (1999) Molecular cloning of a lipolysis-stimulated remnant receptor expressed in the liver. J Biol Chem 274: 13390-13398). Splicing patterns of these genes generate isoforms, similar to those of Ildr2. Each gene's largest isoform includes an extra-cellular Ig-like domain, a single TMD, and a similar set of ICDs in related order. In one isoform of each protein, the TMD and cysteine-rich domains are absent. An evolutionary, regulatory relationship is suggested by the observation that the Ildr2-paralog and lldr1 are adjacent in the zebra fish genome (Zv6 assembly, UCSC Genome Browser). All three genes are abundantly expressed in the brain, liver and pancreas (and islets, where studied), and all are predicted to have 14-3-3 interacting domains (thus far experimentally verified for the human LSR) (Garcia-Ocana A, Takane K K, Syed M A, Philbrick W M, Vasavada R C, et al. (2000) Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia. J Biol Chem 275: 1226-1232). Although 14-3-3 interacting domains may be present on as many as 0.6% of human proteins, their occurrence on all of these Lisch-related proteins is notable, since among known 14-3-3-interacting proteins is phoshodiesterase-3B, which is implicated in diabetes and pancreatic β-cell physiology (Onuma H, Osawa H, Yamada K, Ogura T, Tanabe F, et al. (2002) Identification of the insulin-regulated interaction of phosphodiesterase 3B with 14-3-3 beta protein. Diabetes 51: 3362-3367; Xiang K, Wang Y, Zheng T, Jia W, Li J, et al. (2004) Genome-wide search for type 2 diabetes/impaired glucose homeostasis susceptibility genes in the Chinese: significant linkage to chromosome 6q21-q23 and chromosome 1q21-q24. Diabetes 53: 228-234; Pozuelo Rubio M, Geraghty K M, Wong B H, Wood N T, Campbell D G, et al. (2004) 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking Biochem J 379: 395-408), and others, such as the Cdc25 family members, important in regulating cell proliferation and survival (Meek S E, Lane W S, Piwnica-Worms H (2004) Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins. J Biol Chem 279: 32046-32054; Hermeking H, Benzinger A (2006) 14-3-3 proteins in cell cycle regulation. Semin Cancer Biol 16: 183-192).
Screening Methods to Identify Agents which Modulate Expression of Ildr2 or ILDR2
In certain aspects the invention provides methods to identify agents which modulate expression of Ildr2 or ILDR2, the method comprising determining expression in the absence of a candidate agent, contacting a cell with a candidate agent, determining expression in the presence of the candidate agent, and comparing the expression determined in the presence and the absence of the candidate agent. In certain aspects, the invention provides a method for identifying an agent which modulates expression of an Ildr2 RNA comprising: (a) determining expression of an Ildr2 RNA in a cell, (b) contacting the cell with an agent; and (c) determining expression of the Ildr2 RNA in the presence of the agent, wherein a change in the expression of the Ildr2 RNA in the presence of the agent, compared to the expression of the Ildr2 RNA in the absence of the agent, is indicative of an agent which modulates the expression of the Ildr2 RNA. In certain embodiments, the method comprises: (a) contacting a cell with an agent; (b) determining expression of the Ildr2 RNA in the presence and the absence of the agent; and (c) comparing expression of the Ildr2 RNA in the presence and the absence of the agent, wherein a change in the expression of the Ildr2 RNA in the presence of the agent is indicative of an agent which modulates the level of expression of the RNA. In certain embodiments, the method measures expression of ILDR2 RNA. In certain embodiments, the assay is carried out in a cell which is comprised in an animal. In a non-limiting example the animal is a mouse. In other embodiments, the assay is carried out in a cell which is comprised in a tissue culture and/or a cell line derived from tissues of a mouse, or a human subject. In certain aspects, the cell is comprised in a diabetes-relevant tissue. In other aspects, the cell is derived from any tissue or source which allows to determine modulation of expression of Ildr2 or ILDR2. In non-limiting examples, the cell is a pancreatic cell, an insulin producing beta cell, or a hepatocyte, a hypothalamic or other brain cell, or any combination thereof.
In certain embodiments, the method is carried out in a cell which expresses endogenous Ildr2 or ILDR2. In other embodiments, the method is carried out in a cell comprising an expression vector or a construct comprising nucleic acid which encodes Ildr2 or ILDR2. The nucleic acid encoding Ildr2 or ILDR2 can be a nucleic acid, for example encoding any splice variant, isoform, or a fragment, a genomic DNA, or any portion of the genomic DNA. In certain aspects, the expression vector is introduced by transfection into an autologous cell type. In other aspects, the expression vector is introduced by transfection into a non-autologous cell type. Methods to create expression vectors and constructs are well known in the art. Non-limiting examples of various expression vectors, cells, tissues, and cell lines are described herein. In certain embodiments, the cell can comprise any other suitable nucleic acid or an expression vectors comprising a nucleic acid which encodes such suitable nucleic acid. In non-limiting examples, such suitable nucleic acid can be a nucleic acid which encodes a Ildr2- or ILDR2-interacting, and/or regulatory partner.
In certain embodiments, determining comprises quantitative determination of the level of expression. In other embodiments, determining comprises quantitative determination of the stability or turnover of Ildr2 or ILDR2. Methods for determining expression of a RNA or a protein, including quantitative and/or qualitative determinations, are described herein and well known in the art. In certain embodiments, the methods of the invention determine an increase in the expression. In other embodiments, the methods of the invention determine a decrease in the expression. The expression of a gene can be readily detected, e.g., by quantifying the protein and/or RNA encoded by the gene. Many methods standard in the art can be thus employed, including, but not limited to, immunoassays to detect and/or visualize protein expression, nonlimiting examples include western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunocytochemistry, etc., and/or hybridization assays to detect gene expression by detecting and/or visualizing respectively RNA, including but not limited to mRNA encoding a gene (PCR, northern assays, dot blots, in situ hybridization, etc.). Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Non-limiting exemplary assays are described herein.
In certain embodiments, the methods of the invention can determine changes in the expression, associated with changes in the localization, processing, trafficking, posttranslational modification, or any other cellular modification of Ildr2 or ILDR2. Determining expression of Ildr2 or ILDR2 can be carried out by any suitable method as described herein, or known in the art.
In certain embodiments, the step of contacting a cell with an agent is under conditions suitable for gene or protein expression. In certain embodiments, contacting step is in an aqueous solution comprising a buffer and a combination of salts. In certain embodiments, the aqueous solution approximates or mimics physiologic conditions.
In certain embodiments, once an agent has been identified to modulate expression, and optionally, the structure of the compound has been identified, the agent can be further tested for biological activity in additional assays and/or animal models for type 2 diabetes or metabolic disease. In addition, a lead compound can be used to design analogs, and other structurally similar compounds.
In certain embodiments, the invention provides screening of libraries of agents, including combinatorial libraries, to identify an agent which modulate the expression. Libraries screened using the methods of the present invention can comprise a variety of types of compounds. Non-limiting examples of libraries that can be screened in accordance with the methods of the invention include, but are not limited to, peptoids; random biooligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small molecule libraries, for example but not limited to small organic molecules. In certain embodiments, the compounds in the libraries screened are nucleic acid or peptide molecules. In a non-limiting example, peptide molecules can exist in a phage display library. In other embodiments, the types of compounds include, but are not limited to, peptide analogs including peptides comprising non-naturally occurring amino acids, e.g., D-amino acids, phosphorous analogs of amino acids, such as α-amino phosphoric acids and α-amino phosphoric acids, or amino acids having non-peptide linkages, nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. Libraries of polypeptides or proteins can also be used in the assays of the invention.
In certain embodiments, the combinatorial libraries are small organic molecule libraries including, but not limited to, benzodiazepines, isoprenoids, beta carbalines, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, small inhibitory RNAs short hairpin RNAs, and benzodiazepines. In another embodiment, the combinatorial libraries comprise peptoids; random bio-oligomers; benzodiazepines; diversomers such as hydantoins, benzodiazepines and dipeptides, vinylogous polypeptides; nonpeptidal peptidomirnetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; or carbohydrate libraries. Combinatorial libraries are themselves commercially available from different sources.
In a certain embodiments, the library is preselected so that the compounds of the library are more amenable for cellular uptake. For example, compounds are selected based on specific parameters such as, but not limited to, size, lipophilicity, hydrophilicity, and hydrogen bonding, which enhance the ability of compounds to enter into the cells. In other embodiments, the compounds are analyzed by three-dimensional or four-dimensional computer computation programs.
Methods to synthesize and screen combinatorial libraries are known in the art. In one embodiment, the combinatorial compound library can be synthesized in solution. In other embodiments the combinatorial libraries can be synthesized on solid support. For non-limiting examples of such methods see U.S. Pat. No. 5,866,341 to Spinella et al., U.S. Pat. No. 6,190,619 to Kilcoin et al., U.S. Pat. No. 6,194,612 to Boger et al.; Egner et al., 1995, J. Org. Chem. 60:2652; Anderson et al., 1995, J. Org. Chem. 60:2650; Fitch et al., 1994, J. Org. Chem. 59:7955; Look et al., 1994, J. Org. Chem. 49:7588; Metzger et al., 1993, Angew. Chem., Int. Ed. Engl. 32:894; Youngquist et al., 1994, Rapid Commun. Mass Spect. 8:77; Chu et al., 1995, J. Am. Chem. Soc. 117:5419; Brummel et al., 1994, Science 264:399; and Stevanovic et al., 1993, Bioorg. Med. Chem. Lett. 3:431; Lam et al., 1997, Chem. Rev. 97:41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926 a Nefzi et al., 1997, Chem. Rev. 97:449-472; and references cited therein, all of which are hereby incorporated by reference in their entirety.
Agents that modulate expression, as identified by the methods described herein can be selected and characterized by methods known in the art. For example, if the library comprises arrays or microarrays of agents, wherein each agent has an address or identifier, the agent can be deconvoluted, e.g., by cross-referencing the positive sample to original compound list that was applied to the individual test assays. If the library is a peptide or nucleic acid library, the sequence of the compound can be determined by direct sequencing of the peptide or nucleic acid. Such methods are well known to one of skill in the art. A number of physico-chemical techniques can also be used for the de novo characterization of compounds that modulate expression as determined by the methods of the present invention. Examples of such techniques include, but are not limited to, mass spectrometry, NMR spectroscopy, X-ray crystallography and vibrational spectroscopy.
In certain aspects, the invention provides methods for identifying metabolic or environmental agents and/or stimuli (e.g., exposure to different concentrations of metabolites, nutrients, or the like, or of CO2 and/or O2, stress and different pHs,) that modulate untranslated region-dependent expression of a target gene utilizing the cell-based reporter gene assays described herein. In another embodiment, the environmental stimuli does not include a compound. In non-limiting examples, the metabolic agent is insulin, cAMP, glucose, free fatty acids, cholesterol or a combination thereof.
Antibodies to ILDR2
Using standard immunization protocols, polyclonal rabbit and guinea pig antibodies (Covance Research Products) were generated against the predicted extracellular domain (ECD) of mouse ILDR2 spanning residues 22-186, and intracellular domain (ICD) spanning residues 298-401. α-ICD and α-ECD rabbit antibodies detected the appropriate fusion proteins, showing only minor cross-reactivity. Another set of antibodies to smaller ECD and ICD epitopes (FIG. 41A and FIG. 41B) were generated to detect the localized expression pattern of Ildr2 in pancreatic β cells in non-diabetic mice, as well as an undetectable ILDR2 protein level in diabetic D/D mice—that show reduced β cell replication and reduced islet mass—indicates that Ildr2 can play a critical role in β cell development.
In one aspect, the invention provides antibody that binds to the peptide which is from the extracellular domain (ECD) of mouse ILDR2 spanning residues 22-186 (SEQ ID NO: 3), or a (poly)peptide which comprises the peptide of SEQ ID NO: 7. In another aspect of the invention provides antibody that binds to the peptide which is from the intracellular domain (ICD) of mouse ILDR2 spanning residues 298-401 (SEQ ID NO: 2), or a (poly)peptide which comprises the peptide of SEQ ID NO: 6. In another aspect of the invention provides antibody that binds to the peptide which is from the extracellular domain (ECD) of human ILDR2 spanning residues shown in SEQ ID NO: 5, or a (poly)peptide which comprises the peptide of SEQ ID NO: 9. In another aspect of the invention provides antibody that binds to the peptide which is from the intracellular domain (ICD) of human ILDR2 spanning residues shown in SEQ ID NO: 4, or a (poly)peptide which comprises the peptide of SEQ ID NO: 8. In one aspect, the invention provides antibody that binds to an epitope of the polypeptide of SEQ ID NO: 22.
In another aspect, the antibodies of the invention are isolated. The antibodies of the invention can be monoclonal or polyclonal. Methods for making polyclonal and monoclonal antibodies are well known in the art. Antibodies of the invention can be produced by methods known in the art in any suitable animal host such as but not limited to rabbit, goat, mouse, sheep. In one embodiment, the antibodies can be chimeric, i.e. a combination of sequences of more than one species. In another embodiment, the antibodies can be fully-human or humanized Abs. Humanized antibodies contain complementarity determining regions that are derived from non-human species immunoglobulin, while the rest of the antibody molecule is derived from human immunoglobulin. Fully-human or humanized antibodies avoid certain problems of antibodies that possess non-human regions which can trigger host immune response leading to rapid antibody clearance. In still another embodiment, antibodies of the invention can be produced by immunizing a non-human animal with an immunogenic composition comprising a polypeptide of the invention in the monomeric form. In other embodiments, dimeric or multimeric forms can be used. The immunogenic composition can also comprise other components that can increase the antigenicity of the inventive peptide. In one embodiment the non-human animal is a transgenic mouse model, for e.g., the HuMAb-MouseTM or the Xenomouse®, which can produce human antibodies. Neutralizing antibodies against peptides of interest and the cells producing such antibodies can be identified and isolated by methods know in the art.
Making of monoclonal antibodies is well known in the art. In one embodiment, the monoclonal antibodies of the invention are made by harvesting spleen tissue from a rabbit which produces a polyclonal antibody. Harvested cells are fused with the immortalized myeloma cell line partner. After an initial period of growth of the fused cells, single antibody producing clones are isolated by cell purification, grown and analyzed separately using a binding assay (e.g., ELISA, or Western). Hybridomas can be selected based on the ability of their secreted antibody to bind to a peptide interest, including a polypeptide comprising SEQ ID NOs: 2-9. Variable regions can be cloned from the hybridomas by PCR and the sequence of the epitope binding region can be determined by sequencing methods known in the art.
The invention provides antibodies and antibody fragments of various isotypes. The recombined immunoglobulin (Ig) genes, for example the variable region genes, can be isolated from the deposited hybridomas, by methods known in the art, and cloned into an Ig recombination vector that codes for human Ig constant region genes of both heavy and light chains. The antibodies can be generated of any isotype such as IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, IgA1, IgA2, or sIgA isotype. The invention provides isotypes found in non-human species as well such as but not limited to IgY in birds and sharks. Vectors encoding the constant regions of various isotypes are known and previously described. (See, for example, Preston et al. Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for P. aeruginosa serogroup O6 lipopolysaccharide. Infect Immun. 1998 September; 66(9):4137-42; Coloma et al. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Methods. 1992 Jul. 31; 152(1):89-104; Guttieri et al. Cassette vectors for conversion of Fab fragments into full-length human IgG1 monoclonal antibodies by expression in stably transformed insect cells. Hybrid Hybridomics. 2003 June; 22(3):135-45; McLean et al. Human and murine immunoglobulin expression vector cassettes. Mol Immunol. 2000 October; 37(14):837-45; Walls et al. Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions. Nucleic Acids Res. 1993 Jun. 25; 21(12):2921-9; Norderhaug et al. Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells. J Immunol Methods. 1997 Can 12; 204(1):77-87.)
The antibodies of the invention bind to a polypeptide having the sequence of any of SEQ ID NOs: 2-9, comprised in a longer polypeptide, in a specific manner. In one embodiment, the antibodies, or antibody fragments of the invention bind specifically to a peptide of SEQ ID NO: 2, 3, 4, or 5. In one embodiment, the antibodies, or antibody fragments of the invention bind specifically to a peptide of SEQ ID NO: 6, 7, 8, or 9. For example, antibodies that bind specifically to a peptide that comprises a sequences shown in any of SEQ ID NOs: 2-9 will not bind to polypeptides which do not comprise the amino acid sequence of any of SEQ ID NO: 2-9 to the same extent and with the same affinity as they bind to a peptide that comprises a sequences shown in any of SEQ ID NOs: 2-9. In another embodiment, the antibody, or/and antibody fragments, of the invention can bind specifically to polypeptides which comprise any of SEQ ID NOs: 14-47, but this binding can occur with lesser affinity compared to the binding to a polypeptide that comprises a sequences shown in any of SEQ ID NOs: 2-9. Lesser affinity can include at least 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or 95% less.
The present invention provides specific monoclonal antibodies, including but not limited to rabbit, mouse and human, which recognize a peptide of SEQ ID NO: 2, 3, 4, or 5, including a polypeptide comprising SEQ ID NO: 6, 7, 8, or 9. When used in vivo in humans, human monoclonal antibodies are far less likely to be immunogenic (as compared to antibodies from another species).
Variable region nucleic acids for the heavy and light chains of the antibodies can be cloned into an human Ig expression vector that contain any suitable constant region, for example (i.e., TCAE6) that contains the IgG1 (gamma 1) constant region coding sequences for the heavy chain and the lambda constant region for the light chains. (See, for example, Preston et al. Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for P. aeruginosa serogroup O6 lipopolysaccharide. Infect Immun. 1998 September; 66(9):4137-42.) The variable regions can be placed in any vector that encodes constant region coding sequences. For example, human Ig heavy-chain constant-region expression vectors containing genomic clones of the human IgG2, IgG3, IgG4 and IgA heavy-chain constant-region genes and lacking variable-region genes have been described in Coloma, et al. 1992 J. Immunol. Methods 152:89-104.) These expression vectors can then be transfected into cells (e.g., CHO DG44 cells), the cells are grown in vitro, and IgG1 are subsequently harvested from the supernatant. Resultant antibodies can be generated to posses human variable regions and human IgG1 and lambda constant regions. In other embodiments, the Fc portions of the antibodies of the invention can be replaced so as to produce IgM.
In other embodiments, the antibody of the invention also includes an antibody fragment. It is well-known in the art, only a portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford; and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology, Infection and Immunity (2004) 1st Ed. American Society for Microbiology Press, Washington D.C.). The pFc′ and Fc regions of the antibody, for example, are effectors of the complement cascade and can mediate binding to Fc receptors on phagocytic cells, but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, e.g. an F(ab′)2 fragment, retains both of the antigen binding sites of an intact antibody. An isolated F(ab′)2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, e.g. an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd (heavy chain variable region). The Fd fragments are the major determinant of antibody specificity (a single Fd fragment can be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. An antibody fragment is a polypeptide which can be targeted to the nucleus. Methods to modify polypeptides for targeting to the nucleus are known in the art.
Additional methods of producing and using antibodies and antibody fragments comprising Fab, Fc, pFc′, F(ab′)2 and Fv regions are well known in the art [Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford); and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology, Infection and Immunity (2004) 1st Ed. American Society for Microbiology Press, Washington D.C.].
Usually the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions of the donor antibody. However, in certain embodiments, it can be desirable to modify one or more CDR regions to modify the antigen binding specificity of the antibody and/or reduce the immunogenicity of the antibody. One or more residues of a CDR can be altered to modify binding to achieve a more favored on-rate of binding, a more favored off-rate of binding, or both, such that an idealized binding constant is achieved. Using this strategy, an antibody having high or ultra high binding affinity of can be achieved. Briefly, the donor CDR sequence is referred to as a base sequence from which one or more residues are then altered. Affinity maturation techniques can be used to alter the CDR region(s) followed by screening of the resultant binding molecules for the desired change in binding. The method can also be used to alter the donor CDR to be less immunogenic such that a potential chimeric antibody response is minimized or avoided. Accordingly, as CDR(s) are altered, changes in binding affinity as well as immunogenicity can be monitored and scored such that an antibody optimized for the best combined binding and low immunogenicity are achieved (see, e.g., U.S. Pat. No. 6,656,467 and U.S. Pat. Pub. Nos: US20020164326A1; US20040110226A1; US20060121042A1).
The antibodies of the invention can be used in a variety of applications including but not limited to (a) methods for diagnosing type 2 diabetes or metabolic disease in a subject, wherein the antibody is used to determine different expression of ILDR2 in a blood or other tissue sample from a subject compared to the expression of ILDR2 in a control sample, (b) methods for screening agents, including but not limited to small molecule drugs, biological agents, in order to identify and monitor agents which can modulate the expression, production, localization, and/or stability of ILDR2. Additionally, such antibodies could be used to affect the action or regulate the activity of the native peptide at surface of the cell, or to detect shed molecules in the circulation as a diagnostic.
In one aspect, the antibodies that specifically bind polypeptide of SEQ ID NO: 2-9 or a polypeptide which comprises the corresponding peptide, can be used in a screening method to evaluate agents designed to affect the levels of expression of ILDR2. Because the antibody can be used to quantitate protein levels and expression, protein localization, or protein modification of ILDR2. The effect, including the efficiency and/or potency, of the drug can be addressed by following its effect on the presence, or absence, or change, for example but not limited to change in levels of the ILDR2, which can be detected by the antibody of the invention.
The antibodies of the present invention, including fragments and derivatives thereof, can be labeled. It is, therefore, another aspect of the present invention to provide labeled antibodies that bind specifically to one or more of the polypeptides of the present invention, to one or more of the polypeptides encoded by the isolated nucleic acid molecules of the present invention, or the binding of which can be competitively inhibited by one or more of the polypeptides of the present invention or one or more of the polypeptides encoded by the isolated nucleic acid molecules of the present invention. The choice of label depends, in part, upon the desired use.
For example, when the antibodies of the present invention are used for immunohistochemical staining of tissue samples, the label can usefully be an enzyme that catalyzes production and local deposition of a detectable product. Enzymes useful as conjugates to antibodies to permit antibody detection are well known. Exemplary conjugataes are alkaline phosphatase, p-galactosidase, glucose oxidase, horseradish peroxidase (HRP), and urease. Exemplary substrates for production and deposition of visually detectable products are o-nitrophenyl-beta-D-galactopyranoside (ONPG); o-phenylenediamine dihydrochloride (OPD); p-nitrophenyl phosphate (NPP); p-nitrophenyl-beta-D-galactopryanoside (PNPG); 3′,3′-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol (CN); 5-bromo-4-chloro-3-indolyl-phosphate (BCIP); ABTS®; BluoGal; iodonitrotetrazolium (INT); nitroblue tetrazolium chloride (NBT); phenazine methosulfate (PMS); phenolphthalein monophosphate (PMP); tetramethyl benzidine (TMB); tetranitroblue tetrazolium (TNBT); X-Gal; X-Gluc; and X-Glucoside.
Other substrates can be used to produce luminescent products for local deposition. For example, in the presence of hydrogen peroxide (H2O2), horseradish peroxidase (HRP) can catalyze the oxidation of cyclic diacylhydrazides, such as luminol. Immediately following the oxidation, the luminol is in an excited state (intermediate reaction product), which decays to the ground state by emitting light. Strong enhancement of the light emission is produced by enhancers, such as phenolic compounds. Advantages include high sensitivity, high resolution, and rapid detection without radioactivity and requiring only small amounts of antibody. See, e.g., Thorpe et al., Methods Enzymol. 133: 331-53 (1986); Kricka et al., J. Immmunoassay 17(1): 67-83 (1996); and Lundqvist et al., J. Biolumin. Chemiluimin. 10(6): 353-9 (1995). Kits for such enhanced chemiluminescent detection (ECL) are available commercially. The antibodies can also be labeled using colloidal gold.
As another example, when the antibodies of the present invention are used, e.g., for flow cytometric detection, for scanning laser cytometric detection, or for fluorescent immunoassay, they can usefully be labeled with fluorophores. There are a wide variety of fluorophore labels that can usefully be attached to the antibodies of the present invention. For flow cytometric applications, both for extracellular detection and for intracellular detection, common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, CyS, fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-CyS, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7.
Other fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, all of which are also useful for fluorescently labeling the antibodies of the present invention. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin.
When the antibodies of the present invention are used, e.g., for western blotting applications, they can usefully be labeled with radioisotopes, such as 33P, 32P, 35S, 3H, and 125I. As another example, when the antibodies of the present invention are used for radioimmunotherapy, the label can usefully be 228Th, 227Ac, 225Ac, 223Ra, 213Bi, 212Pb, 212Bi, 211At, 203Pb, 194Os, 188Re, 186Re, 153Sm, 149Tb, 131I, 125I, 111In, 105Rh, 99mTc, 97Ru, 90Y, 90Sr, 88Y, 72Se, 67Cu, or 47Sc.
As another example, when the antibodies of the present invention are to be used for in vivo diagnostic use, they can be rendered detectable by conjugation to MRI contrast agents, such as gadolinium diethylenetriaminepentaacetic acid (DTPA), Lauffer et al., Radiology 207(2): 529-38 (1998), or by radioisotopic labeling.
The antibodies of the present invention, including fragments and derivatives thereof, can also be conjugated to toxins, in order to target the toxin's ablative action to cells that display and/or express the polypeptides of the present invention. The antibody in such immunotoxins is conjugated to Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, or ricin. See Hall (ed.), Immunotoxin Methods and Protocols (Methods in Molecular Biology, vol. 166), Humana Press (2000); and Frankel et al. (eds.), Clinical Applications of Immunotoxins, Springer-Verlag (1998).
The antibodies of the present invention can usefully be attached to a substrate, and it is, therefore, another aspect of the invention to provide antibodies that bind specifically to one or more of the polypeptides of the present invention, to one or more of the polypeptides encoded by the isolated nucleic acid molecules of the present invention, or the binding of which can be competitively inhibited by one or more of the polypeptides of the present invention or one or more of the polypeptides encoded by the isolated nucleic acid molecules of the present invention, attached to a substrate. Substrates can be porous or nonporous, planar or nonplanar. For example, the antibodies of the present invention can usefully be conjugated to filtration media, such as NHS-activated Sepharose or CNBr-activated Sepharose for purposes of immunoaffinity chromatography. For example, the antibodies of the present invention can usefully be attached to paramagnetic microspheres by, for example, biotin-streptavidin interaction. The microsphere can then be used for isolation of one or more cells that express or display the polypeptides of the present invention. As another example, the antibodies of the present invention can be attached to the surface of a microtiter plate for ELISA.
As noted herein, the antibodies of the present invention can be produced in prokaryotic and eukaryotic cells. It is, therefore, another aspect of the present invention to provide cells that express the antibodies of the present invention, including hybridoma cells, Beta cells, plasma cells, and host cells recombinantly modified to express the antibodies of the present invention.
In yet a further aspect, the present invention provides aptamers evolved to bind specifically to one or more of the ILDR2 proteins of the present invention or to polypeptides encoded by the nucleic acids of the invention.
In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which can be used to alter the biological properties of the antibodies of this invention including methods which can increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that can render it more suitable for a particular application.
Cellular Biology of ILDR2 (Ildr2)
Embodiments and aspects described herein refer specifically to Ildr2, however, any of the described assays, techniques, reagents, experiments and so forth are equally applicable to determining and characterizing function and cellular biology of other ILDR2 homologues and orthologues, including but not limited to the human orthologue Ildr2.
In certain aspects, the invention provides that ILDR2 promotes Beta cell growth, and can regulate peripheral metabolism through its effects on liver function. Both of these effects can be conveyed via the CNS/hypothalamus where ILDR2 is expressed. There are precedents for such effects on liver glucose metabolism and islet Beta cell function. ILDR2 function can be determined using assays of protein biosynthesis, processing, sub-cellular localization, signaling properties. Structure/function relationships are analyzed by way of gain- and loss-of-function experiments in appropriate cellular contexts.
In certain embodiments, the invention provides that highest levels of Ildr2 expression are found in liver, brain, Beta cell/islet, and skeletal muscle. The metabolic properties of these organs are distinct, and make it difficult to identify an overarching function of the ILDR2 protein. Ki67 labeling studies indicate that Beta cell proliferation is reduced in the early post-natal period in DD (hypomorphic) congenics, indicating function for Ildr2 in the regulation of 0 cell mass. Thus, ILDR2 modulates pancreatic Beta cell proliferation directly, or indirectly. ILDR2 cellular biological features can be determined by assays described herein and any other suitable method known in the art, in physiologically relevant cell types.
In certain aspects the invention provides antisera and antibodies against epitopes of predicted intra and extracellular domains that detect ILDR2 in immunoprecipitation, immunoblot and immunohistochemistry assays. These antibodies can be used to determine the cellular properties of the endogenous protein.
In other aspects the invention provides reagents to study the properties of Ildr2 in gain-of-function experiments. Non-limiting examples of such reagents are FLAG epitope-tagged mammalian expression vectors. An ILDR2-GFP fusion protein has been constructed and can be used to analyze sub-cellular localization. ILDR2- and/or C1ORF32-fusion proteins to any other fluorescent protein variant, or any other protein reporter, or protein tag can also be generated. Also provided are mammalian expression vectors with N-terminal and C-terminal epitope tags and adenoviruses encoding WT Ildr2. Ildr2 siRNA constructs have been tested and shown effective in HEK 293 cells. These probes can be engineered into adenoviral vectors for efficient gene knockdown in cultured cells and mice. siRNA-resistant rescue vectors can be generated in which synonymous nucleotide changes are introduced in the Ildr2 cDNA to render it resistant to siRNA-mediated degradation. These constructs can be used to validate the specificity of the Ildr2 siRNA. For most experiments described, mammalian expression vectors provide adequate expression levels, but to detect effects of ILDR2 on biological processes where high transfection and expression efficiency is needed, an adenovirus can be used.
Expression Vectors, Host Cells and Recombinant Methods of Producing Polypeptides
Another aspect of the present invention provides vectors that comprise one or more of the isolated nucleic acid molecules of the present invention, and host cells in which such vectors have been introduced.
The vectors can be used, inter alia, for propagating the nucleic acid molecules of the present invention in host cells (cloning vectors), for shuttling the nucleic acid molecules of the present invention between host cells derived from disparate organisms (shuttle vectors), for inserting the nucleic acid molecules of the present invention into host cell chromosomes (insertion vectors), for expressing sense or antisense RNA transcripts of the nucleic acid molecules of the present invention in vitro or within a host cell, and for expressing polypeptides encoded by the nucleic acid molecules of the present invention, alone or as fusion proteins with heterologous polypeptides (expression vectors). Vectors are by now well known in the art, and are described, inter alia, in Jones et al. (eds.), Vectors: Cloning Applications: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd. (1998); Jones et al. (eds.), Vectors: Expression Systems: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd. (1998); Gacesa et al., Vectors: Essential Data, John Wiley & Sons Ltd. (1995); Cid-Arregui (eds.), Viral Vectors: Basic Science and Gene Therapy, Eaton Publishing Co. (2000); Sambrook (2001), supra; Ausubel (1999), supra. Furthermore, a variety of vectors are available commercially. Use of existing vectors and modifications thereof are well within the skill in the art.
Nucleic acid sequences can be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. Expression control sequences are sequences that control the transcription, post-transcriptional events and translation of nucleic acid sequences. Such operative linking of a nucleic sequence of this invention to an expression control sequence, of course, includes, if not already part of the nucleic acid sequence, the provision of a translation initiation codon, ATG or GTG, in the correct reading frame upstream of the nucleic acid sequence.
A wide variety of host/expression vector combinations can be employed in expressing the nucleic acid sequences of this invention. Useful expression vectors, for example, can consist of segments of chromosomal, non-chromosomal and synthetic nucleic acid sequences.
In one embodiment, prokaryotic cells can be used with an appropriate vector. Prokaryotic host cells are often used for cloning and expression. In one embodiment, prokaryotic host cells include E. coli, Pseudomonas, Bacillus and Streptonzyces. In another embodiment, bacterial host cells are used to express the nucleic acid molecules and polypeptides of the invention. Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli, Bacillus or Streptoinyces, including pBluescript, pGEX-2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, λGT10 and λGT11, and other phages, e.g., M13 and filamentous single stranded phage DNA. Where E. coli is used as host, selectable markers are, analogously, chosen for selectivity in gram negative bacteria: e.g., typical markers confer resistance to antibiotics, such as ampicillin, tetracycline, chloramphenicol, kanamycin, streptomycin and zeocin; auxotrophic markers can also be used.
In other embodiments, eukaryotic host cells, such as yeast, insect, mammalian or plant cells, can be used. Yeast cells, can be useful for eukaryotic genetic studies, due to the ease of targeting genetic changes by homologous recombination and the ability to easily complement genetic defects using recombinantly expressed proteins. Yeast cells are useful for identifying interacting protein components, e.g. through use of a two-hybrid system. In one embodiment, yeast cells are useful for protein expression. Vectors of the present invention for use in yeast can contain an origin of replication suitable for use in yeast and a selectable marker that is functional in yeast. Yeast vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp and YEp series plasmids), Yeast Centromere plasmids (the YCp series plasmids), Yeast Artificial Chromosomes (YACs) which are based on yeast linear plasmids, denoted YLp, pGPD-2, 2 μplasmids and derivatives thereof, and improved shuttle vectors such as those described in Gietz et al., Gene, 74: 527-34 (1988) (YIplac, YEplac and YCplac). Selectable markers in yeast vectors include a variety of auxotrophic markers, the most common of which are (in Saccharomyces cerevisiae) URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations, such as ura3-52, his3-D1, leu2-D1, trp1-D1 and lys2-201.
Insect cells can be chosen for high efficiency protein expression. Where the host cells are from Spodoptera frugiperda, e.g., Sf9 and Sf21 cell lines, and ExpresSFTM cells (Protein Sciences Corp., Meriden, Conn., USA), the vector replicative strategy can be based upon the baculovirus life cycle. Baculovirus transfer vectors can be used to replace the wild-type AcMNPV polyhedrin gene with a heterologous gene of interest. Sequences that flank the polyhedrin gene in the wild-type genome can be positioned 5′ and 3′ of the expression cassette on the transfer vectors. Following co-transfection with AcMNPV DNA, a homologous recombination event occurs between these sequences resulting in a recombinant virus carrying the gene of interest and the polyhedrin or p10 promoter. Selection can be based upon visual screening for lacZ fusion activity.
The host cells can also be mammalian cells, which can be useful for expression of proteins intended as pharmaceutical agents, and for screening of potential agonists and antagonists of a protein or a physiological pathway. Mammalian vectors intended for autonomous extrachromosomal replication can include a viral origin, such as the SV40 origin, the papillomavirus origin, or the EBV origin for long term episomal replication. Vectors intended for integration, and thus replication as part of the mammalian chromosome, can include an origin of replication functional in mammalian cells, such as the SV40 origin. Vectors based upon viruses, such as adenovirus, adeno-associated virus, vaccinia virus, and various mammalian retroviruses, can replicate according to the viral replicative strategy. Selectable markers for use in mammalian cells include, include but are not limited to, resistance to neomycin (G418), blasticidin, hygromycin and zeocin, and selection based upon the purine salvage pathway using HAT medium.
Expression in mammalian cells can be achieved using a variety of plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses). Useful vectors for insect cells include baculoviral vectors and pVL 941.
Plant cells can also be used for expression, with the vector replicon derived from a plant virus (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) and selectable markers chosen for suitability in plants.
It is known that codon usage of different host cells can be different. For example, a plant cell and a human cell can exhibit a difference in codon preference for encoding a particular amino acid. As a result, human mRNA can not be efficiently translated in a plant, bacteria or insect host cell. Therefore, another embodiment of this invention is directed to codon optimization. The codons of the nucleic acid molecules of the invention can be modified to resemble genes naturally contained within the host cell without altering the amino acid sequence encoded by the nucleic acid molecule.
Any of a wide variety of expression control sequences can be used in these vectors to express the nucleic acid molecules of this invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Expression control sequences that control transcription include, e.g., promoters, enhancers and transcription termination sites. Expression control sequences in eukaryotic cells that control post-transcriptional events include splice donor and acceptor sites and sequences that modify the half-life of the transcribed RNA, e.g., sequences that direct poly(A) addition or binding sites for RNA-binding proteins. Expression control sequences that control translation include ribosome binding sites, sequences which direct targeted expression of the polypeptide to or within cellular compartments, and sequences in the 5′ and 3′ untranslated regions that modify the rate or efficiency of translation.
Examples of useful expression control sequences for a prokaryote, e.g., E. coli, will include a promoter, often a phage promoter, such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter (in E. coli cells engineered to express the T7 polymerase), the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, and the araBAD operon. Prokaryotic expression vectors can further include transcription terminators, such as the aspA terminator, and elements that facilitate translation, such as a consensus ribosome binding site and translation termination codon, Schomer et al., Proc. Natl. Acad. Sci. USA 83: 8506-8510 (1986).
Expression control sequences for yeast cells can include a yeast promoter, such as the CYC1 promoter, the GAL1 promoter, the GAL10 promoter, ADH1 promoter, the promoters of the yeast α-mating system, or the GPD promoter, and can have elements that facilitate transcription termination, such as the transcription termination signals from the CYC1 or ADH1 gene.
Expression vectors useful for expressing proteins in mammalian cells will include a promoter active in mammalian cells. These promoters include, but are not limited to, those derived from mammalian viruses, such as the enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus (CMV), the enhancer-promoter sequences from the Rous sarcoma virus long terminal repeat (RSV LTR), the enhancer-promoter from SV40 and the early and late promoters of adenovirus. Other expression control sequences include the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase. Other expression control sequences include those from the gene comprising the OSNA of interest. Often, expression is enhanced by incorporation of polyadenylation sites, such as the late SV40 polyadenylation site and the polyadenylation signal and transcription termination sequences from the bovine growth hormone (BGH) gene, and ribosome binding sites. Furthermore, vectors can include introns, such as intron II of rabbit β-globin gene and the SV40 splice elements.
Nucleic acid vectors also include a selectable or amplifiable marker gene and means for amplifying the copy number of the gene of interest. Such marker genes are well known in the art. Nucleic acid vectors can also comprise stabilizing sequences (e.g., ori- or ARS-like sequences and telomere-like sequences), or can alternatively be designed to favor directed or non-directed integration into the host cell genome. In one embodiment, nucleic acid sequences of this invention are inserted in frame into an expression vector that allows a high level expression of an RNA which encodes a protein comprising the encoded nucleic acid sequence of interest. Nucleic acid cloning and sequencing methods are well known to those of skill in the art and are described in an assortment of laboratory manuals, including Sambrook (1989), supra, Sambrook (2000), supra; and Ausubel (1992), supra, Ausubel (1999), supra. Product information from manufacturers of biological, chemical and immunological reagents also provide useful information.
Expression vectors can be constitutive or inducible. Inducible vectors include naturally inducible promoters, such as the trc promoter, which is regulated by the lac operon, and the pL promoter, which is regulated by tryptophan, the MMTV-LTR promoter, which is inducible by dexamethasone, or can contain synthetic promoters and/or additional elements that confer inducible control on adjacent promoters. Examples of inducible synthetic promoters are the hybrid Plac/ara-1 promoter and the PLtetO-1 promoter. The PLtetO-1 promoter takes advantage of the high expression levels from the PL promoter of phage lambda, but replaces the lambda repressor sites with two copies of operator 2 of the Tn10 tetracycline resistance operon, causing this promoter to be tightly repressed by the Tet repressor protein and induced in response to tetracycline (Tc) and Tc derivatives such as anhydrotetracycline. Vectors can also be inducible because they contain hormone response elements, such as the glucocorticoid response element (GRE) and the estrogen response element (ERE), which can confer hormone inducibility where vectors are used for expression in cells having the respective hormone receptors. To reduce background levels of expression, elements responsive to ecdysone, an insect hormone, can be used instead, with coexpression of the ecdysone receptor.
In one embodiment of the invention, expression vectors can be designed to fuse the expressed polypeptide to small protein tags that facilitate purification and/or visualization. Such tags include a polyhistidine tag that facilitates purification of the fusion protein by immobilized metal affinity chromatography, for example using NiNTA resin (Qiagen Inc., Valencia, Calif., USA) or TALONTM resin (cobalt immobilized affinity chromatography medium, Clontech Labs, Palo Alto, Calif., USA). The fusion protein can include a chitin-binding tag and self-excising intein, permitting chitin-based purification with self-removal of the fused tag (IMPACTTM system, New England Biolabs, Inc., Beverley, Mass., USA). Alternatively, the fusion protein can include a calmodulin-binding peptide tag, permitting purification by calmodulin affinity resin (Stratagene, La Jolla, Calif., USA), or a specifically excisable fragment of the biotin carboxylase carrier protein, permitting purification of in vivo biotinylated protein using an avidin resin and subsequent tag removal (Promega, Madison, Wis., USA). As another useful alternative, the polypeptides of the present invention can be expressed as a fusion to glutathione-S-transferase, the affinity and specificity of binding to glutathione permitting purification using glutathione affinity resins, such as Glutathione-Superflow Resin (Clontech Laboratories, Palo Alto, Calif., USA), with subsequent elution with free glutathione. Other tags include, for example, the Xpress epitope, detectable by anti-Xpress antibody (Invitrogen, Carlsbad, Calif., USA), a myc tag, detectable by anti-myc tag antibody, the V5 epitope, detectable by anti-V5 antibody (Invitrogen, Carlsbad, Calif., USA), FLAG® epitope, detectable by anti-FLAG® antibody (Stratagene, La Jolla, Calif., USA), and the HA epitope, detectable by anti-HA antibody.
For secretion of expressed polypeptides, vectors can include appropriate sequences that encode secretion signals, such as leader peptides. For example, the pSecTag2 vectors (Invitrogen, Carlsbad, Calif., USA) are 5.2 kb mammalian expression vectors that carry the secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of recombinant proteins from a variety of mammalian cell lines.
Expression vectors can also be designed to fuse proteins encoded by the heterologous nucleic acid insert to polypeptides that are larger than purification and/or identification tags. Useful protein fusions include those that permit display of the encoded protein on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as those that have a green fluorescent protein (GFP)-like chromophore, fusions to the IgG Fc region, and fusions for use in two hybrid systems.
Vectors for phage display fuse the encoded polypeptide to, e.g., the gene III protein (pIII) or gene VIII protein (pVIII) for display on the surface of filamentous phage, such as M13. See Barbas et al., Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001); Kay et al. (eds.), Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, Inc., (1996); Abelson et al. (eds.), Combinatorial Chemistry (Methods in Enzymology, Vol. 267) Academic Press (1996). Vectors for yeast display, e.g. the pYD1 yeast display vector (Invitrogen, Carlsbad, Calif., USA), use the α-agglutinin yeast adhesion receptor to display recombinant protein on the surface of S. cerevisiae. Vectors for mammalian display, e.g., the pDisplayTM vector (Invitrogen, Carlsbad, Calif., USA), target recombinant proteins using an N-terminal cell surface targeting signal and a C-terminal transmembrane anchoring domain of platelet derived growth factor receptor.
A wide variety of vectors now exist that fuse proteins encoded by heterologous nucleic acids to the chromophore of the substrate-independent, intrinsically fluorescent green fluorescent protein from Aequorea Victoria (“GFP”) and its variants. The GFP-like chromophore can be selected from GFP-like chromophores found in naturally occurring proteins, such as A. Victoria GFP (GENBANK accession number AAA27721), Renilla reniformis GFP, FP583 (GENBANK accession no. AF168419) (DsRed), FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423), and FP506 (AF168422), and need include only so much of the native protein as is needed to retain the chromophore's intrinsic fluorescence. Methods for determining the minimal domain required for fluorescence are known in the art. See Li et al., J. Biol. Chem. 272: 28545-28549 (1997). Alternatively, the GFP-like chromophore can be selected from GFP-like chromophores modified from those found in nature. The methods for engineering such modified GFP-like chromophores and testing them for fluorescence activity, both alone and as part of protein fusions, are well known in the art. See Heim et al., Curr. Biol. 6: 178-182 (1996) and Palm et al., Methods Enzymol. 302: 378-394 (1999). A variety of such modified chromophores are now commercially available and can readily be used in the fusion proteins of the present invention. These include EGFP (“enhanced GFP”), EBFP (“enhanced blue fluorescent protein”), BFP2, EYFP (“enhanced yellow fluorescent protein”), ECFP (“enhanced cyan fluorescent protein”) or Citrine. EGFP (see, e.g, Cormack et al., Gene 173: 33-38 (1996); U.S. Pat. Nos. 6,090,919 and 5,804,387, the disclosures of which are incorporated herein by reference in their entireties) is found on a variety of vectors, both plasmid and viral, which are available commercially (Clontech Labs, Palo Alto, Calif., USA); EBFP is optimized for expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, can be expressed in bacteria (see, e.g, Heim et al., Curr. Biol. 6: 178-182 (1996) and Cormack et al., Gene 173: 33-38 (1996)). Vectors containing these blue-shifted variants are available from Clontech Labs (Palo Alto, Calif., USA). Vectors containing EYFP, ECFP (see, e.g., Heim et al., Curr. Biol. 6: 178-182 (1996); Miyawaki et al., Nature 388: 882-887 (1997)) and Citrine (see, e.g., Heikal et al., Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000)) are also available from Clontech Labs. The GFP-like chromophore can also be drawn from other modified GFPs, including those described in U.S. Pat. Nos. 6,124,128; 6,096,865; 6,090,919; 6,066,476; 6,054,321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668; and 5,625,048, the disclosures of which are incorporated herein by reference in their entireties. See also Conn (ed.), Green Fluorescent Protein (Methods in Enzymology, Vol. 302), Academic Press, Inc. (1999); Yang, et al., J Biol Chem, 273: 8212-6 (1998); Bevis et al., Nature Biotechnology, 20:83-7 (2002). The GFP-like chromophore of each of these GFP variants can usefully be included in the fusion proteins of the present invention.
Polypeptides, Including Fragments Mutant Proteins, Homologous Proteins, Allelic Variants, Analogs and Derivatives
Another aspect of the invention relates to polypeptides encoded by the nucleic acid molecules described herein. In one embodiment, the polypeptide is an ILDR2 polypeptide. A polypeptide as defined herein can be produced recombinantly, as discussed supra, can be isolated from a cell that naturally expresses the protein, or can be chemically synthesized following the teachings of the specification and using methods well known to those having ordinary skill in the art. One skilled in the art understands that polypeptides (for example, ILDR2) can be obtained in several ways, which include but are not limited to, expressing a nucleotide sequence encoding the protein of interest, or fragment thereof, by genetic engineering methods.
In one embodiment, the nucleic acid is expressed in an expression cassette, for example, to achieve overexpression in a cell. The nucleic acids of the invention can be an RNA, cDNA, cDNA-like, or a DNA of interest in an expressible format, such as an expression cassette, which can be expressed from the natural promoter or an entirely heterologous promoter. The nucleic acid of interest can encode a protein, and may or may not include introns. Any recombinant expression system can be used, including, but not limited to, bacterial, mammalian, yeast, insect, or plant cell expression systems.
Polypeptides of the present invention can also comprise a part or fragment of a ILDR2. In one embodiment, the fragment is derived from a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2-9, or 14-47. Polypeptides of the present invention comprising a part or fragment of an entire ILDR2 protein can or can not be ILDR2 proteins. A polypeptide that is not an ILDR2 protein, whether it is a fragment, analog, mutant protein, homologous protein or derivative, is nevertheless useful, especially for immunizing animals to prepare anti-ILDR2 protein antibodies. In one embodiment, the part or fragment is an ILDR2 protein. Methods of determining whether a polypeptide of the present invention is a ILDR2 protein are described herein.
Polypeptides of the present invention comprising fragments of at least 8 contiguous amino acids, often at least 15 contiguous amino acids, are useful as immunogens for raising antibodies that recognize polypeptides of the present invention. See, e.g., Lerner, Nature 299: 592-596 (1982); Shinnick et al., Annu Rev. Microbiol. 37: 425-46 (1983); Sutcliffe et al., Science 219: 660-6 (1983). As further described in the references cited herein, 8-mers, conjugated to a carrier, such as a protein, prove immunogenic and are capable of eliciting antibody for the conjugated peptide; accordingly, fragments of at least 8 amino acids of the polypeptides of the present invention have utility as immunogens.
Polypeptides comprising fragments of at least 8, 9, 10 or 12 contiguous amino acids are also useful as competitive inhibitors of binding of the entire polypeptide, or a portion thereof, to antibodies (as in epitope mapping), and to natural binding partners, such as subunits in a multimeric complex or to receptors or ligands of the subject protein; this competitive inhibition permits identification and separation of molecules that bind specifically to the polypeptide of interest. See U.S. Pat. Nos. 5,539,084 and 5,783,674, incorporated herein by reference in their entireties.
The polypeptides of the present invention thus can be at least 6 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 12 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, at least 25 amino acids in length, at least 30 amino acids in length, at least 35 amino acids in length, at least 50 amino acids in length, at least 75 amino acids in length, at least 100 amino acids in length, or at least 150 amino acids in length. Polypeptides of the present invention can also be larger and comprise a full-length ILDR2 protein and/or an epitope tag and/or a fusion protein.
One having ordinary skill in the art can produce fragments by truncating the nucleic acid molecule, encoding the polypeptide and then expressing it recombinantly. Alternatively, one can produce a fragment by chemically synthesizing a portion of the full-length polypeptide. One can also produce a fragment by enzymatically cleaving a recombinant polypeptide or an isolated naturally occurring polypeptide. Methods of producing polypeptide fragments are well known in the art. See, e.g., Sambrook (1989), supra; Sambrook (2001), supra; Ausubel (1992), supra; and Ausubel (1999), supra. In one embodiment, a polypeptide comprising only a fragment can be produced by chemical or enzymatic cleavage of a ILDR2 polypeptide.
Polypeptides of the present invention are also inclusive of mutants, fusion proteins, homologous proteins and allelic variants.
A mutant protein can have the same or different properties compared to a naturally occurring polypeptide and comprises at least one amino acid insertion, duplication, deletion, rearrangement or substitution compared to the amino acid sequence of a native polypeptide. Small deletions and insertions can often be found that do not alter the function of a protein. The mutant protein can be a polypeptide that comprises at least one amino acid insertion, duplication, deletion, rearrangement or substitution compared to the amino acid sequence of SEQ ID NO: 2-9, or 14-47. Accordingly, in one embodiment, the mutant protein is one that exhibits at least 60% sequence identity, at least 70%, or at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97%, sequence identity at least 985, sequence identity at least 99% or sequence identity at least 99.5% to an ILDR2 protein.
A mutant protein can be produced by isolation from a naturally occurring mutant cell, tissue or organism. A mutant protein can be produced by isolation from a cell, tissue or organism that has been experimentally mutagenized. Alternatively, a mutant protein can be produced by chemical manipulation of a polypeptide, such as by altering the amino acid residue to another amino acid residue using synthetic or semi-synthetic chemical techniques. In one embodiment, a mutant protein is produced from a host cell comprising a mutated nucleic acid molecule compared to the naturally occurring nucleic acid molecule. For instance, one can produce a mutant protein of a polypeptide by introducing one or more mutations into a nucleic acid molecule of the invention and then expressing it recombinantly. These mutations can be targeted, in which encoded amino acids are altered, or can be untargeted, in which random encoded amino acids within the polypeptide are altered. Mutant proteins with random amino acid alterations can be screened for a biological activity or property. Multiple random mutations can be introduced into the gene by methods well known to the art, e.g., by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis and site-specific mutagenesis. Methods of producing mutant proteins with targeted or random amino acid alterations are well known in the art. See, e.g., Sambrook (1989), supra; Sambrook (2001), supra; Ausubel (1992), supra; and Ausubel (1999), as well as U.S. Pat. No. 5,223,408, which is herein incorporated by reference in its entirety.
The invention also contemplates polypeptides that are homologous to a polypeptide of the invention. By homologous polypeptide it is means one that exhibits significant sequence identity to an ILDR2 protein. By significant sequence identity it is meant that the homologous polypeptide exhibits at least exhibits at least 60% sequence identity, at least 70%, or at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97%, sequence identity at least 985, sequence identity at least 99% or sequence identity at least 99.5% to an ILDR2 protein. In one embodiment, the amino acid substitutions of the homologous polypeptide are conservative amino acid substitutions.
Homologous polypeptides of the present invention can be naturally occurring and derived from another species, especially one derived from another primate, such as chimpanzee, gorilla, rhesus macaque, or baboon, wherein the homologous polypeptide comprises an amino acid sequence that exhibits significant sequence identity to a polypepetide of the invention. The homologous polypeptide can also be a naturally occurring polypeptide from a human, when the ILDR2 protein is a member of a family of polypeptides. The homologous polypeptide can also be a naturally occurring polypeptide derived from a non-primate, mammalian species, including without limitation, domesticated species, e.g., dog, cat, mouse, rat, rabbit, guinea pig, hamster, cow, horse, goat or pig. The homologous polypeptide can also be a naturally occurring polypeptide derived from a non-mammalian species, such as birds or reptiles. The naturally occurring homologous protein can be isolated directly from humans or other species. Alternatively, the nucleic acid molecule encoding the naturally occurring homologous polypeptide can be isolated and used to express the homologous polypeptide recombinantly. The homologous polypeptide can also be one that is experimentally produced by random mutation of a nucleic acid molecule and subsequent expression of the nucleic acid molecule. Alternatively, the homologous polypeptide can be one that is experimentally produced by directed mutation of one or more codons to alter the encoded amino acid of an ILDR2 protein.
Relatedness of proteins can also be characterized using a second functional test, the ability of a first protein competitively to inhibit the binding of a second protein to an antibody. It is, therefore, another aspect of the present invention to provide isolated polpeptide not only identical in sequence to those described herein, but also to provide isolated polypeptide (“cross-reactive proteins”) that can competitively inhibit the binding of antibodies to all or to a portion of various of the isolated polypeptides of the present invention. Such competitive inhibition can readily be determined using immunoassays well known in the art.
As discussed herein, single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes, and the sequence determined from one individual of a species can differ from other allelic forms present within the population. Thus, polypeptides of the present invention are also inclusive of those encoded by an allelic variant of a nucleic acid molecule encoding an ILDR2 protein.
Polypeptides of the present invention are also inclusive of derivative polypeptides encoded by a nucleic acid molecule according to the invention. Also inclusive are derivative polypeptides having an amino acid sequence selected from the group consisting of an ILDR2 protein or a polypeptide of SEQ ID NO: 2-9, or 14-47 and which has been acetylated, carboxylated, phosphorylated, glycosylated, ubiquitinated or other post-translational modifications. In another embodiment, the derivative has been labeled with, e.g., radioactive isotopes such as 125I, 32P, 35S, and 3H. In another embodiment, the derivative has been labeled with fluorophores, chemiluminescent agents, enzymes, and antiligands that can serve as specific binding pair members for a labeled ligand.
Polypeptide modifications are well known to those of skill and have been described in detail in the scientific literature. Several common modifications, such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance Creighton, Protein Structure and Molecular Properties, 2nd ed., W. H. Freeman and Company (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, in Johnson (ed.), Posttranslational Covalent Modification of Proteins, pgs. 1-12, Academic Press (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Ann. N.Y. Acad. Sci. 663: 48-62 (1992).
One can determine whether a polypeptide of the invention will be post-translationally modified by analyzing the sequence of the polypeptide to determine if there are peptide motifs indicative of sites for post-translational modification. There are a number of computer programs that permit prediction of post-translational modifications. See, e.g., expasy with the extension.org of the world wide web (accessed Nov. 11, 2002), which includes PSORT, for prediction of protein sorting signals and localization sites, SignalP, for prediction of signal peptide cleavage sites, MITOPROT and Predotar, for prediction of mitochondrial targeting sequences, NetOGlyc, for prediction of type O-glycosylation sites in mammalian proteins, big-PI Predictor and DGPI, for prediction of prenylation-anchor and cleavage sites, and NetPhos, for prediction of Ser, Thr and Tyr phosphorylation sites in eukaryotic proteins. Other computer programs, such as those included in GCG, also can be used to determine post-translational modification peptide motifs.
Examples of types of post-translational modifications include, but are not limited to: (Z)-dehydrobutyrine; 1-chondroitin sulfate-L-aspartic acid ester; 1′-glycosyl-L-tryptophan; 1′-phospho-L-histidine; 1-thioglycine; 2′-(S-L-cysteinyl)-L-histidine; 2′-[3-carboxamido (trimethylammonio)propyl]-L-histidine; 2′-alpha-mannosyl-L-tryptophan; 2-methyl-L-glutamine; 2-oxobutanoic acid; 2-pyrrolidone carboxylic acid; 3′-(1′-L-histidyl)-L-tyrosine; 3′-(8alpha-FAD)-L-histidine; 3′-(S-L-cysteinyl)-L-tyrosine; 3′,3′′,5′-triiodo-L-thyronine; 3′-4′-phospho-L-tyrosine; 3-hydroxy-L-proline; 3′-methyl-L-histidine; 3-methyl-L-lanthionine; 3′-phospho-L-histidine; 4′-(L-tryptophan)-L-tryptophyl quinone; 42 N-cysteinyl-glycosylphosphatidylinositolethanolamine; 43-(T-L-histidyl)-L-tyrosine; 4-hydroxy-L-arginine; 4-hydroxy-L-lysine; 4-hydroxy-L-proline; 5′-(N6-L-lysine)-L-topaquinone; 5-hydroxy-L-lysine; 5-methyl-L-arginine; alpha-1-microglobulin-Ig alpha complex chromophore; bis-L-cysteinyl bis-L-histidino diiron disulfide; bis-L-cysteinyl-L-N3′-histidino-L-serinyl tetrairon′ tetrasulfide; chondroitin sulfate D-glucuronyl-D-galactosyl-D-galactosyl-D-xylosyl-L-serine; //D-alanine; D-allo-isoleucine; D-asparagine; dehydroalanine; dehydrotyrosine; dermatan 4-sulfate D-glucuronyl-D-galactosyl-D-galactosyl-D-xylosyl-L-serine; D-glucuronyl-N-glycine; dipyrrolylmethanemethyl-L-cysteine; D-leucine; D-methionine; D-phenylalanine; D-serine; D-tryptophan; glycine amide; glycine oxazolecarboxylic acid; glycine thiazolecarboxylic acid; heme P450-bis-L-cysteine-L-tyrosine; heme-bis-L-cysteine; hemediol-L-aspartyl ester-L-glutamyl ester; hemediol-L-aspartyl ester-L-glutamyl ester-L-methionine sulfonium; heme-L-cysteine; heme-L-histidine; heparan sulfate D-glucuronyl-D-galactosyl-D-galactosyl-D-xylosyl-L-serine; heme P450-bis-L-cysteine-L-lysine; hexakis-L-cysteinyl hexairon hexasulfide; keratan sulfate D-glucuronyl-D-galactosyl-D-galactosyl-D-xylosyl-L-threonine; L oxoalanine-lactic acid; L phenyllactic acid; 1′-(8alpha-FAD)-L-histidine; L-2′.4′,5′-topaquinone; L-3′,4′-dihydroxyphenylalanine; L-3′.4′.5′-trihydroxyphenylalanine; L-4′-bromophenylalanine; L-6′-bromotryptophan; L-alanine amide; L-alanyl imidazolinone glycine; L-allysine; L-arginine amide; L-asparagine amide; L-aspartic 4-phosphoric anhydride; L-aspartic acid 1-amide; L-beta-methylthioaspartic acid; L-bromohistidine; L-citrulline; L-cysteine amide; L-cysteine glutathione disulfide; L-cysteine methyl disulfide; L-cysteine methyl ester; L-cysteine oxazolecarboxylic acid; L-cysteine oxazolinecarboxylic acid; L-cysteine persulfide; L-cysteine sulfenic acid; L-cysteine sulfinic acid; L-cysteine thiazolecarboxylic acid; L-cysteinyl homocitryl molybdenum-heptairon-nonasulfide; L-cysteinyl imidazolinone glycine; L-cysteinyl molybdopterin; L-cysteinyl molybdopterin guanine dinucleotide; L-cystine; L-erythro-beta-hydroxyasparagine; L-erythro-beta-hydroxyaspartic acid; L-gamma-carboxyglutarnic acid; L-glutamic acid 1-amide; L-glutamic acid 5-methyl ester; L-glutamine amide; L-glutamyl 5-glycerylphosphorylethanolarnine; L-histidine amide; L-isoglutamyl-polyglutamic acid; L-isoglutamyl-polyglycine; L-isoleucine amide; L-lanthionine; L-leucine amide; L-lysine amide; L-lysine thiazolecarboxylic acid; L-lysinoalanine; L-methionine amide; L-methionine sulfone; L-phenyalanine thiazolecarboxylic acid; L-phenylalanine amide; L-proline amide; L-selenocysteine; L-selenocysteinyl molybdopterin guanine dinucleotide; L-serine amide; L-serine thiazolecarboxylic acid; L-seryl imidazolinone glycine; L-T-bromophenylalanine; L-T-bromophenylalanine; L-threonine amide; L-thyroxine; L-tryptophan amide; L-tryptophyl quinone; L-tyrosine amide; L-valine amide; meso-lanthionine; N-(L-glutamyl)-L-tyrosine; N-(L-isoaspartyl)-glycine; N-(L-isoaspartyl)-L-cysteine; N,N,N-trimethyl-L-alanine; N,N-dimethyl-L-proline; N2-acetyl-L-lysine; N2-succinyl-L-tryptophan; N4-(ADP-ribosyl)-L-asparagine; N4-glycosyl-L-asparagine; N4-hydroxymethyl-L-asparagine; N4-methyl-L-asparagine; N5-methyl-L-glutamine; N6-1-carboxyethyl-L-lysine; N6-(4-amino hydroxybutyl)-L-lysine; N6-(L-isoglutamyl)-L-lysine; N6-(phospho-5′-adenosine)-L-lysine; N6-(phospho-5′-guanosine)-L-lysine; N6,N6,N6-trimethyl-L-lysine; N6,N6-dimethyl-L-lysine; N6-acetyl-L-lysine; N6-biotinyl-L-lysine; N6-carboxy-L-lysine; N6-formyl-L-lysine; N6-glycyl-L-lysine; N6-lipoyl-L-lysine; N6-methyl-L-lysine; N6-methyl-N6-poly(N-methyl-propylamine)-L-lysine; N6-mureinyl-L-lysine; N6-myristoyl-L-lysine; N6-palmitoyl-L-lysine; N6-pyridoxal phosphate-L-lysine; N6-pyruvic acid 2-iminyl-L-lysine; N6-retinal-L-lysine; N-acetylglycine; N-acetyl-L-glutamine; N-acetyl-L-alanine; N-acetyl-L-aspartic acid; N-acetyl-L-cysteine; N-acetyl-L-glutamic acid; N-acetyl-L-isoleucine; N-acetyl-L-methionine; N-acetyl-L-proline; N-acetyl-L-serine; N-acetyl-L-threonine; N-acetyl-L-tyrosine; N-acetyl-L-valine; N-alanyl-glycosylphosphatidylinositolethanolamine; N-asparaginyl-glycosylphosphatidylinositolethanolamine; N-aspartyl-glycosylphosphatidylinositolethanolamine; N-formylglycine; N-formyl-L-methionine; N-glycyl-glycosylphosphatidylinositolethanolamine; N-L-glutamyl-poly-L-glutamic acid; N-methylglycine; N-methyl-L-alanine; N-methyl-L-methionine; N-methyl-L-phenylalanine; N-myristoyl-glycine; N-palmitoyl-L-cysteine; N-pyruvic acid 2-iminyl-L-cysteine; N-pyruvic acid 2-iminyl-L-valine; N-seryl-glycosylphosphatidylinositolethanolamine; N-seryl-glycosyOSPhingolipidinositolethanolamine; O-(ADP-ribosyl)-L-serine; O-(phospho-5′-adenosine)-L-threonine; O-(phospho-5′-DNA)-L-serine; O-(phospho-5′-DNA)-L-threonine; 0-(phospho-5′rRNA)-L-serine; O-(phosphoribosyl dephospho-coenzyme A)-L-serine; O-(sn-1-glycerophosphoryl)-L-serine; O4′-(8alpha-FAD)-L-tyrosine; O4′-(phospho-5′-adenosine)-L-tyrosine; O4′-(phospho-5′-DNA)-L-tyrosine; O4′-(phospho-5′-RNA)-L-tyrosine; O4′-(phospho-5′-uridine)-L-tyrosine; O4-glycosyl-L-hydroxyproline; O4′-glycosyl-L-tyrosine; O4′-sulfo-L-tyrosine; O5-glycosyl-L-hydroxylysine; O-glycosyl-L-serine; O-glycosyl-L-threonine; omega-N-(ADP-ribosyl)-L-arginine; omega-N-omega-N′-dimethyl-L-arginine; omega-N-methyl-L-arginine; omega-N-omega-N-dimethyl-L-arginine; omega-N-phospho-L-arginine; O'octanoyl-L-serine; O-palmitoyl-L-serine; O-palmitoyl-L-threonine; O-phospho-L-serine; O-phospho-L-threonine; O-phosphopantetheine-L-serine; phycoerythrobilin-bis-L-cysteine; phycourobilin-bis-L-cysteine; pyrroloquinoline quinone; pyruvic acid; S hydroxycinnamyl-L-cysteine; S-(2-aminovinyl)methyl-D-cysteine; S-(2-aminovinyl)-D-cysteine; S-(6-FW-L-cysteine; S-(8alpha-FAD)-L-cysteine; S-(ADP-ribosyl)-L-cysteine; S-(L-isoglutamyl)-L-cysteine; S-12-hydroxyfamesyl-L-cysteine; S-acetyl-L-cysteine; S-diacylglycerol-L-cysteine; S-diphytanylglycerot diether-L-cysteine; S-famesyl-L-cysteine; S-geranylgeranyl-L-cysteine; S-glycosyl-L-cysteine; S-glycyl-L-cysteine; S-methyl-L-cysteine; S-nitrosyl-L-cysteine; S-palmitoyl-L-cysteine; S-phospho-L-cysteine; S-phycobiliviolin-L-cysteine; S-phycocyanobilin-L-cysteine; S-phycoerythrobilin-L-cysteine; S-phytochromobilin-L-cysteine; S-selenyl-L-cysteine; S-sulfo-L-cysteine; tetrakis-L-cysteinyl diiron disulfide; tetrakis-L-cysteinyl iron; tetrakis-L-cysteinyl tetrairon tetrasulfide; trans-2,3-cis 4-dihydroxy-L-proline; tris-L-cysteinyl triiron tetrasulfide; tris-L-cysteinyl triiron trisulfide; tris-L-cysteinyl-L-aspartato tetrairon tetrasulfide; tris-L-cysteinyl-L-cysteine persulfido-bis-L-glutamato-L-histidino tetrairon disulfide trioxide; tris-L-cysteinyl-L-N3′-histidino tetrairon tetrasulfide; tris-L-cysteinyl-L-NM′-histidino tetrairon tetrasulfide; and tris-L-cysteinyl-L-serinyl tetrairon tetrasulfide.
Additional examples of post translational modifications can be found in web sites such as the Delta Mass database based on Krishna, R. G. and F. Wold (1998). Posttranslational Modifications. Proteins—Analysis and Design. R. H. Angeletti. San Diego, Academic Press. 1: 121-206.; Methods in Enzymology, 193, J. A. McClosky (ed) (1990), pages 647-660; Methods in Protein Sequence Analysis edited by Kazutomo Imahori and Fumio Sakiyama, Plenum Press, (1993) “Post-translational modifications of proteins” R. G. Krishna and F. Wold pages 167-172; “GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources” Cooper et al. Nucleic Acids Res. 29; 332-335 (2001) “O-GLYCBASE version 4.0: a revised database of O-glycosylated proteins” Gupta et al. Nucleic Acids Research, 27: 370-372 (1999); and “PhosphoBase, a database of phosphorylation sites: release 2.0.”, Kreegipuu et al. Nucleic Acids Res 27(1):237-239 (1999) see also, WO 02/211 39A2, the disclosure of which is incorporated herein by reference in its entirety.
Disease states are often accompanied by alterations in the post-translational modifications of proteins. Thus, in another embodiment, the invention provides polypeptides from diseased cells or tissues that have altered post-translational modifications compared to the post-translational modifications of polypeptides from normal cells or tissues. A number of altered post-translational modifications are known. One common alteration is a change in phosphorylation state, wherein the polypeptide from the diseased cell or tissue is hyperphosphorylated or hypophosphorylated compared to the polypeptide from a normal tissue, or wherein the polypeptide is phosphorylated on different residues than the polypeptide from a normal cell. Another common alteration is a change in glycosylation state, wherein the polypeptide from the diseased cell or tissue has more or less glycosylation than the polypeptide from a normal tissue, and/or wherein the polypeptide from the diseased cell or tissue has a different type of glycosylation than the polypeptide from a non-diseased cell or tissue.
Another post-translational modification that can be altered in diseased cells is prenylation. Prenylation is the covalent attachment of a hydrophobic prenyl group (farnesyl or geranylgeranyl) to a polypeptide. Prenylation is required for localizing a protein to a cell membrane and is often required for polypeptide function. For instance, the Ras superfamily of GTPase signalling proteins must be prenylated for function in a cell. See, e.g., Prendergast et al., Semin. Cancer Biol. 10: 443-452 (2000) and Khwaja et al., Lancet 355: 741-744 (2000).
Other post-translation modifications that can be altered in diseased cells include, without limitation, polypeptide methylation, acetylation, arginylation or racemization of amino acid residues. In these cases, the polypeptide from the diseased cell can exhibit increased or decreased amounts of the post-translational modification compared to the corresponding polypeptides from non-diseased cells.
Other polypeptide alterations in diseased cells include abnormal polypeptide cleavage of proteins and aberrant protein-protein interactions. Abnormal polypeptide cleavage can be cleavage of a polypeptide in a diseased cell that does not usually occur in a normal cell, or a lack of cleavage in a diseased cell, wherein the polypeptide is cleaved in a normal cell. Aberrant protein-protein interactions can be covalent cross-linking or non-covalent binding between proteins that do not normally bind to each other. Alternatively, in a diseased cell, a protein can fail to bind to another protein to which it is bound in a non-diseased cell. Alterations in cleavage or in protein-protein interactions can be due to over- or underproduction of a polypeptide in a diseased cell compared to that in a normal cell, or can be due to alterations in post-translational modifications of one or more proteins in the diseased cell. See, e.g., Henschen-Edman, Ann. N.Y. Acad. Sci. 936: 580-593 (2001).
Alterations in polypeptide post-translational modifications, as well as changes in polypeptide cleavage and protein-protein interactions, can be determined by any method known in the art. For instance, alterations in phosphorylation can be determined by using anti-phosphoserine, anti-phosphothreonine or anti-phosphotyrosine antibodies or by amino acid analysis. Glycosylation alterations can be determined using antibodies specific for different sugar residues, by carbohydrate sequencing, or by alterations in the size of the glycoprotein, which can be determined by, e.g., SDS polyacrylamide gel electrophoresis (PAGE). Other alterations of post-translational modifications, such as prenylation, racemization, methylation, acetylation and arginylation, can be determined by chemical analysis, protein sequencing, amino acid analysis, or by using antibodies that bind a post-translational modification. Changes in protein-protein interactions and in polypeptide cleavage can be analyzed by any method known in the art including, without limitation, non-denaturing PAGE (for non-covalent protein-protein interactions), SDS PAGE (for covalent protein-protein interactions and protein cleavage), chemical cleavage, protein sequencing or immunoassays.
In another embodiment, the invention provides polypeptides that have been post-translationally modified. In one embodiment, polypeptides can be modified enzymatically or chemically, by addition or removal of a post-translational modification. For example, a polypeptide can be glycosylated or deglycosylated enzymatically. Similarly, polypeptides can be phosphorylated using a purified kinase, such as a MAP kinase (e.g, p38, ERK, or JNK) or a tyrosine kinase (e.g., Src or erbB2). A polypeptide can also be modified through synthetic chemistry. Alternatively, one can isolate the polypeptide of interest from a cell or tissue that expresses the polypeptide with the desired post-translational modification. In another embodiment, a nucleic acid molecule encoding the polypeptide of interest is introduced into a host cell that is capable of post-translationally modifying the encoded polypeptide in the desired fashion. If the polypeptide does not contain a motif for a desired post-translational modification, one can alter the post-translational modification by mutating the nucleic acid sequence of a nucleic acid molecule encoding the polypeptide so that it contains a site for the desired post-translational modification. Amino acid sequences that can be post-translationally modified are known in the art. See, e.g., the programs described herein on the Expasy website. The nucleic acid molecule can also be introduced into a host cell that is capable of post-translationally modifying the encoded polypeptide. Similarly, one can delete sites that are post-translationally modified by mutating the nucleic acid sequence so that the encoded polypeptide does not contain the post-translational modification motif, or by introducing the native nucleic acid molecule into a host cell that is not capable of post-translationally modifying the encoded polypeptide.
Polypeptides are not always entirely linear. For instance, polypeptides can be branched as a result of ubiquitination, and they can be circular, with or without branching, as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides can be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications can be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.
Useful post-synthetic (and post-translational) modifications include conjugation to detectable labels, such as fluorophores. A wide variety of amine-reactive and thiol-reactive fluorophore derivatives have been synthesized that react under nondenaturing conditions with N-terminal amino groups and epsilon amino groups of lysine residues, on the one hand, and with free thiol groups of cysteine residues, on the other.
Kits are available commercially that permit conjugation of proteins to a variety of amine-reactive or thiol-reactive fluorophores: Molecular Probes, Inc. (Eugene, Oreg., USA), e.g., offers kits for conjugating proteins to Alexa Fluor 350, Alexa Fluor 430, Fluorescein-EX, Alexa Fluor 488, Oregon Green 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, and Texas Red-X A wide variety of other amine-reactive and thiol-reactive fluorophores are available commercially (Molecular Probes, Inc., Eugene, Oreg., USA), including Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA).
The polypeptides of the present invention can also be conjugated to fluorophores, other proteins, and other macromolecules, using bifunctional linking reagents. Common homobifunctional reagents include, e.g., APG, AEDP, BASED, BMB, BMDB, BMH, BMOE, BM[PEO]3, BM[PEO]4, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP (Lomant's Reagent), DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, Sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS (available from Pierce, Rockford, Ill., USA); common heterobifunctional cross-linkers include ABH, AMAS, ANB-NOS, APDP, ASBA, BMPA, BMPH, BMPS, EDC, EMCA, EMCH, EMCS, KMUA, KMUH, GMBS, LC-SMCC, LC-SPDP, MBS, M2C2H, MPBH, MSA, NHS-ASA, PDPH, PMPI, SADP, SAED, SAND, SANPAH, SASD, SATP, SBAP, SFAD, SIA, SLAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-HSAB, Sulfo-KMUS, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-NHS-LC-ASA, Sulfo-SADP, Sulfo-SANPAH, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB, Sulfo-LC-SMPT, SVSB, TFCS (available Pierce, Rockford, Ill., USA).
Polypeptides of the present invention, including full length polypeptides, fragments and fusion proteins, can be conjugated, using such cross-linking reagents, to fluorophores that are not amine- or thiol-reactive. Other labels that usefully can be conjugated to polypeptides of the present invention include radioactive labels, echosonographic contrast reagents, and MRI contrast agents.
Polypeptides of the present invention, including full length polypeptide, fragments and fusion proteins, can also usefully be conjugated using cross-linking agents to carrier proteins, such as KLH, bovine thyroglobulin, and even bovine serum albumin (BSA), to increase immunogenicity for raising anti-ILDR2 protein antibodies.
Polypeptides of the present invention, including full length polypeptide, fragments and fusion proteins, can also usefully be conjugated to polyethylene glycol (PEG); PEGylation increases the serum half life of proteins administered intravenously for replacement therapy. Delgado et al., Crit. Rev. Ther. Drug Carrier Syst. 9(3-4): 249-304 (1992); Scott et al., Curr. Pharm. Des. 4(6): 423-38 (1998); DeSantis et al., Curr. Opin. Biotechnol. 10(4): 324-30 (1999). PEG monomers can be attached to the protein directly or through a linker, with PEGylation using PEG monomers activated with tresyl chloride (2,2,2-trifluoroethanesulphonyl chloride) permitting direct attachment under mild conditions.
Polypeptides of the present invention are also inclusive of analogs of a polypeptide encoded by a nucleic acid molecule according to the invention. In one embodiment, this polypeptide is an ILDR2 protein. In another embodiment the analog polypeptide comprises one or more substitutions of non-natural amino acids or non-native inter-residue bonds compared to the naturally occurring polypeptide. In one embodiment, the analog is structurally similar to an ILDR2 protein, but one or more peptide linkages is replaced by a linkage selected from the group consisting of —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH-(cis and trans), —COCH2—, —CH(OH)CH2— and —CH2SO—. In another embodiment, the analog comprises substitution of one or more amino acids of a ILDR2 protein with a D-amino acid of the same type or other non-natural amino acid in order to generate more stable peptides. D-amino acids can readily be incorporated during chemical peptide synthesis: peptides assembled from D-amino acids are more resistant to proteolytic attack; incorporation of D-amino acids can also be used to confer specific three-dimensional conformations on the peptide. Other amino acid analogues that can be added during chemical synthesis include ornithine, norleucine, phosphorylated amino acids (for example, phosphoserine, phosphothreonine, phosphotyrosine), L-malonyltyrosine, a non-hydrolyzable analog of phosphotyrosine (see, e.g., Kole et al., Biocheem. Biophlys. Res. Com. 209: 817-821 (1995)), and various halogenated phenylalanine derivatives.
Non-natural amino acids can be incorporated during solid phase chemical synthesis or by recombinant techniques. Solid phase chemical synthesis of peptides is well established in the art. Procedures are described, inter alia, in Chan et al. (eds.), Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series), Oxford Univ. Press (March 2000); Jones, Amino Acid and Peptide Synthesis (Oxford Chemistry Primers, No 7), Oxford Univ. Press (1992); and Bodanszky, Principles of Peptide Synthesis (Springer Laboratory), Springer Verlag (1993).
Amino acid analogues having detectable labels are also usefully incorporated during synthesis to provide derivatives and analogs. Biotin, for example can be added using biotinoyl-(9-fluorenylmethoxycarbonyl)-L-lysine (FMOC biocytin) (Molecular Probes, Eugene, Oreg., USA). Biotin can also be added enzymatically by incorporation into a fusion protein of a E. coli BirA substrate peptide. The FMOC and tBOC derivatives of dabcyl-L-lysine (Molecular Probes, Inc., Eugene, Oreg., USA) can be used to incorporate the dabcyl chromophore at selected sites in the peptide sequence during synthesis. The aminonaphthalene derivative EDANS, the most common fluorophore for pairing with the dabcyl quencher in fluorescence resonance energy transfer (FRET) systems, can be introduced during automated synthesis of peptides by using EDANS-FMOC-L-glutamic acid or the corresponding tBOC derivative (both from Molecular Probes, Inc., Eugene, Oreg., USA). Tetramethylrhodamine fluorophores can be incorporated during automated FMOC synthesis of peptides using (FMOC)-TMR-L-lysine (Molecular Probes, Inc. Eugene, Oreg., USA).
Other useful amino acid analogues that can be incorporated during chemical synthesis include aspartic acid, glutamic acid, lysine, and tyrosine analogues having allyl side-chain protection (Applied Biosystems, Inc., Foster City, Calif., USA); the allyl side chain permits synthesis of cyclic, branched-chain, sulfonated, glycosylated, and phosphorylated peptides.
A large number of other FMOC-protected non-natural amino acid analogues capable of incorporation during chemical synthesis are available commercially, including, e.g., Fmoc-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, Fmoc-3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid, Fmoc-3-exo-aminobicyclo[2.2.1]heptane-2-exo-carboxylic acid, Fmoc-3-endo-amino-bicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid, Fmoc-3-exo-amino-bicyclo[2.2.1]hept-5-ene-2-exo-carboxylic acid, Fmoc-cis-2-amino-1-cyclohexanecarboxylic acid, Fmoc-trans-2-amino-1-cyclohexanecarboxylic acid, Fmoc-1-amino-1-cyclopentanecarboxylic acid, Fmoc-cis-2-amino-1-cyclopentanecarboxylic acid, Fmoc-1-amino-1-cyclopropanecarboxylic acid, Fmoc-D-2-amino-4-(ethylthio)butyric acid, Fmoc-L-2-amino-4-(ethylthio)butyric acid, Fmoc-L-buthionine, Fmoc-S-methyl-L-Cysteine, Fmoc-2-aminobenzoic acid (anthranillic acid), Fmoc-3-aminobenzoic acid, Fmoc-4-aminobenzoic acid, Fmoc-2-aminobenzophenone-2′-carboxylic acid, Fmoc-N-(4-aminobenzoyl)-β-alanine, Fmoc-2-amino4,5-dimethoxybenzoic acid, Fmoc-4-aminohippuric acid, Fmoc-2-amino-3-hydroxybenzoic acid, Fmoc-2-amino-5-hydroxybenzoic acid, Fmoc-3-amino4-hydroxybenzoic acid, Fmoc4-amino-3-hydroxybenzoic acid, Fmoc-4-amino-2-hydroxybenzoic acid, Fmoc-5-amino-2-hydroxybenzoic acid, Fmoc-2-amino-3-methoxybenzoic acid, Fmoc4-amino-3-methoxybenzoic acid, Fmoc-2-amino-3-methylbenzoic acid, Fmoc-2-amino-5-methylbenzoic acid, Fmoc-2-amino-6-methylbenzoic acid, Fmoc-3-amino-2-methylbenzoic acid, Fmoc-3-amino4-methylbenzoic acid, Fmoc-4-amino-3-methylbenzoic acid, Fmoc-3-amino-2-naphtoic acid, Fmoc-D,L-3-amino-3-phenylpropionic acid, Fmoc-L-Methyldopa, Fmoc-2-amino-4,6-dimethyl-3-pyridinecarboxylic acid, Fmoc-D,L-amino-2-thiophenacetic acid, Fmoc-4-(carboxymethyl)piperazine, Fmoc-4-carboxypiperazine, Fmoc-4-(carboxymethyl)homopiperazine, Fmoc-4-phenyl-4-piperidinecarboxylic acid, Fmoc-L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, Fmoc-L-thiazolidine4-carboxylic acid, available from—The Peptide Laboratory (Richmond, Calif., USA).
Non-natural residues can also be added biosynthetically by engineering a suppressor tRNA by chemical aminoacylation with the desired unnatural amino acid. Conventional site-directed mutagenesis is used to introduce the chosen stop codon UAG at the site of interest in the protein gene. When the acylated suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon to give a protein containing that amino acid at the specified position. Liu et al., Proc. Natl Acad. Sci. USA 96(9): 4780-5 (1999); Wang et al., Science 292(5516): 498-500 (2001).
Fusion Proteins
Another aspect of the present invention relates to the fusion of a polypeptide of the present invention to heterologous polypeptides. In one embodiment, the polypeptide of the present invention is an ILDR2 protein or is a mutant protein, homologous polypeptide, analog or derivative thereof.
The fusion proteins of the present invention will include at least one fragment of a polypeptide of the present invention, which fragment is at least 6 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 12 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, at least 25 amino acids in length, at least 30 amino acids in length, at least 35 amino acids in length, at least 50 amino acids in length, at least 75 amino acids in length, at least 100 amino acids in length, or at least 150 amino acids in length. Fusions proteins that include the entirety of a polypeptide of the present invention are also useful.
The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and can be at least 15, 20, or 25 amino acids in length. Fusions that include larger polypeptides, such as the IgG Fc region, and even entire proteins (such as GFP chromophore-containing proteins) can be useful.
Heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those designed to facilitate purification and/or visualization of recombinantly-expressed proteins. See, e.g., Ausubel, Chapter 16, (1992), supra. Although purification tags can also be incorporated into fusions that are chemically synthesized, chemical synthesis can also provides sufficient purity. Such tags can retain their utility even when the protein is produced by chemical synthesis, and when so included render the fusion proteins of the present invention useful as directly detectable markers of the presence of a polypeptide of the invention.
Heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those that facilitate secretion of recombinantly expressed proteins into the periplasmic space or extracellular milieu for prokaryotic hosts or into the culture medium for eukaryotic cells through incorporation of secretion signals and/or leader sequences. For example, a His6 tagged protein (SEQ ID NO: 146) can be purified on a Ni affinity column and a GST fusion protein can be purified on a glutathione affinity column. Similarly, a fusion protein comprising the Fc domain of IgG can be purified on a Protein A or Protein G column and a fusion protein comprising an epitope tag such as myc can be purified using an immunoaffinity column containing an anti-c-myc antibody. The epitope tag can be separated from the protein encoded by the essential gene by an enzymatic cleavage site that can be cleaved after purification. See also the discussion of nucleic acid molecules encoding fusion proteins that can be expressed on the surface of a cell.
Other useful fusion proteins of the present invention include those that permit use of the polypeptide of the present invention as bait in a yeast two-hybrid system. See Bartel et al. (eds.), The Yeast Two-Hybrid System, Oxford University Press (1997); Zhu et al., Yeast Hybrid Technologies, Eaton Publishing (2000); Fields et al., Trends Genet. 10(8): 286-92 (1994); Mendelsohn et al, Curr. Opin. Biotechnol. 5(5): 482-6 (1994) Luban et al., Curr. Opin. Biotechnol. 6(1): 59-64 (1995); Allen et al., Trends Biochem. Sci. 20(12): 511-6 (1995); Drees, Curr. Opin. Cliem. Biol. 3(1): 64-70 (1999); Topcu et al, Pharm. Res. 17(9): 1049-55 (2000); Fashena et al., Gene 250(1-2): 1-14 (2000); Colas et al., Nature 380, 548-550 (1996); Norman, T. et al., Science 285, 591-595 (1999); Fabbrizio et al., Oncogene 18, 4357-4363 (1999); Xu et al., Proc Natl Acad Sci USA. 94, 12473-12478 (1997); Yang, et al., Nuc. Acids Res. 23, 1152-1156 (1995); Kolonin et al., Proc Natl Acad Sci USA 95, 14266-14271 (1998); Cohen et al., Proc Natl Acad Sci USA 95, 14272-14277 (1998); Uetz, et al. Nature 403, 623-627(2000); Ito, et al., Proc Natl Acad Sci USA 98, 4569-4574 (2001). Such fusion can be made to E. coli LexA or yeast GAL4 DNA binding domains. Related bait plasmids are available that express the bait fused to a nuclear localization signal.
Other useful fusion proteins include those that permit display of the encoded polypeptide on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region, as described herein.
The polypeptides of the present invention can also usefully be fused to protein toxins, such as Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, ricin, in order to effect ablation of cells that bind or take up the proteins of the present invention.
Fusion partners include, inter alia, myc, hemagglutinin (HA), GST, immunoglobulins, p-galactosidase, biotin trpE, protein A, β-lactamase, α-amylase, maltose binding protein, alcohol dehydrogenase, polyhistidine (for example, six histidine (SEQ ID NO: 146) at the amino and/or carboxyl terminus of the polypeptide), lacZ, green fluorescent protein (GFP), yeast a mating factor, GALA transcription activation or DNA binding domain, luciferase, and serum proteins such as ovalbumin, albumin and the constant domain of IgG. See, e.g., Ausubel (1992), supra and Ausubel (1999), supra. Fusion proteins can also contain sites for specific enzymatic cleavage, such as a site that is recognized by enzymes such as Factor XIII, trypsin, pepsin, or any other enzyme known in the art. Fusion proteins can be made by recombinant nucleic acid methods or chemically synthesized using techniques well known in the art (e.g., a Merrifield synthesis), or produced by chemical cross-linking.
Another advantage of fusion proteins is that the epitope tag can be used to bind the fusion protein to a plate or column through an affinity linkage for screening binding proteins or other molecules that bind to the ILDR2 protein.
The polypeptides of the present invention can readily be used as specific immunogens to raise antibodies that specifically recognize polypeptides of the present invention including ILDR2 proteins and their allelic variants and homologues. The antibodies can be used to specifically to assay for the polypeptides of the present invention with the use of several techniques, for example ELISA, immunohistochemistry, laser scanning cytometry, flow cytometry, immunoprecipitation, immunoblotting and for detection of ILDR2 proteins or for use as specific agonists or antagonists of ILDR2 proteins.
One can determine whether polypeptides of the present invention including ILDR2 proteins, mutant proteins, homologous proteins or allelic variants or fusion proteins of the present invention are functional by methods known in the art. For instance, residues that are tolerant of change while retaining function can be identified by altering the polypeptide at known residues using methods known in the art, such as alanine scanning mutagenesis, Cunningham et al., Science 244(4908): 1081-5 (1989); transposon linker scanning mutagenesis, Chen et al., Gene 263(1-2): 39-48 (2001); combinations of homolog- and alanine-scanning mutagenesis, Jin et al., J. Mol. Biol. 226(3): 851-65 (1992); combinatorial alanine scanning, Weiss et al., Proc. Natl. Acad. Sci USA 97(16): 8950-4 (2000), followed by functional assay. Transposon linker scanning kits are available commercially (New England Biolabs, Beverly, Mass., USA, catalog. no. E7-1025; EZ::TNTM In-Frame Linker Insertion Kit, catalogue no. EZI04KN, (Epicentre Technologies Corporation, Madison, Wis., USA).
Purification of the polypeptides or fusion proteins of the present invention is well known and within the skill of one having ordinary skill in the art. See, e.g., Scopes, Protein Purification, 2d ed. (1987). Purification of recombinantly expressed polypeptides is described herein. Purification of chemically-synthesized peptides can readily be effected, e.g., by HPLC.
Accordingly, it is an aspect of the present invention to provide the isolated polypeptides or fusion proteins of the present invention in pure or substantially pure form in the presence of absence of a stabilizing agent. Stabilizing agents include both proteinaceous and non-proteinaceous material and are well known in the art. Stabilizing agents, such as albumin and polyethylene glycol (PEG) are known and are commercially available.
Although high levels of purity can be useful when the isolated polypeptide or fusion protein of the present invention are used as therapeutic agents, such as in vaccines and replacement therapy, the isolated polypeptides of the present invention are also useful at lower purity. For example, partially purified polypeptides of the present invention can be used as immununogens to raise antibodies in laboratory animals. The purified and substantially purified polypeptides of the present invention are in compositions that lack detectable ampholytes, acrylamide monomers, bis-acrylamide monomers, and polyacrylamide.
The polypeptides or fusion proteins of the present invention can usefully be attached to a substrate. The substrate can be porous or solid, planar or non-planar; the bond can be covalent or noncovalent. For example, the peptides of the invention can be stabilized by covalent linkage to albumin. See, U.S. Pat. No. 5,876,969, the contents of which are hereby incorporated in its entirety.
For example, the polypeptides or fusion proteins of the present invention can usefully be bound to a porous substrate or a membrane such as nitrocellulose, polyvinylidene fluoride (PVDF), or cationically derivatized, hydrophilic PVDF. When bound the polypeptides or fusion proteins of the present invention can be used to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized polypeptide or fusion protein of the present invention.
As another example, the polypeptides or fusion proteins of the present invention can usefully be bound to a substantially nonporous substrate, such as plastic, to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized protein of the present invention. Such plastics include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof; when the assay is performed in a standard microtiter dish, the plastic can be polystyrene.
The polypeptides and fusion proteins of the present invention can also be attached to a substrate suitable for use as a surface enhanced laser desorption ionization source; so attached, the polypeptide or fusion protein of the present invention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound polypeptide or fusion protein to indicate biologic interaction there between. The polypeptides or fusion proteins of the present invention can also be attached to a substrate suitable for use in surface plasmon resonance detection; so attached, the polypeptide or fusion protein of the present invention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound polypeptide or fusion protein to indicate biological interaction there between.
Purification of Recombinant Proteins
An ILDR2 protein can be purified from any human or non-human cell which expresses the polypeptide, including those which have been transfected with expression constructs that express an ILDR2. A purified ILDR2 protein can be separated from other compounds which normally associate with ILDR2 protein, in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art. For protein recovery, isolation and/or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired polypeptide molecule (for example, an ILDR2 protein) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
Alternative Transcripts
In another aspect, the present invention provides splice variants of genes and proteins encoded thereby. The identification of a splice variant which encodes an amino acid sequence with a region that can be targeted for the generation of reagents for use in detection and/or treatment of diabetes or metabolic disease. The amino acid sequence can lead to a unique protein structure, protein subcellular localization, biochemical processing or function of the splice variant. This information can be used to directly or indirectly facilitate the generation of additional or therapeutics or diagnostics. The nucleotide sequence in this splice variant can be used as a nucleic acid probe for the diagnosis and/or treatment of diabetes or metabolic disease.
Specifically, the newly identified sequences can enable the production of antibodies or compounds directed against the region for use as a therapeutic or diagnostic. Alternatively, the newly identified sequences can alter the biochemical or biological properties of the encoded protein in such a way as to enable the generation of improved or different therapeutics targeting this protein.
Tissues, Cells, Cell Lines: Protein Synthesis, Processing, Degradation
Ildr2 is expressed as several variably spliced isoforms with specificity by strain and organ. In certain aspects, the invention provides a full-length cDNA cloned in a mammalian expression vector, adding C-terminal and/or N-terminal tags—as noted—to facilitate detection following transfection. In certain embodiments, transient transfection assays can be carried out in β-TC3 insulinoma cells and SV40-transformed hepatocytes (Rother, 1998, J Biol Chem 273:17491-17497) followed by immunoprecipitation with anti-HA antiserum and immunoblot with anti-Ildr2 antiserum. These cell lines have been chosen because they maintain at least some physiologic properties of β cells and hepatocytes. Moreover, they are well characterized, easy to maintain, and handle transfecting/transducing them with a variety of expression and viral vectors. These lines were successfully transfected with full length Ildr2 constructs. Using these lines, experiments can be performed in the presence and absence of cycloheximide to block protein synthesis and visualize on the blots the molecular weight of the expressed products, how rapidly they are degraded, and whether they differ in different cell types. Transient transfection assays can be used for this type of experiment because they are easier and prevent clonal artifact. Although transfection efficiency is irrelevant in this context, this technique can be optimized in these cell types. Using modified lipofection reagents, 30-40% efficiency can be achieved in SV40 hepatocytes. Using the Amaxa system, up to 80-90% of β-TC3 cells can be transfected.
In alternative embodiments of the methods of the present invention, different insulinoma cells, such as Ins1, MIN-6 or HIT can be transfected. In other embodiments, screening methods of the invention, or basic studies of (cell) biology of ILDR2 or C1ORF32 can be carried out in HEK293 or 3T3 cells. The former cells have the advantage of being easily transfectable but—HEK293 being a human kidney-derived cell line—Ildr2 processing can or can not reflect that in murine Ildr2 target tissues. To circumvent this problem, murine 3T3 cells, or any other suitable cell type can be used.
Sub-Cellular Localization
Ildr2 is predicted to encode a single membrane-spanning domain, with a large extracellular domain and a C-terminal intracellular domain. In GT1-7, a mouse hypothalamic neuronal cell line, Ildr2-isoform 2 localized solely to the ER membrane. In Hepa1c1c7, a mouse hepatoma cell line, Ildr2-isoform 4 localized solely to the ER membrane. Localization of ILDR2 can be addressed using the cell types described herein, and confocal microscopy. In certain embodiments, cycloheximide can be used to determine whether ILDR2 localization changes as a function of protein turnover. Time-lapse microscopy will be used to visualize protein fate in the presence of cycloheximide. The GFP or mYFP tag is located at the C-terminus or N-terminus of ILDR2. Thus, if ILDR2 is cleaved during its intracellular journey, this construct will only allow detection of the C-terminal domain. To circumvent this potential problem, immunocytochemistry will be performed with HA antiserum in cells transfected with Ildr2 constructs bearing a double tag N-terminal (HA) and C-terminal (FLAG-tag). In one embodiment, ILDR2 can be processed as a single peptide with a stable sub-cellular localization. In this case, the Ildr2-GFP or the Ildr2-mYFP construct and the double-tag construct will yield overlapping patterns of sub-cellular localization. In another embodiment, ILDR2 can be processed into different peptides, each with a distinct sub-cellular localization in a manner that may be similar to Tubby (Santagata et al, 2001, Science 292:2041-2050; Boggon et al, 1999, Science 286:2119-2125) and SREBP1C proteins, which are proteolytically cleaved to activate their transcriptional functions can be considered (Horton et al, 2002, J Clin Invest 109:1125-1131). In this case, the subcellular localization of the HA-tagged and FLAG-tagged constructs will differ, and only the FLAG-tagged construct will overlap with Ildr2-GFP or Ildr2-mYFP—appropriate cellular markers can be used to identify cellular compartments to which ILDR2 localizes; ILDR2 sub-cellular localization, as a single peptide, or as multiple processed products, changes in response to various cues—the effect of various hormonal and metabolic treatments on this process can be examined. In non-limiting examples, in Beta cells, the effects of glucose and cAMP can be determined, while in liver the effect of insulin and cAMP can be determined. In both cell types, the effects of FFA and lipoproteins can be determined. As a control for these experiments, Foxo-GFP, which undergoes rapid sub-cellular re-localization in response to these various agents, can be used. Actual experimental details (dose response, time course, etc) will be patterned according to prior experience in this area (Nakae et al, 2001, J Clin Invest 108:1359-1367; Nakae et al, 2000, Embo J 19:989-996).
Phosphorylation
Many proteins with metabolic functions are modified via phosphorylation by tyrosine and serine/threonine kinases. As indicated, the putative intracellular domain of ILDR2 contains several putative sites for Ser/Thr kinases. Using 32P-orthophosphate labeling of intact cells, it can be determined whether ILDR2 is phosphorylated in vivo and whether changes in the cell's metabolic status affect ILDR2 phosphorylation. The initial experiments will be carried out by in vivo labeling followed by immunoprecipitation and autoradiography. If required, phospho-peptide maps will be employed (Accili et al, 1991, J Biol Chem 266:434-439) and mass spectrometry to identify individual phosphorylation sites. If ILDR2 phosphorylation changes with the cell's hormonal/nutritional status, further experiments will be conducted to identify phosphorylation sites on ILDR2 and relevant kinases. There are a number of potential Ser/Thr phosphorylation sites in the intracellular domain of ILDR2 (FIG. 12). Of special interest are four PKA sites (at amino acid residue 307, 352, 399, 403), an Akt site at position 618, and a CDK site at position 550. Given that PKA and Akt are activated in response to glucagon and insulin signaling, respectively, it will be of interest to determine whether these agents affect ILDR2 phosphorylation. If so, these sites will be mutated to probe their involvement in ILDR2 phosphorylation and function. Similarly, it will be important to test ILDR2 phosphorylation as function of cell cycle progression, given preliminary data that in dd mice (with low ILDR2 levels) replication of Beta cells is decreased. If there are changes in ILDR2 phosphorylation as function of cell cycle progression, the CDK phosphorylation site can be mutated to determine whether ILDR2 function is affected. One of the two non-conservative nucleotide substitutions identified in DD mice abolishes a potential Ck1 site (T572A). Thus, the phosphorylation state of the WT vs T572A mutant ILDR2 will be compared to determine whether (a) the site is phosphorylated and (b) its mutation into a non-phosphorylatable amino acid changes localization, signaling or bioeffects of Ildr2. Candidate phosphorylation sites described herein will be replaced by non-phosphorylatable amino acids (alanine) to generate phosphorylation-deficient mutants, or by charged amino acids (aspartic or glutamic acid) to mimic the phosphorylated state and generate “constitutively phosphorylated” mutants
Readout Assays of Ildr2 Gain-of-Function
In certain aspects, the basic cell biology of Ildr2 can be characterized. In other aspects, transgenic and knockout mice can be generated and characterized by methods and techniques as described herein, and also known in the art.
In certain aspects, the invention provides that Ildr2 function is related to decrease in Beta cell mass, which is secondary to reduced proliferation. In other aspects, the invention provides that ILDR2 has a role to bind lipids—based upon close sequence homology to LSR (lipolysis-stimulated receptor). To further characterize these, β-TC3 cells (very low in endogenous Ildr2) will be transfected with WT (B6-derived) HA-Ildr2, and Beta cell proliferation will be measured. Gain of Ildr2 function can result in increased Beta cell proliferation. To carry out these experiments it can be necessary to achieve high transfection frequency to measure an effect in an unselected cell population. In non-limiting examples, transfection efficiency can be monitored using tagged constructs, or/and carrying out immunocytochemistry (for HA-tagged constructs) or fluorescence (for GFP-tagged constructs) with Ki67 or BrdU immunocytochemistry to co-localize transfected Ildr2 with in actively replicating cells. Ildr2-expressing cells will stain positive for Ki67 or BrdU enable measurement of replication rates using pulse-chase experiments. Because β-TC3 cells express very low levels of endogenous Ildr2, transfection of recombinant Ildr2 can result in a gain-of-function that may not be apparent in other Beta cell lines expressing higher levels of Ildr2 where pathways may active due to endogenous Ildr2. Tet-dependent β-TC3 clones exist in which addition of tetracycline to the medium results in rapid cell cycle arrest (Efrat et al, 1998, Proc Natl Acad Sci USA 85:9037-9041). Thus, if the replication rates of β-TC3 are unaffected by Ildr2 in regular culture conditions, the ability of Ildr2 over-expression to promote cell cycle progression in Tet-arrested β-TC3 cells can be studied.
To examine the mechanism of Ildr2-induced changes in cellular proliferation, markers of cell cycle progression, including Foxo1/3, p27kip, p21 and pRb will be analyzed (Okamoto et al, 2006, J Clin Invest 116:775-782; Buteau et al, 2006, Diabetes 55:1190-1196; Kitamura et al, 2005, Cell Metab 2:153-163; Kitamura et al, 2002, J Clin Invest 110:1839-1847). ILDR2 can also affect proliferation by reducing apoptosis. Rate of apoptosis can be determined in cultured β cells, and in vivo. In certain aspects, the invention provides that DD mice, have reduced Beta cell proliferation in the early post-natal stage. A physiologic remodeling of β-cell mass occurs in rodents at this stage (Scaglia et al, 1997, Endocrinology 138:1736-1741), due to a wave of apoptosis. ILDR2 can be involved in this process. Apoptosis markers such as Fas1, Caspase-3, −8, Bax and Bim will be examined.
In addition to cell replication, insulin secretion assays in response to glucose and other secretagogues, as well as mitochondrial function experiments to measure mitochondrial integrity will be performed (Buteau et al, 2006, Diabetes 55:1190-1196). Because insulin secretion and β cell proliferation are linked (Okamoto et al, 2006, J Clin Invest 116:775-782), ILDR2 can affect primarily secretion, which secondarily impairs β cell proliferation. The expression of markers of terminally differentiated Beta cells, such as MafA, a transcription factor expressed at low levels in Beta-TC3 cells, which makes them an ideal system to study MafA induction (Kitamura 2005) will be determined. Foxo1-3, Pdx1, Nkx2.2 and Hnf4 will be measured. ILDR2 can beneficially affect stimulus/secretion coupling in the β cell, and thus upregulate expression of relevant transcription factors.
Signaling Pathways Activated by ILDR2 and Protein/Protein Interactions
In certain aspects, the invention provides that ILDR2 function affects signaling pathways in insulinoma cells. Following Ildr2 over-expression activation of candidate pathways, including but not limited to PI 3-kinase/Akt, mTOR/S6k, AMPK/Acc, cAMP/PKA pathways will be measured (Buteau et al, 2006, Diabetes 55:1190-1196; Kitamura et al, 2005, Cell Metab 2:153-163). These assays can be carried out in an unselected population of cells after transient transfection. In other embodiments, similar experiments can be carried in cells transduced with Ildr2 adenovirus (Kitamura et al, 2005, Cell Metab 2:153-163).
Loss-of-Function Experiments
In other aspects, the invention provides methods to determine the effect of Ildr2 reduction or ablation on the aforementioned parameters and characteristics in islet cells. Because Beta-TC3 cells express low endogenous Ildr2 levels and are not suitable for this purpose, these experiments will be carried out in MIN-6 cells. To carry out these experiments, high-efficiency transfection with the Amaxa system, or siRNA adenovirus will be used (Matsumoto et al, 2006, J Clin Invest 116:2464-2472). As control, transfections of mutant siRNA or siRNA-resistant Ildr2 will be used. In certain aspects, the invention provides that gain of Ildr2 function increases cellular proliferation and loss of Ildr2 function decreases it. In certain embodiments, the invention provides methods to determine Ildr2 function in primary cultures of mouse islets transduced with adenoviral constructs (Kitamura et al, 2005, Cell Metab 2:153-163).
ILDR2 Functions in the Hepatocyte
In liver, the outcome of functional experiments is more complex. Proliferation of hepatocytes, while important in many pathophysiologic conditions, is not considered a predisposing factor in diabetes/insulin resistance. Thus, the actions of ILDR2 in hepatocytes must be deduced from other assays. The phenotypes of the ENU Ildr2-null mice (and a transgenic or conditional knockout mouse) will guide experimental approach to ILDR2 function in hepatocytes. In certain aspects, the invention provides methods to carry out gain-of-function experiments in hepatocytes to study Ildr2's cell biological properties: localization, processing, signaling properties. These experiments will employ SV40-transformed hepatocytes, a cell type that retains many of the properties of terminally differentiated hepatocytes (Rother et al, 1998, J Biol Chem 273:17491-17497; Kim et al, 2001, Endocrinology 142:3354-3360; Park et al, 1999, Biochemistry 38:7517-7523). Processing, turnover, localization and phosphorylation can be examined as described herein and by any other suitable method known in the art. Among the signaling pathways that can be studied following Ildr2 over-expression are: cAMP and insulin signaling, as well as adiponectin, lipids (FFA) and bile acids-activated signaling. Candidate effectors of ILDR2 signaling and/or, Srebp1c include PI 3-kinase, mTOR/S6 kinase, AMP kinase, Ppar induction. The biological responses that can be measured include glucose production, glycogen synthesis, TG content and synthesis, ApoB and LDL/VLDL secretion (Han et al, 2006, Cell Metab 3:257-266; Matsumoto et al, 2006, J Clin Invest 116:2464-2472). The liver, in which there are large differences in B6 v. DBA expression of Ildr2, affects Beta cells by a metabolic, e.g. lipoprotein, or endocrine pathway, hepatokine production, or by agents in these pathways. Liver-mediated effects on Beta cell development/function can be examined by co-culture of congenic line or knockout hepatocytes with suitable Beta cell line, expression arrays, and analysis of isolated liver proteins by 2-D gel and mass spectrometry.
Ildr2 Alternatively Spliced Isoforms
Ildr2 is expressed as several different transcripts. Notably, the abundance and assortment of transcripts varies from cell type to cell type, and by strain. Complete transcripts from 7 isoforms were isolated. However, isoforms 5,6,7 were only isolated in trace quantities from cDNA libraries. Isoform 1 contains the ten exons intact, while the others have missing or truncated exons. Complete transcripts for isoforms 1-4 were isolated and partial transcripts in trace quantities were isolated from pooled DBA cDNA libraries for isoforms 5-7.
Evaluating the full spectrum of the functions of these various isoforms can be carried out by methods as described herein and by any suitable methods know in the art (Liu et al, 1998, Mamm Genome 9:780-781; Chua et al, 1997, Genomics 45:264-270). One determination includes whether these spliced isoforms are translated. A protein isoform expression survey using western blot analysis will be carried out. If different molecular species are observed, tissue expression and mRNA variants will be monitored. Some of these isoforms have reduced stability, and that alternative message splicing provides a mechanism to indirectly regulate ILDR2 levels by altering its post-transcriptional or translational degradation. Certain isoforms are secreted and can be detected in the circulation, acting as a decoy receptor for a putative ILDR2 ligand. This will easily become apparent from western blot surveys of various tissues/cell types and incubation media in different conditions, as described herein. To address the issue of secreted isoforms, serum protein will also be included in the tissue survey. The turnover rates of the most prominent splice variants will be investigated using pulse-chase experiments with cycloheximide, and survey their intracellular localization by immunocytochemistry.
The putative transmembrane structure of ILDR2 shows that ILDR2 can be a cell surface receptor. This is supported by the presence of several Ig repeats in the putative extracellular domain, a defining feature of cell adhesion molecules and various cell surface receptors. Methods of identifying ligands for cell surface receptors are well known in the art and can be readily used to identify a ligand for ILDR2 or ILDR2 homologs.
Molecular Basis of Decreased Ildr2 Expression in DD Congenic Mice
In certain aspects, the invention provides that the DBA allele decreases Ildr2 expression levels through a cis-acting DNA element(s). The mechanism can be explained by: (a) reduced gene transcription; (b) decreased mRNA stability, and/or (c) increased protein degradation; these are not mutually exclusive. In other aspects, the invention provides that the DBA allele of Ildr2 results in reduced protein levels in hepatocytes, Beta cells and the brain. Understanding the relevant mechanism(s) will help to elucidate the molecular physiology of ILDR2.
The Ildr2 gene encodes large, alternatively spliced transcripts. Coding (exon 9) and non-coding (mainly 3′ UTR) sequence changes can be evaluated in the DDA vs. BBA strains as candidate mutations causing alterations of mRNA levels. Because the extent of the decrease in mRNA levels is different from tissue to tissue (Table 4), tissue-specific factors can contribute to the process. Because the largest differences in mRNA levels were found in the liver, cis-acting variations in Ildr2 can be examined in this tissue. The results described herein show that the region downstream of exon 8 is implicated in conveying diabetes susceptibility. Because this is a region of sequence overlap within Ildr2 in the congenic lines described herein such analysis can be used to determine whether the 5′UTR is cis-acting region that can contribute variation to differences in gene expression among the congenic lines. For examples, regulatory DNA elements acting upstream of the transcription start site may interact with elements downstream of exon 8 to decrease mRNA transcription/stability. These experiments can determine whether the low levels of Ildr2 transcripts seen in liver are due to decreased transcription. mRNA stability and decay can be also analyzed.
Chances in Gene Transcription
The promoter regions of Ildr2 in DD and BB mice are extremely well conserved. Although, there are no nucleotide substitutions detected in the 10 kb upstream of the transcription start site, cis-acting elements controlling Ildr2 expression have not been mapped and may reside outside the sequenced regions. In one embodiment, in vivo run-on studies using livers of DD vs DB mice can be performed to determine if the two alleles are transcribed at different rates. Because the mRNA levels in liver differ >10-fold between the two strains (Table 4), one can detect a difference, if indeed mRNA transcription is responsible for the molecular phenotype. Methods known in the art can be used to address these questions (McKeon et al, 1997, Biochem Biophys Res Commun 240:701-706; McKeon et al, 1990, Mol Endocrinol 4:647-656). In another embodiment, primary hepatocytes from the two strains can be prepared and run-on experiments can be performed in this culture system, which is more amenable to hormonal/metabolic control (i.e., it can be determined if the process is critically dependent on various hormone/metabolic cues). Comparison of a strain that segregates for DBA alleles only in exons 8-10+3′ UTR (e.g. 1jcdt) to one in which the entire Ildr2 gene is DBA (1jc) can allow apportioning effects via the 5′ promoter region.
In Vivo Analysis of Ildr2 Function in Mice
In certain aspects, the invention provides that loss or reduction of Ildr2 function predisposes to diabetes in mice, of a susceptible genetic background by impairing β cell proliferation and hepatic metabolism. In other aspect, the invention provides that loss or reduction of ILDR2 function predisposes human subject to diabetes or metabolic disease.
In certain aspects, the invention provides that loss-of-function conveyed by the DBA allele of Ildr2 is the cause of diabetes susceptibility in DD mice. Thus, conference of diabetes susceptibility can be achieved by introducing loss of Ildr2 function in diabetes-susceptible strains.
ENU mutagenesis provides a powerful tool to introduce mutations in the mouse genome. In certain embodiments, the invention provides an ENU-mutagenized mouse (C3HeB/FeJ) segregating for a W87* (stop) mutation in Ildr2. The ENU amber mutation in exon 2 of Ildr2 can produce a completely inactive allele. Because, the mutation is on a C3HeB/FeJ background, a C57BL/6J conditional knockout of Ildr2 can be made with or without a knockout vicinal genes. In other embodiments, the invention provides methods to characterize ILDR2 knockout mice by a number of metabolic abnormalities related to diabetes. In certain embodiments, characterization can be made by measuring the β cell response, hepatic glucose, or lipid metabolism.
ENU-mutagenized mice, as well as knockout strains which can be generated as described herein and by methods known in the art, can be characterized at various developmental stages using several parameters. Exemplary parameters are somatic growth curves, body composition, plasma glucose and insulin levels in fasted and fed states, lipid profile (triglycerides, cholesterol, FFAs), glucose tolerance tests, insulin release tests, pyruvate challenge, glucose clamps, functional, histological and immunohistochemical characterization of pancreatic islets as indicated below. Assays and techniques to carry out these characterizations are described herein and known in the art.
Non-limiting methods include calorimetry and euglycemic hyperinsulinemic clamp studies. Euglycemic hyperinsulinemic clamp studies—euglycemic clamps will be performed in conscious, unrestrained, catheterized mice as previously described (Okamoto et al, 2005, J Clin Invest 115:1314-1322). A solution of glucose (10%) will be infused at a variable rate as required to maintain euglycemia (7 mM). Mice will receive a constant infusion of HPLC-purified [3-3H] and insulin (18 mU/kg body wt/min). Thereafter, plasma will be collected to determine glucose levels at times 10, 20, 30, 40, 50, 60, 70, 80, and 90 min, as well as the specific activities of [3-3H] glucose and tritiated water at times 30, 40, 50, 60, 70, 80, and 90 min. Steady-state conditions can be achieved for both plasma glucose concentration and specific activity by 30 minutes in these studies. [U-14C] lactate (5 μCi bolus/0.25 μCi/min) will be infused during the last 10 min of the study.
β-Cell “Phenotyping”.
Numerous assays have been described herein and are known in the art to evaluate β-cell function in mouse models of diabetes. Ki67 immunoreactivity will be used to assess Beta cell proliferation. Detection of apoptosis can be carried out using immunohistochemistry with caspase-3. Because apoptosis occurs at specific developmental stages, time course analysis can be performed in 1 to 4 week-old mice. Islets can be isolated from mice by in vivo collagenase perfusion, and insulin release under different experimental conditions can be determined. If mutations result in developmental abnormalities, embryonic analysis can be performed by delivering embryos at various gestational stages by Caesarian section. The analysis can comprise identification of the pancreatic buds, dissection, histological or morphometric analysis of islet number, size and composition. Electron microscopy can be performed as described (Cinti et al, 1998, Diabetologia 41:171-177).
In certain embodiments, the −/−ENU mice, can be characterized by stressing the 13 cells using low dose streptozotocin, dexamethasone, dietary manipulations, etc.
Targeted Mutations
Targeted mutations in animals can be generated with ENU mice segregating on the basis of a stop codon in exon 2.
Conventional Knock-Out
A gene targeting vector, as described herein, can be designed to carry out a conventional gene inactivation experiment. The vector can be used for both ubiquitous and conditional inactivation of Ildr2. For conventional gene knockout, the sequence flanked by loxP sites can be excised in vitro, using transfections of ES cells carrying the gene-targeted allele (Bruning et al, 1998, Mol Cell 2:559-569), or by intercrossing mice carrying a floxed allele with “deleter” cre transgenics, leading to removal of the lox-flanked sequence in germ cells (Okamoto et al, 2004, J Clin Invest 114:214-223; Bruning et al, 1998, Mol Cell 2:559-569; Han et al, 2006, Cell Metab 3:257-266; Xuan et al, 2002, J Clin Invest 110:1011-1019; Okamoto et al, 2005, J Clin Invest 115:1314-1322).
Conditional Knock-Out
Cre-loxP technology known in the art can be used to introduce mutations in an organ or in a developmental stage-specific fashion. As described herein, Ildr2 ablation in β cells can affect their ability to proliferate, thus modulating diabetes susceptibility in vivo. Conditional Ildr2 knockouts can be generated at various developmental stages during endocrine pancreas differentiation using crosses of mice homozygous for a floxed Ildr2 allele with Neurogenin 3-cre, Pdx-cre and Insulin-cre transgenic mice. Each cre transgenic can cause Ildr2 inactivation at a different stage in pancreas development, and can thus provide insight into the developmental role of Ildr2 in this process.
Pdx-Cre Knock-Out
In certain embodiments, Pdx-Cre can be used to inactivate Ildr2 in pancreatic progenitors, prior to the differentiation of the endocrine, exocrine and ductal lineages. If Ildr2 plays a role in the determination of the pancreatic lineages, ablation of Ildr2 driven by this Cre mice can result in widespread alterations of exocrine and endocrine cell number, characteristics, as well as islet number, size, distribution.
Neuroenin 3-Cre Knock-Out
In other embodiments, Neurogenin 3-Cre mice can be generated to direct ablation of Ildr2 in the endocrine progenitor cell in the pancreas and entero-endocrine system, after the endocrine/exocrine split has occurred, but prior to final specification of individual islet cell types. If ILDR2 plays a role in endocrine cell differentiation, the effects of its ablation can be determined in non-β cell types (α, δ, ε, PP). This can also drive inactivation of ILDR2 in entero-endocrine cells and result in inactivation of Ildr2 in incretin-producing cells (K and L cells in the gut). Because incretin production is observed in diabetes, incretin response can be characterized in Neurogenin3-Cre/Ildr2 knockouts (Buteau et al, 2006, Diabetes 55:1190-1196).
Insulin-Cre Knock-Out
In other embodiments, Insulin-cre can inactivate Ildr2 in terminally differentiated Beta cells. As such, the phenotype of these mice can reflect the function of Ildr2 in daily maintenance of the phenotype/function of Beta cells. This phenotype can resemble aspects of the diabetes susceptibility seen in DD mice. In certain embodiments, stress on the Beta cell can be imposed using standard approaches such as low-dose streptozotocin, high-dose dexamethasone, high-fat, high-sucrose diet, and partial pancreatectomy.
Conditional Knock-Out in Liver
In other embodiments, Albumin-cre and α1-antitrypsin/cre mice can be used to generate Ildr2 knock out in the liver. Albumin-cre and α1-antitrypsin/cre mice have been used to ablate genes in hepatocytes, with the α1-antitrypsin/cre line being useful for earlier-onset ablation during fetal development, and the albumin-cre mice being useful for post-natal knockout (Postic et al, 2000, Genesis 26:149-150). Analyses of the knockout can be performed by protein- and mRNA-based expression assays.
The characterization of any of the knock out mice described herein, can include hepatic metabolism, hepatic glucose production (GTTs, hyperinsulinemic/euglycemic clamps, gene expression, pyruvate challenge tests) and lipid metabolism (Total and Hdl cholesterol, hepatic TG content, gene expression, ApoB levels and secretion using Triton inhibition of lipoprotein clearance; VLDL and LDL measurements by FPLC and ultracentrifugation will help identify variations in lipoprotein composition). The role of altered lipid metabolism in ILDR2 function can be examined the liver conditional Ildr2 knockout mice.
Ttr-Cre Knock-Out
In certain aspects, the invention provides unique liver/β-cell combination of expression driven by the transthyretin promoter to probe the role of the Beta cell/liver axis in metabolic control (Okamoto et al, 2004, J Clin Invest 114:214-223; Okamoto et al, 2006, J Clin Invest 116:775-782; Okamoto et al, 2005, J Clin Invest 115:1314-1322; Nakae et al, 2002, Nat Genet 32:245-253). Because Ildr2 is prominently expressed in liver and Beta cells, it can be useful to the generate of a double knockout driven by Ttr-cre to studying role the role of Ildr2 in these tissues.
Genetic and Environmental Interactions of the Ildr2 Mutation
In addition to analyzing Ildr2 mutant mice according to genetic background, the invention provides methods to determine the contribution of Ildr2 loss-of-function to other forms of insulin-resistant diabetes. In certain aspects, dietary manipulations such as high fat and “Surwit” high fat-high sucrose diets can be used to examine the contribution of Ildr2 to the environmental determinants of diabetes. The genetic component can be assessed by crossing Ildr2 knockouts with Insulin Receptor heterozygous knockouts as a model of insulin resistance (Kido et al, 2000, J Clin Invest 105:199-205), or Irs2 knockouts (Kitamura et al, 2002, J Clin Invest 110:1839-1847), as a model of β-cell failure (Accili 2004, Diabetes 53:1633-1642).
Metabolic Characterization
Metabolic characterization can be carried out for β cells, hepatocytes and other cell, tissue or organ of interest. Non-limiting examples of such tissues or organs are muscle, brain or the gut.
Conditional Activation of Ildr2
Phenotypical analysis of mice carrying the ENU amber mutation can yield preliminary insights into the developmental phenotypes of Ildr2-deficient animals. Such Ildr2-nullizygous mice can be tailored to develop normally and show increased susceptibility to diabetes at early post-natal stages. Ildr2 function can then be restored to alleviate or cure the disease. For example, if C57BL/6 Ildr2-deficient mice are viable and develop diabetes postnatally, tissue-specific reactivation of Ildr2 expression can be used to rescue the phenotype. In certain embodiments, the invention provides a conditional re-activatable Ildr2 allele generated by inserting a loxP-flanked STOP cassette consisting of an artificial splice acceptor site and a neomycin selection marker cassette into the first intron of the Ildr2 gene (FIG. 20). In this approach the presence of the STOP cassette in intron 1 can cause splicing to this artificial exon and termination of transcription by the triple SV40 polyA signal to efficiently prevent expression of the Ildr2 allele in the absence of cre (Hingorani et al, 2003, Cancer Cell 4:437-450; Ventura et al, 2007, Nature 445:661-665). Ildr2 function can then be restored in a tissue-specific manner employing the cre lines used for conditional inactivation of the gene. In other aspects, the invention provides animals carrying one or more re-activatable alleles described herein.
Methods of Treatment
The invention provides for methods of treating a metabolic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In some embodiments, the metabolic disease is a fatty liver disease, dyslipidemia, metabolic syndrome, a cardiovascular disease, obesity, a leptin disorder, or any combination of the listed diseases. The invention provides methods for decreasing lipid levels in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In some embodiments, the lipid level is a cholesterol level, triglyceride level, ApoB level, LDL-cholesterol level, VLDL-cholesterol level, small LDL-particle level, small VLDL-particle level, non-HDL-cholesterol level, phospholipid level, or fatty acid level, or any combination lipid level listed herein. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the nucleic acid is administered to the subject by an adenovirus or a adeno-associated virus. In one embodiment, the agent is an ILDR2 protein, an ILDR2 polypeptide, an ILDR2 isoform, or an ILDR2 functional fragment. In one embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor.
For example, several well-established metabolic disease treatments as well as treatments for decreasing lipid levels ranging from non-pharmaceutical to pharmaceutical intervention are known in the art. Non-pharmaceutical interventions include, but are not limited to, dietary restriction, exercise, psychiatric treatment, and surgical treatments to reduce food consumption (e.g., bariatric surgery) or remove fat (e.g., liposuction). Present pharmacological interventions can induce a weight loss of between 5 to 15 kg. Appetite suppressants and energy expenditure or nutrient-modifying agents are the main focus of pharmacological intervention. Dexfenfluramine (Redux), sibutramine (Meridia), beta3-adrenergic agonists, sympathomimetic adrenergic agents (such as amphetamines (dextroamphetamine)), phentermine, benzphetamine, phendimetrazine, mazindol, diethylpropion, phenylpropanolamine, serotonin (5-HT) reuptake inhibitors (such as sibutramine), and gastrointestinal lipases (such as orlistat) are examples of such pharmacological interventions. See also, Bays, (2004) Obesity Research 12(8):1197-1211, and Klonoff et al., J Diabetes Sci Technol. 2008 September; 2(5):913-8, the contents of each which are incorporated by reference in their entireties. However, if the medication is discontinued, renewed weight gain can ensue. Surgical treatments are comparatively successful, but are complicated, expensive, and have significant risks. Surgical treatments are reserved for patients with extreme obesity and/or with serious medical complications.
The amount which will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques. In vitro or in vivo assays can also be used to identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the severity of the obesity or the obesity-associated disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems, such as a db/db mouse, an ob/ob mouse, or a High-fat-diet-fed mouse model (e.g., the Diet Induced Obesity (DIO) mouse model).
In some embodiments, the subject is already suspected to have a metabolic disease. In other embodiments, the subject is being treated for a metabolic disease, before being treated according to the methods of the invention. In other embodiments, the subject is not being treated for a metabolic disease, before being treated according to the methods of the invention.
The present invention also provides a kit for treating a metabolic disease in a subject. In one embodiment, the kit for treating a metabolic disease comprises an agent which increases expression of Ildr2 mRNA or ILDR2 protein to administer to a subject and instructions of use. In one aspect, the invention provides an agent which increases expression of the Ildr2 mRNA or ILDR2 protein. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding a ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or a ILDR2 functional fragment. In another embodiment, the agent is an ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or a ILDR2 functional fragment. In another embodiment, the agent is a peptide having SEQ ID NO: 2-9. In one embodiment, the agent is an inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1. In one embodiment, the inhibitor of PERK, IRE1a, active ATF6, or spliced XBP1 is a RNAi. In one embodiment, the agent is an ER stress inhibitor.
The present invention also provide a method of determining the presence of, or predisposition to, a metabolic disease in a subject. In one embodiment, the presence of, or predisposition to a metabolic disease in a subject is determined by extracting a sample from a subject and detecting the presence, absence or reduction of an ILDR2 protein or nucleic acid in the sample, wherein absence, or reduction of the ILDR2 protein or nucleic acid indicates the presence of, or predisposition to, a metabolic disease. In a further embodiment, the method further comprises administering of an agent which increases expression of ILDR2 mRNA or ILDR2 protein to the subject where an ILDR2 protein or nucleic acid was not detected. In one embodiment, a reduction of an ILDR2 protein or nucleic acid in the sample comprises detecting a lower amount of an ILDR2 protein or nucleic acid in the sample than the amount of an ILDR2 protein or nucleic acid in a control sample. In one embodiment, the control sample is from a subject without a metabolic disease. In one embodiment, the ILDR2 protein or nucleic acid is detected by incubating the sample with an agent that binds to an ILDR2 protein or nucleic acid. In a further embodiment, the agent is an antibody to an ILDR2 protein.
The present invention also provides a diagnostic kit for determining the presence of, or predisposition to, a metabolic disease, the kit comprising an agent that binds to an ILDR2 protein or nucleic acid, and instructions for use. In one embodiment, the agent is an antibody to an ILDR2 protein.
In one embodiment, the subject is an animal. In another embodiment, the subject is an animal that has or is diagnosed with a disease of the digestive system. In one embodiment, the subject is a human. In other embodiments, the subject is a mammal. In one embodiment, the subject is a dog. In another embodiment, the subject is a cat. In some embodiments, the subject is a rodent, such as a mouse or a rat. In some embodiments, the subject is a cow, pig, sheep, goat, cat, horse, dog, and/or any other species of animal used as livestock or kept as pets.
Molecules of the Invention
As used herein, a “ILDR2 molecule” refers to a ILDR2 protein, or a fragment thereof. A “ILDR2 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a ILDR2 protein, or fragment thereof. For example, an ILDR2 molecule can include the amino acid sequence shown in SEQ ID NO: 22. For example, an ILDR2 molecule can be encoded by a recombinant nucleic acid encoding a ILDR2 protein, or fragment thereof. The ILDR2 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a ILDR2 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. An ILDR2 molecule can include a fragment or portion of a ILDR2 protein. An ILDR2 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms. In one embodiment, an ILDR2 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 129 wherein the variant has a nucleotide sequence identity to SEQ ID NO: 129 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the ILDR2 protein comprises a protein or polypeptide encoded by a ILDR2 nucleic acid sequence, such as the sequence shown in SEQ ID NO: 129.
The nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. For example, a nucleic acid encoding a ILDR2 protein can comprise a recombinant nucleic acid encoding such a protein. The nucleic acid can be a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences). It can be double-stranded or single-stranded.
The invention further provides for nucleic acids that are complementary to a ILDR2 molecule. Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions. Non-limiting examples of stringent hybridization conditions include temperatures above 30° C., above 35° C., in excess of 42° C., and/or salinity of less than about 500 mM, or less than 200 mM. Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.
According to the invention, protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
In one embodiment, an ILDR2 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding an ILDR2 protein, such as the sequences shown in SEQ ID NO: 129. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of an ILDR2 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 22. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NO: 22 or having at least from about 50.1% to about 55% identity to SEQ ID NO: 22, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 22, or having from at least about 60.1% to about 65% identity to SEQ ID NO: 22, or having from about 65.1% to about 70% identity TO SEQ ID NO: 22, or having at least from about 70.1% to about 75% identity to SEQ ID NO: 22, or having at least from about 75.1% to about 80% identity to SEQ ID NO: 22, or having at least from about 80.1% to about 85% identity to SEQ ID NO: 22, or having at least from about 85.1% to about 90% identity to SEQ ID NO: 22, or having at least from about 90.1% to about 95% identity to SEQ ID NO: 22, or having at least from about 95.1% to about 97% identity to SEQ ID NO: 22, or having at least from about 97.1% to about 99% identity to SEQ ID NO: 22. In another embodiment, An ILDR2 molecule can be a fragment of a ILDR2 protein.
In one embodiment, an ILDR2 molecule, according to the methods described herein can be administered to a subject as a recombinant protein. In another embodiment, a ILDR2 molecule, can be administered to a subject as a modified recombinant protein. In a further embodiment, an ILDR2 molecule, according to the methods described herein can be administered to a subject by delivery of a nucleic acid encoding a ILDR2 protein, or fragment thereof. For example, nucleic acids can be delivered to a subject using a viral vector.
The invention provides for a nucleic acid encoding an ILDR2 protein, or fragment thereof.
For example, the polypeptide sequence of human ILDR2 is depicted in SEQ ID NO: 22. The nucleotide sequence of human ILDR2 is shown in SEQ ID NO: 129. Sequence information related to ILDR2 is accessible in public databases by GENBANK Accession numbers NP 955383.1 (protein) and NM_99351.2 (nucleic acid).
SEQ ID NO: 22 is the human wild type amino acid sequence corresponding to ILDR2 (residues 1-639):









  1
mdrvllrwis lfwltamveg lqvtvpdkkk vamlfqptvl rchfstsshq pavvqwkfks



 


 61
ycqdrmgesl gmsstraqsl skrnlewdpy ldcldsrrtv rvvaskqgst vtlgdfyrgr


 


121
eitivhdadl qigklmwgds glyyciittp ddlegkneds vellvlgrtg lladllpsfa


 


181
veimpewvfv glvllgvflf fvlvgicwcq ccphscccyv rcpccpdscc cpqalyeagk


 


241
aakagyppsv sgvpgpysip svplggapss gmlmdkphpp plapsdstgg shsvrkgyri


 


301
qadkerdsmk vlyyvekela qfdparrmrg rynntisels slheedsnfr qsfhqmrskq


 


361
fpvsgdlesn pdywsgvmgg ssgasrgpsa meynkedres frhsqprsks emlsrknfat


 


421
gvpavsmdel aafadsygqr prradgnshe arggsrfers esrahsgfyq ddsleeyygq


 


481
rsrsrepltd adrgwafspa rrrpaedahl prlvsrtpgt apkydhsylg sarergarpe


 


541
gasrggslet pskrsaqlgp rsasyyawsp pgtykagssq ddqedasdda lppyselelt


 


601
rgpsyrgrdl pyhsnsekkr kkepakktnd fptrmslvv






SEQ ID NO: 129 is the human wild type nucleotide sequence corresponding to ILDR2 (nucleotides 1-508):









   1
gttccagcca tttcccactt tcctcactcc gtaattcggc tgggaagttg gggaagatgg



 


  61
atagggtctt gctgaggtgg atttctctct tctggctaac agccatggtc gaaggccttc


 


 121
aggtcacagt gcccgacaag aagaaggtgg ccatgctctt ccagcccact gtgcttcgct


 


 181
gccacttctc aacatcctcc catcagcctg cagttgtgca gtggaagttc aagtcctact


 


 241
gccaggatcg catgggagaa tccttgggca tgtcctctac ccgggcccaa tctctcagca


 


 301
agagaaacct ggaatgggac ccctacttgg attgtttgga cagcaggagg actgttcgag


 


 361
tagtagcttc aaaacagggc tcgactgtca ccctgggaga tttctacagg ggcagagaga


 


 421
tcacgattgt tcatgatgca gatcttcaaa ttggaaagct tatgtgggga gacagcggac


 


 481
tctattactg tattatcacc accccagatg acctggaggg gaaaaatgag gactcagtgg


 


 541
aactgctggt gttgggcagg acagggctgc ttgctgatct cttgcccagt tttgctgtgg


 


 601
agattatgcc agagtgggtg tttgttggcc tggtgctcct gggcgtcttc ctcttcttcg


 


 661
tcctggtggg gatctgctgg tgccagtgct gccctcacag ctgctgctgc tatgtccgct


 


 721
gcccatgctg cccagattcc tgctgctgcc ctcaagcctt gtatgaagca gggaaagcag


 


 781
caaaggccgg gtaccctccc tctgtctccg gtgtccccgg cccttactcc atcccctctg


 


 841
tccctttggg aggagccccc tcatctggca tgctgatgga caagccgcat ccacctccct


 


 901
tggcaccaag tgactccact ggaggaagcc acagtgttcg caaaggttac cggatccagg


 


 961
ctgacaaaga gagagactcc atgaaggtcc tgtactatgt tgagaaggag ctggctcagt


 


1021
ttgatccagc cagaaggatg agaggcagat ataacaacac catctcagaa ctcagctccc


 


1081
tacatgagga ggacagcaat ttccgccagt ctttccatca gatgagaagc aagcagttcc


 


1141
ctgtgtctgg ggacttggag agcaatcctg actattggtc aggtgtcatg ggaggcagca


 


1201
gtggggcaag ccgcgggccc tcagccatgg agtataacaa agaggatcga gagagcttca


 


1261
ggcacagcca gccgcgctcc aagtcggaga tgctgtcgcg gaagaacttc gccacggggg


 


1321
tgccggccgt ttccatggac gagctggcgg ccttcgctga ctcctacggc cagcggcccc


 


1381
gccgggcaga cggcaacagt cacgaggcgc ggggcgggag ccgcttcgag cgctcggagt


 


1441
cgcgggcgca cagcggcttc taccaggacg actccttgga ggagtactac ggtcagcgca


 


1501
gccgcagccg cgagcccctg accgatgctg accgcggctg ggccttcagc cccgcgcgcc


 


1561
gcagacccgc cgaggacgcg cacctgccgc ggctggtgag ccgcacgcca ggcaccgcac


 


1621
ccaaatacga ccactcgtac ctgggcagcg cgcgggagcg ccaggcgcgg cccgagggcg


 


1681
ccagccgcgg tggcagcctg gagacgccat ccaagcggag cgcgcagctc ggcccgcgca


 


1741
gcgcctccta ctacgcttgg tcgccgcccg gcacctacaa ggccggctcg tcgcaggacg


 


1801
accaggagga cgcgtccgac gacgcgctgc cgccctacag cgagctggag ctgacccgcg


 


1861
gcccgtccta ccgcggccgc gacctgccct accacagcaa ctcggagaag aagaggaaaa


 


1921
aggagcccgc caagaaaacc aatgactttc caaccaggat gtcccttgtg gtctgatgtt


 


1981
gtcaacattt ctctggataa tgagaaatca gacatggact acggggacaa gacacaaatc


 


2041
taagaaccag caggcccagg accttctctg gccatcacct tggaagattt gctgatctct


 


2101
gctttggcaa gggatggcag gcagccttta agggaggctg atttcaaacc tctgtgccca


 


2161
tctaactagt ttgagaagct taccaagaaa gcaagaatgt gtgagaacat tcctacatac


 


2221
agagtttctc aactatagcg tttatcctgc ccagcctcct cccttaacag aaccaggact


 


2281
ccatttgcaa ttctgaaaga gagttagctc tggactgcta aactccagaa attgcctatg


 


2341
cctacaatat gcttttctat acctcctgtg ctatacttag agacagaaga atttattact


 


2401
actattagaa ggccttcttc tgacaaggga agatagcttc aagtcaaaat atacctttta


 


2461
tccccatcac tttacagtca ctagtcaatg actgttgtta cactaaaatc aaaaggcctt


 


2521
tggtgagctc agtgacagtg acctctggga caatcacaga aatgacttca ctgctgttct


 


2581
gaatgacaat tcttaagtgg ctaggacaaa gcaaaagcga gtataccttt ttgaaaagct


 


2641
gtctaagtgg tatttccttt tccattctga gaacgtaaac tgctttttcc ttttctgctg


 


2701
cacatgtcaa tatcggagtc ttagacatta agggctcttc tcttcctccc ctctcctgga


 


2761
cttcccacag gttggtgcca cacacacagc cctgcctccc tctgcactct gattagattg


 


2821
tcattgaatg ccttgtgata aatgcttaaa atatacacat gaaagagaag agggaggaaa


 


2881
gaggagaaag cagtaatgca tatagaaaag aatgagaagg aatttaaaag ggaaataaca


 


2941
tcatctcatt atattttgaa tgtggaccat ttcacccaca aacttcactc agtcttttcc


 


3001
ggttttgtgc ttcacttgcc gttaattgtt tctgccatcc cagttctgcc attctaggac


 


3061
atgggggatg tggaacatac agcatttggc ctgactagac tgccactatg gctgctttca


 


3121
agagattaga gatactgctt tctcaggaag gagtacttcc tattcccacc cttgcctaaa


 


3181
tgatagattt tgcctaaatc ccaaagctag atctctggat tttatcgttt gtgtagatag


 


3241
caaaaatggc cacgaactct ttccttctca tcaagaggtg ccatcttttt tccaacccct


 


3301
tgaatctgga gttggctatg tgatttgatt tagccagtag cccaacaaat gtgacacaag


 


3361
cagagacttg aaaagttctt gtgcatgggg cttgtcctct tttgctgctc ttgggaacct


 


3421
tgcaactacc atcaggtgaa caagcctgga ctatcctgct gcatgacaaa agaaaagggc


 


3481
ccagttaccc ttgtcaccct atctgacagc ccgtcaactt ccagctgatg cagacatggg


 


3541
tgagtccagg tgataccaat agaagaactg cctagctgaa cccagcccaa atttctgatt


 


3601
cctactcgag gcgtgagaag ttggtgcatt ctttatgttc atttctcttt aagaacaaac


 


3661
ctgactcgtt cttgtctgaa tgttcatccc tgaacctctt aatccatcca aacttgtgtt


 


3721
tctcatagcc tccactattg ttgacaaatt tttatcaaag cttttcaccc tgcactcttg


 


3781
tggagtggag gataatgctt gactttgcta tcccttaatc cagcagtggt ttcttccctg


 


3841
tctatggatc cgtggacaac ctctgaagat ctcttcttta ccatcctttt ggtcttctcc


 


3901
agagccaccc tactggggct agaccctatt ctcaaagtac cattcctcta gtatccattt


 


3961
gtacttcatg acatttccaa aaaaagtcct atgtttgcaa tgaaataaga aagtggctgg


 


4021
gtgagggtcg gagggatgag ctggtatgtg tcattgcttg gagaattgac ctaccaaagg


 


4081
acttccgtgt tgctttggcc agtcccagag aaatcaggag aacagttgaa cccagtgctg


 


4141
gcacttccaa gggctggaag acacaagcca taaccctggt gctgagtttt agacttgctg


 


4201
gtgtccctgg cctctgaaag cctaggtcta gcctgtctgt ttggacccca gttcagatgg


 


4261
aaaagatgat aaaaaacatt tcttagtcac cagctttgga tttcaacttg ctcaggcaat


 


4321
tttggagaat attgggtagc tgtggtagct attatcgctt tatactgagc actgtgtcag


 


4381
gcttttcacc accaaagagc ctcatagcac cagctgcaga gaccaaaaat aatgtttttt


 


4441
taaagctacg gacgtatgat tttggtgaag gttgagggta gcaatgggaa agaaagaatt


 


4501
attaaaattg gaaacctgca attccaaaga caacaaaact acagatagac tgaaaagtac


 


4561
aaagaagata gcaggctact gaattaacct tgggagttgg gaccaggttg tcctttgtag


 


4621
aactggataa atcattcaga cttccaggct ttcagtagag agaaaaagca gttgtttctg


 


4681
ggtatatgga caggagttaa ggctgagttg acaagtcaaa acttcttgtc ctcagcaccc


 


4741
ctgactttcc tctatgtgtc cttttgtttc ttctcctttg aatagtgtgt cctgcacagg


 


4801
aaatggttat atttgttagc ttctttccta ggtcttattg gagtacaaag taaatcttgt


 


4861
gtaagacata atctctgtcc actaggaccc cgtaatttaa taggggaaat aagacatgct


 


4921
caagaaagga gattttatac atagagtatg aaatagtgct atggataaat tataataaaa


 


4981
ccagagattt agttttttta aaaaatgaga atactttgat attaaagtgt tgtatgtgtt


 


5041
tgtccatcat cttatttaaa catagacttg gtgatctgaa aagccaatac tcaaaagtct


 


5101
gaactgaaaa gaggtgaatt aggatcggga aagggtgagc agcaggggtc ccagggggat


 


5161
gatgcataag ctgctgtgct tgcctgtgag tcactactga gtcaggaaca cgctggagga


 


5221
gggagtgtgg atgcaggtgg cagggaggtg tcccctggta gatgagctgc ttctctaggc


 


5281
catgcatgga ttcattagga agttggagac aatggccatg gacctggtgc atggcagcta


 


5341
ttccatccaa gcacgttcac aggggagctc agcgtggctg ctcctggggc tcagttctgc


 


5401
ggctgtgagt gctgctgccc atattcacca acacagggca ggcctcacgg agatgctagg


 


5461
cctcacatcc cccttctcat gatcctcact gtgcacttga cataggatta ggcatactgg


 


5521
agatgagaaa aggctgccac ccaaacccaa ggcacctgac cacatctgta aatatttctg


 


5581
aatagtccac aaaatttcac ataggtagtc tgattagatc ttgcctttga gagaagctga


 


5641
agtcacagat actgttgtaa tttaccaccg cccctcaccc aatttttttt aataggtgaa


 


5701
gaaaccatca ctgccattaa tgaagtcaca aacctattag gtctttagac tcccaacctc


 


5761
tggatctttt ctgctgatta gtgtttccca aaattgccta accacaagaa ttaacttgat


 


5821
agctgctgtt aaaaaggtat tgttggaccc tgttttggag attgattggg tgggtctaga


 


5881
gccagaattc atatttttaa tatgcattcc aggagactcc tgtgatcaga tgcatttgga


 


5941
aatcattgca ctaagtcata cctctgggta ctccaaacag ctagtcctga ggcttccttg


 


6001
ggccttagaa ttttttcttc aaatgtcctg gtgaggtccc tctcaatcct ttggggctgg


 


6061
ctgtggtgag tcactcagaa gtctggctgt gacctggatg ggctcaccag agtacgctag


 


6121
tggtagtggg aaaacaggca gagagaaagg agtgtcagga gcactcccag ggaggctgtt


 


6181
gtagatattt ccattcccag aacagtgatc tattgtgaca gtctcagaac agacaacaag


 


6241
aattacaggt aattttctca ttctcttgat atatttttag caaaacttaa atcatgaata


 


6301
gaaggaaaag atgccattgg ggaaatagaa aaactcaatc attttataaa gcatacaaat


 


6361
cataaggatg actggccaat agcactccca ctttggtctt acctaaagtt gggtggacaa


 


6421
gaataataaa agtcctcatt ttatatcctt ccaaaatcag atttaaatgc tgccagcatc


 


6481
ttaatggaag tctgaaattg attgatagga tgtagaaatc caaattcact aaaatagggg


 


6541
gccagctaca taaagtccta gaaggaaaaa gtgcctcgct tttttctgcc attatcctac


 


6601
cccctagtca tctggggaat tgatctatga agcttgaaga aggggcattt aacatcagag


 


6661
tggtgcaagg gcagtgttga gatgctttaa gcagcagcct gagctttagc actatttgaa


 


6721
ggggagaagg ttaatactaa taatatttgt gttattttta tgatatatta ctgtttacag


 


6781
aacactttca tttgatccca acatcaactg ctgtgataga ggcagggcag atgttgtggt


 


6841
ctcattacat agaatgtaaa actgaggttg aaaaatacta agtgacttgt ctgtagtcaa


 


6901
atggttttta aaattataaa gccaggcctt ctgactgtct tgtctggtgt cctttccaat


 


6961
tccttaaata ctcatgggac tggaatctgg gtattccaga ttccagtttc tcttcacagc


 


7021
cagacatctg gtgagaagag ccgtagactt gatgcttgtt catatgtcat ggatgtggcg


 


7081
aagccatgaa gacagatact gttgctgctt catccaacta agcaccattc attcctcaaa


 


7141
tgctaatcta agagggagtt gtagcttcac tcaaggagag tttcgttttc tttttctttc


 


7201
tttttttttt ttttgagaca gggtcttgct ctgtggccca tggtgcagtg cagtgcagtg


 


7261
gtgctatcag ctcactgcag tctcaaactc ctagctcaag caatcctcct ccctcagcct


 


7321
cccaagtagg taggactaca gatatatgcc accacgtcca gcaaattttg tttgtttgta


 


7381
gagatggggt cttgctatat ttcccaggct tgtctcaaac tcctggcctc aagtgatcct


 


7441
cccaccttgg ctgcctaaag tgctggtatt acagacatga gccactgaac ccagctgaga


 


7501
gcctcacttt catcacctgt gctgtgaggg gtaatatatg cttcaggttt tctggagaat


 


7561
ccttcttgca gagaagtttc tgaatgaaac gacagattca tctggattca gaactccagg


 


7621
cagaagctgc ttaacagcaa aaatctggca tcttcactac attttaagat tttaggtaga


 


7681
actaagaggg atcagatata gaggaataag gaatgtgaga aggaaaaaga tatagtagtt


 


7741
tagctaaatt tttcttagag tttcttggtg gggctggcca tgaagtaact agtctgactc


 


7801
atttcttctg ggaaggctaa aagagacaca gatagcttct cttttacctt ggctttaagg


 


7861
aaaagccatt ttattaacaa aagtattaga cacgactgca taagaaattt gctgtgtgag


 


7921
aataaagaac aagggagtag gagggtggga cagagaaggg tgagaagttg gcttcgtgag


 


7981
ggccacctgt cagttgtctt tgtgccttgt gacatcaaaa ctgaaatgtt tgtattactg


 


8041
ttgtccatga cttttttttt ctgtgtcaga catacaaatt gaatttggtt gtaatgtttt


 


8101
aaacgtaata aagaattctt acctaca






The invention also provides for a nucleic acid encoding a PERK protein, or fragment thereof. PERK is also known as, “eukaryotic translation initiation factor 2-alpha kinase 3” (“EIF2AK3”), “PEK” and “WRS”. “PERK” refers to all PERK orthologs, including, but not limited to, those found in mice and humans.
For example, the polypeptide sequence of human PERK is depicted in SEQ ID NO: 130. The nucleotide sequence of human PERK is shown in SEQ ID NO: 131. Sequence information related to PERK is accessible in public databases by GENBANK Accession numbers NP 004827.4 (protein) and NM_004836.5 (nucleic acid).
SEQ ID NO: 130 is the human wild type amino acid sequence corresponding to PERK (residues 1-1116):









1
meraispgll vralllllll lglaartvaa grarglpapt aeaafglgaa aaptsatrvp



 


61
aagavaaaev tvedaealpa aagegeprgp epddetelrp rgrslviist ldgriaaldp


 


121
enhgkkqwdl dvgsgslvss slskpevfgn kmiipsldga lfqwdqdres metvpftves


 


181
llessykfgd dvvlvggksl ttyglsaysg kvryicsalg crqwdsdeme geedilllqr


 


241
tqktvravgp rsgnekwnfs vghfelryip dmetragfie stfkpnente eskiisdvee


 


301
qeaaimdivi kvsvadwkvm afskkgghle weyqfctpia sawllkdgkv ipislfddts


 


361
ytsnddvled eediveaarg atensvylgm yrgglylqss vrisekfpss pkalesvtne


 


421
naiiplptik wkplihspsr tpvlvgsdef dkclsndkfs heeysngals ilqypydngy


 


481
ylpyykrern krstqitvrf ldnphynkni rkkdpvlllh wwkeivatil fciiattfiv


 


541
rrlfhphphr qrkesetqcq tenkydsvsg eandsswndi knsgyisryl tdfepiqclg


 


601
rggfgvvfea knkvddcnya ikrirlpnre larekvmrev kalaklehpg ivryfnawle


 


661
appekwgekm deiwlkdest dwplsspspm dapsvkirrm dpfatkehie iiapspqrsr


 


721
sfsvgiscdq tsssesqfsp lefsgmdhed isesvdaayn lqdscltdcd vedgtmdgnd


 


781
eghsfelcps easpyvrsre rtsssivfed sgcdnasske epktnrlhig nhcankltaf


 


841
kptsskssse atlsispprp ttlsldltkn tteklqpssp kvylyiqmql crkenlkdwm


 


901
ngrctieere rsvclhiflq iaeaveflhs kglmhrdlkp snifftmddv vkvgdfglvt


 


961
amdqdeeeqt vltpmpayar htgqvgtkly mspeqihgns yshkvdifsl glilfellyp


 


1021
fstqmervrt ltdvrnlkfp plftqkypce yvmvqdmlsp spmerpeain iienavfedl


 


1081
dfpgktvlrq rsrslsssgt khsrqsnnsh splpsn






SEQ ID NO: 131 is the human wild type nucleotide sequence corresponding to PERK (nucleotides 1-4665):









1
ggaaagtcca ccttccccaa caaggccagc ctgggaacat ggagtggcag cggccgcagc



 


61
caatgagaga gcaaacgcgc ggaaagtttg ctcaatgggc gatgtccgag ataggctgtc


 


121
actcaggtgg cagcggcaga ggccgggctg agacgtggcc aggggaacac ggctggctgt


 


181
ccaggccgtc ggggcggcag tagggtccct agcacgtcct tgccttcttg ggagctccaa


 


241
gcggcgggag aggcaggcgt cagtggctgc gcctccatgc ctgcgcgcgg ggcgggacgc


 


301
tgatggagcg cgccatcagc ccggggctgc tggtacgggc gctgctgctg ctgctgctgc


 


361
tgctggggct cgcggcaagg acggtggccg cggggcgcgc ccgtggcctc ccagcgccga


 


421
cggcggaggc ggcgttcggc ctcggggcgg ccgctgctcc cacctcagcg acgcgagtac


 


481
cggcggcggg cgccgtggct gcggccgagg tgactgtgga ggacgctgag gcgctgccgg


 


541
cagccgcggg agagcaggag cctcggggtc cggaaccaga cgatgagaca gagttgcgac


 


601
cgcgcggcag gtcattagta attatcagca ctttagatgg gagaattgct gccttggatc


 


661
ctgaaaatca tggtaaaaag cagtgggatt tggatgtggg atccggttcc ttggtgtcat


 


721
ccagccttag caaaccagag gtatttggga ataagatgat cattccttcc ctggatggag


 


781
ccctcttcca gtgggaccaa gaccgtgaaa gcatggaaac agttcctttc acagttgaat


 


841
cacttcttga atcttcttat aaatttggag atgatgttgt tttggttgga ggaaaatctc


 


901
tgactacata tggactcagt gcatatagtg gaaaggtgag gtatatctgt tcagctctgg


 


961
gttgtcgcca atgggatagt gacgaaatgg aacaagagga agacatcctg cttctacagc


 


1021
gtacccaaaa aactgttaga gctgtcggac ctcgcagtgg caatgagaag tggaatttca


 


1081
gtgttggcca ctttgaactt cggtatattc cagacatgga aacgagagcc ggatttattg


 


1141
aaagcacctt taagcccaat gagaacacag aagagtctaa aattatttca gatgtggaag


 


1201
aacaggaagc tgccataatg gacatagtga taaaggtttc ggttgctgac tggaaagtta


 


1261
tggcattcag taagaaggga ggacatctgg aatgggagta ccagttttgt actccaattg


 


1321
catctgcctg gttacttaag gatgggaaag tcattcccat cagtcttttt gatgatacaa


 


1381
gttatacatc taatgatgat gttttagaag atgaagaaga cattgtagaa gctgccagag


 


1441
gagccacaga aaacagtgtt tacttgggaa tgtatagagg ccagctgtat ctgcagtcat


 


1501
cagtcagaat ttcagaaaag tttccttcaa gtcccaaggc tttggaatct gtcactaatg


 


1561
aaaacgcaat tattccttta ccaacaatca aatggaaacc cttaattcat tctccttcca


 


1621
gaactcctgt cttggtagga tctgatgaat ttgacaaatg tctcagtaat gataagtttt


 


1681
ctcatgaaga atatagtaat ggtgcacttt caatcttgca gtatccatat gataatggtt


 


1741
attatctacc atactacaag agggagagga acaaacgaag cacacagatt acagtcagat


 


1801
tcctcgacaa cccacattac aacaagaata tccgcaaaaa ggatcctgtt cttcttttac


 


1861
actggtggaa agaaatagtt gcaacgattt tgttttgtat catagcaaca acgtttattg


 


1921
tgcgcaggct tttccatcct catcctcaca ggcaaaggaa ggagtctgaa actcagtgtc


 


1981
aaactgaaaa taaatatgat tctgtaagtg gtgaagccaa tgacagtagc tggaatgaca


 


2041
taaaaaactc tggatatata tcacgatatc taactgattt tgagccaatt caatgcctgg


 


2101
gacgtggtgg ctttggagtt gtttttgaag ctaaaaacaa agtagatgac tgcaattatg


 


2161
ctatcaagag gatccgtctc cccaataggg aattggctcg ggaaaaggta atgcgagaag


 


2221
ttaaagcctt agccaagctt gaacacccgg gcattgttag atatttcaat gcctggctcg


 


2281
aagcaccacc agagaagtgg caagaaaaga tggatgaaat ttggctgaaa gatgaaagca


 


2341
cagactggcc actcagctct cctagcccaa tggatgcacc atcagttaaa atacgcagaa


 


2401
tggatccttt cgctacaaaa gaacatattg aaatcatagc tccttcacca caaagaagca


 


2461
ggtctttttc agtagggatt tcctgtgacc agacaagttc atctgagagc cagttctcac


 


2521
cactggaatt ctcaggaatg gaccatgagg acatcagtga gtcagtggat gcagcataca


 


2581
acctccagga cagttgcctt acagactgtg atgtggaaga tgggactatg gatggcaatg


 


2641
atgaggggca ctcctttgaa ctttgtcctt ctgaagcttc tccttatgta aggtcaaggg


 


2701
agagaacctc ctcttcaata gtatttgaag attctggctg tgataatgct tccagtaaag


 


2761
aagagccgaa aactaatcga ttgcatattg gcaaccattg tgctaataaa ctaactgctt


 


2821
tcaagcccac cagtagcaaa tcttcttctg aagctacatt gtctatttct cctccaagac


 


2881
caaccacttt aagtttagat ctcactaaaa acaccacaga aaaactccag cccagttcac


 


2941
caaaggtgta tctttacatt caaatgcagc tgtgcagaaa agaaaacctc aaagactgga


 


3001
tgaatggacg atgtaccata gaggagagag agaggagcgt gtgtctgcac atcttcctgc


 


3061
agatcgcaga ggcagtggag tttcttcaca gtaaaggact gatgcacagg gacctcaagc


 


3121
catccaacat attctttaca atggatgatg tggtcaaggt tggagacttt gggttagtga


 


3181
ctgcaatgga ccaggatgag gaagagcaga cggttctgac cccaatgcca gcttatgcca


 


3241
gacacacagg acaagtaggg accaaactgt atatgagccc agagcagatt catggaaaca


 


3301
gctattctca taaagtggac atcttttctt taggcctgat tctatttgaa ttgctgtatc


 


3361
cattcagcac tcagatggag agagtcagga ccttaactga tgtaagaaat ctcaaatttc


 


3421
caccattatt tactcagaaa tatccttgtg agtacgtgat ggttcaagac atgctctctc


 


3481
catcccccat ggaacgacct gaagctataa acatcattga aaatgctgta tttgaggact


 


3541
tggactttcc aggaaaaaca gtgctcagac agaggtctcg ctccttgagt tcatcgggaa


 


3601
caaaacattc aagacagtcc aacaactccc atagcccttt gccaagcaat tagccttaag


 


3661
ttgtgctagc aaccctaata ggtgatgcag ataatagcct acttcttaga atatgcctgt


 


3721
ccaaaattgc agacttgaaa agtttgttct tcgctcaatt tttttgtgga ctactttttt


 


3781
tatatcaaat ttaagctgga tttgggggca taacctaatt tgagccaact cctgagtttt


 


3841
gctatactta aggaaagggc tatctttgtt ctttgttagt ctcttgaaac tggctgctgg


 


3901
ccaagcttta tagccctcac catttgccta aggaggtagc agcaatccct aatatatata


 


3961
tatagtgaga actaaaatgg atatattttt ataatgcaga agaaggaaag tccccctgtg


 


4021
tggtaactgt attgttctag aaatatgctt tctagagata tgatgatttt gaaactgatt


 


4081
tctagaaaaa gctgactcca tttttgtccc tggcgggtaa attaggaatc tgcactattt


 


4141
tggaggacaa gtagcacaaa ctgtataacg gtttatgtcc gtagttttat agtcctattt


 


4201
gtagcattca atagctttat tccttagatg gttctagggt gggtttacag ctttttgtac


 


4261
ttttacctcc aataaaggga aaatgaagct ttttatgtaa attggttgaa aggtctagtt


 


4321
ttgggaggaa aaaagccgta gtaagaaatg gatcatatat attacaacta acttcttcaa


 


4381
ctatggactt tttaagccta atgaaatctt aagtgtctta tatgtaatcc tgtaggttgg


 


4441
tacttccccc aaactgatta taggtaacag tttaatcatc tcacttgcta acatgttttt


 


4501
atttttcact gtaaatatgt ttatgtttta tttataaaaa ttctgaaatc aatccatttg


 


4561
ggttggtggt gtacagaaca cacttaagtg tgttaacttg tgacttcttt caagtctaaa


 


4621
tgatttaata aaactttttt taaattaaaa aaaaaaaaaa aaaaa






The invention also provides for a nucleic acid encoding a IRE1a protein, or fragment thereof. IRE1a is also known as, “endoplasmic reticulum to nucleus signaling 1” (“ERN1”), “IRE1”, “IRE1P”, “IRE1a”, and “hIRE1p”. “IRE1a” refers to all IRE1a orthologs, including, but not limited to, those found in mice and humans.
For example, the polypeptide sequence of human IRE1a is depicted in SEQ ID NO: 132. The nucleotide sequence of human IRE1a is shown in SEQ ID NO: 133. Sequence information related to IRE1 a is accessible in public databases by GENBANK Accession numbers NP 001424.3 (protein) and NM_001433.3 (nucleic acid).
SEQ ID NO: 132 is the human wild type amino acid sequence corresponding to IRE1a (residues 1-977):









1
mparrlllll tlllpglgif gststvtlpe tllfvstldg slhayskrtg sikwtlkedp



 


61
vlgvpthvee paflpdpndg slytlgsknn egltklpfti pelvgaspcr ssdgilymgk


 


121
kgdiwyvidl ltgekqqtls safadslcps tsllylgrte ytitmydtkt relrwnatyf


 


181
dyaaslpedd vdykmshfvs ngdglvvtvd sesgdvlwiq nyaspvvafy vwqreglrkv


 


241
mhinvavetl ryltfmsgev gritkwkypf pketeakskl tptlyvgkys tslyaspsmv


 


301
hegvavvprg stlpllegpq tdgvtigdkg ecvitpstdv kfdpglkskn klnylrnywl


 


361
lighhetpls astkmlerfp nnlpkhrenv ipadsekksf eevinlvdqt senapttvsr


 


421
dveekpahap arpeapvdsm lkdmatiils tflligwvaf iityplsmhq qqqlqhqqfq


 


481
kelekiqllq qqqqqlpfhp pgdtaqdgel ldtsgpyses sgtsspstsp rasnhslcsg


 


541
ssaskagssp sleqddgdee tsvvivgkis fcpkdvlghg aegtivyrgm fdnrdvavkr


 


601
ilpecfsfad revqllresd ehpnviryfc tekdrqfqyi aielcaatlq eyveqkdfah


 


661
lglepitllq qttsglahlh slnivhrdlk phnilismpn ahgkikamis dfglckklav


 


721
grhsfsrrsg vpgtegwiap emlsedcken ptytvdifsa gcvfyyvise gshpfgkslq


 


781
rqanillgac sldclhpekh edviarelie kmiamdpqkr psakhvlkhp ffwslekqlq


 


841
ffqdvsdrie kesldgpivk qlerggravv kmdwrenitv plqtdlrkfr tykggsvrdl


 


901
lramrnkkhh yrelpaevre tlgslpddfv cyftsrfphl lahtyramel csherlfqpy


 


961
yfheppepqp pvtpdal






SEQ ID NO: 133 is the human wild type nucleotide sequence corresponding to IRE1a (nucleotides 1-4005):









1
tgcctagtca gttctgcgtc cgctgaggct cggtcaccgc ctcgctgtcg tcgcggcgcc



 


61
cccgccccgt cctctgtccg taccgccccc ggagccaggg ccgagtcctc gccatgccgg


 


121
cccggcggct gctgctgctg ctgacgctgc tgctgcccgg cctcgggatt tttggaagta


 


181
ccagcacagt gacgcttcct gaaaccttgt tgtttgtgtc aacgctggat ggaagtttgc


 


241
atgctgtcag caagaggaca ggctcaatca aatggacttt aaaagaagat ccagtcctgc


 


301
aggtcccaac acatgtggaa gagcctgcct ttctcccaga tcctaatgat ggcagcctgt


 


361
atacgcttgg aagcaagaat aatgaaggcc tgacgaaact tccttttacc atcccagaat


 


421
tggtgcaggc atccccatgc cgaagttcag atggaatcct ctacatgggt aaaaagcagg


 


481
acatctggta tgttattgac ctcctgaccg gagagaagca gcagactttg tcatcggcct


 


541
ttgcagatag tctctgccca tcaacctctc ttctgtatct tgggcgaaca gaatacacca


 


601
tcaccatgta cgacaccaaa acccgagagc tccggtggaa tgccacctac tttgactatg


 


661
cggcctcact gcctgaggac gacgtggact acaagatgtc ccactttgtg tccaatggtg


 


721
atgggctggt ggtgactgtg gacagtgaat ctggggacgt cctgtggatc caaaactacg


 


781
cctcccctgt ggtggccttt tatgtctggc agcgggaggg tctgaggaag gtgatgcaca


 


841
tcaatgtcgc tgtggagacc ctgcgctatc tgaccttcat gtctggggag gtggggcgca


 


901
tcacaaagtg gaagtacccg ttccccaagg agacagaggc caagagcaag ctgacgccca


 


961
ctctgtatgt tgggaaatac tctaccagcc tctatgcctc tccctcaatg gtacacgagg


 


1021
gggttgctgt cgtgccccgc ggcagcacac ttcctttgct ggaagggccc cagactgatg


 


1081
gcgtcaccat tggggacaag ggggagtgtg tgatcacgcc cagcacggac gtcaagtttg


 


1141
atcccggact caaaagcaag aacaagctca actacttgag gaattactgg cttctgatag


 


1201
gacaccatga aaccccactg tctgcgtcta ccaagatgct ggagagattt cccaacaatc


 


1261
tacccaaaca tcgggaaaat gtgattcctg ctgattcaga gaaaaagagc tttgaggaag


 


1321
ttatcaacct ggttgaccag acttcagaaa acgcacctac caccgtgtct cgggatgtgg


 


1381
aggagaagcc cgcccatgcc cctgcccggc ccgaggcccc cgtggactcc atgcttaagg


 


1441
acatggctac catcatcctg agcaccttcc tgctgattgg ctgggtggcc ttcatcatca


 


1501
cctatcccct gagcatgcat cagcagcagc agctccagca ccagcagttc cagaaggaac


 


1561
tggagaagat ccagctcctg cagcagcagc agcagcagct gcccttccac ccacctggag


 


1621
acacggctca ggacggcgag ctcctggaca cgtctggccc gtactcagag agctcgggca


 


1681
ccagcagccc cagcacgtcc cccagggcct ccaaccactc gctctgctcc ggcagctctg


 


1741
cctccaaggc tggcagcagc ccctccctgg aacaagacga tggagatgag gaaaccagcg


 


1801
tggtgatagt tgggaaaatt tccttctgtc ccaaggatgt cctgggccat ggagctgagg


 


1861
gcacaattgt gtaccggggc atgtttgaca accgcgacgt ggccgtgaag aggatcctcc


 


1921
ccgagtgttt tagcttcgca gaccgtgagg tccagctgtt gcgagaatcg gatgagcacc


 


1981
cgaacgtgat ccgctacttc tgcacggaga aggaccggca attccagtac attgccatcg


 


2041
agctgtgtgc agccaccctg caagagtatg tggagcagaa ggactttgcg catctcggcc


 


2101
tggagcccat caccttgctg cagcagacca cctcgggcct ggcccacctc cactccctca


 


2161
acatcgttca cagagaccta aagccacaca acatcctcat atccatgccc aatgcacacg


 


2221
gcaagatcaa ggccatgatc tccgactttg gcctctgcaa gaagctggca gtgggcagac


 


2281
acagtttcag ccgccgatct ggggtgcctg gcacagaagg ctggatcgct ccagagatgc


 


2341
tgagcgaaga ctgtaaggag aaccctacct acacggtgga catcttttct gcaggctgcg


 


2401
tcttttacta cgtaatctct gagggcagcc acccttttgg caagtccctg cagcggcagg


 


2461
ccaacatcct cctgggtgcc tgcagccttg actgcttgca cccagagaag cacgaagacg


 


2521
tcattgcacg tgaattgata gagaagatga ttgcgatgga tcctcagaaa cgcccctcag


 


2581
cgaagcatgt gctcaaacac ccgttcttct ggagcctaga gaagcagctc cagttcttcc


 


2641
aggacgtgag cgacagaata gaaaaggaat ccctggatgg cccgatcgtg aagcagttag


 


2701
agagaggcgg gagagccgtg gtgaagatgg actggcggga gaacatcact gtccccctcc


 


2761
agacagacct gcgtaaattc aggacctata aaggtggttc tgtcagagat ctcctccgag


 


2821
ccatgagaaa taagaagcac cactaccggg agctgcctgc agaggtgcgg gagacgctgg


 


2881
ggtccctccc cgacgacttc gtgtgctact tcacatctcg cttcccccac ctcctcgcac


 


2941
acacctaccg ggccatggag ctgtgcagcc acgagagact cttccagccc tactacttcc


 


3001
acgagccccc agagccccag cccccagtga ctccagacgc cctctgagcg agggcggccc


 


3061
ctctgttctg gtggccccag ctgtgactga gggcctggtc accacaatta gagcttgatg


 


3121
cctcccggct ttgcagggag accaggcttc ccaaaccaag tgccttgagc tgcctgctct


 


3181
gcagcccaca gaggacagtg ctgaccccag gaagtgggag aagtggcccc tcgtgaccta


 


3241
cagggaactg ggaagatgct ggccccaaaa gccttacggt catgatgtct gcaaaggagg


 


3301
gcctcagaga cagcgcgagt agcaccccca gccatctact ggataaactt gcttcagact


 


3361
ttttaaattc ctgcttaatg tcagtctaca ggcctttcag gaagggagag gagggaatcg


 


3421
tacattttgc ttgcgtgctg ggacagctag gctgagatgc accaagtaca gccttcactg


 


3481
gagaccggaa ttgagaggtg ggggatgctg aggaggggga ggacggagtt cagagggtgt


 


3541
cgtcctgcag tgtgagattt ctcattgatc acagatgtgc ccagagtagc ccaggtcact


 


3601
gttaactagt gtttctgcag aggcagcagg agccatgagc atgaggtgtg gcattaggga


 


3661
ctggtcagct atgcatgctg gcaggtgggg ttgtgtctgc aggtctcaga aatgaagagg


 


3721
ctgctctgtt ctggaggcag ccgtggccca gtgccagtgg ccagaacagt ggcctttggt


 


3781
gggtgtgtcc cgggccatct cggggtggtg ctcaggagcg cctggggcaa gaggtaaaga


 


3841
gttccctggc cttcaaggag agcagcgaag acccagacag gggccagcct tcaggaccag


 


3901
agggaggccg ccgaatggga ccctcctggt caccaggaga aagccctggg ccagcgagta


 


3961
ggcagtcaaa ctccttcgtc cccaaggccg gtggaacaag aggct






The invention also provides for a nucleic acid encoding a ATF6 protein, or fragment thereof. ATF6 is also known as, “activating transcription factor 6” (“ATF6”) and “ATF6A”. “ATF6” refers to all ATF6 orthologs, including, but not limited to, those found in mice and humans.
For example, the polypeptide sequence of human ATF6 is depicted in SEQ ID NO: 134. The nucleotide sequence of human PERK is shown in SEQ ID NO: 135. Sequence information related to ATF6 is accessible in public databases by GENBANK Accession numbers NP 031374.2 (protein) and NM_007348.3 (nucleic acid).
SEQ ID NO: 134 is the human wild type amino acid sequence corresponding to ATF6 (residues 1-670):









1
mgepagvagt mespfspglf hrldedwdsa lfaelgyftd tdelqleaan etyennfdnl



 


61
dfdldlmpwe sdiwdinnqi ctvkdikaep qplspasssy syssprsvds ysstqhvpee


 


121
ldlssssqms plslygensn slssaeplke dkpvtgprnk tengltpkkk iqvnskpsiq


 


181
pkplllpaap ktqtnssvpa ktiiiqtvpt lmplakqqpi islqpaptkg qtvllsqptv


 


241
vqlqapgvlp saqpvlavag gvtqlpnhvv nvvpapsans pvngklsvtk pvlqstmrnv


 


301
gsdiavlrrq qrmiknresa cqsrkkkkey mlglearlka alseneqlkk engtlkrqld


 


361
evvsenqrlk vpspkrrvvc vmivlafiil nygpmsmleq dsrrmnpsys panqrrhllg


 


421
fsakeaqdts dgiiqknsyr ydhsysndka lmvlteepll yippppcqp1 intteslrin


 


481
helrgwvhrh evertksrrm tnnqqktril qgaleqgsns qlmavqytet tssisrnsgs


 


541
elqvyyaspr syqdffeair rrgdtfyvvs frrdhlllpa tthnkttrpk msivlpaini


 


601
nenvingqdy evmmqidcqv mdtrilhiks ssvppylrdq qrnqtntffg sppaateath


 


661
vvstipeslq






SEQ ID NO: 135 is the human wild type nucleotide sequence corresponding to ATF6 (nucleotides 1-7563):









1
aaaagtagtt tgtctttact aggccaccgt ctcgtcagcg ttacggagta ttttgtccgc



 


61
ctgccgccgc cgtcccagat attaatcacg gagttccagg gagaaggaac ttgtgaaatg


 


121
ggggagccgg ctggggttgc cggcaccatg gagtcacctt ttagcccggg actctttcac


 


181
aggctggatg aagattggga ttctgctctc tttgctgaac tcggttattt cacagacact


 


241
gatgagctgc aattggaagc agcaaatgag acgtatgaaa acaattttga taatcttgat


 


301
tttgatttgg atttgatgcc ttgggagtca gacatttggg acatcaacaa ccaaatctgt


 


361
acagttaaag atattaaggc agaacctcag ccactttctc cagcctcctc aagttattca


 


421
gtctcgtctc ctcggtcagt ggactcttat tcttcaactc agcatgttcc tgaggagttg


 


481
gatttgtctt ctagttctca gatgtctccc ctttccttat atggtgaaaa ctctaatagt


 


541
ctctcttcag cggagccact gaaggaagat aagcctgtca ctggtcctag gaacaagact


 


601
gaaaatggac tgactccaaa gaaaaaaatt caggtgaatt caaaaccttc aattcagccc


 


661 
aagcctttat tgcttccagc agcacccaag actcaaacaa actccagtgt tccagcaaaa



 


721
accatcatta ttcagacagt accaacgctt atgccattgg caaagcagca accaattatc



 


781
agtttacaac ctgcacccac taaaggccag acggttttgc tgtctcagcc tactgtggta


 


841
caacttcaag cacctggagt tctgccctct gctcagccag tccttgctgt tgctggggga


 


901
gtcacacagc tccctaatca cgtggtgaat gtggtaccagc cccttcagc gaatagccca


 


961
gtgaatggaa aactttccgt gactaaacct gtcctacaaa gtaccatgag aaatgtcggt


 


1021
tcagatattg ctgtgctaag gagacagcaa cgtatgataa aaaatcgaga atccgcttgt


 


1081
cagtctcgca agaagaagaa agaatatatg ctagggttag aggcgagatt aaaggctgcc


 


1141
ctctcagaaa acgagcaact gaagaaagaa aatggaacac tgaagcggca gctggatgaa


 


1201
gttgtgtcag agaaccagag gcttaaagtc cctagtccaa agcgaagagt tgtctgtgtg


 


1261
atgatagtat tggcatttat aatactgaac tatggaccta tgagcatgtt ggaacaggat


 


1321
tccaggagaa tgaaccctag tgtgagccct gcaaatcaaa ggaggcacct tctaggattt


 


1381
tctgctaaag aggcacagga cacatcagat ggtattatcc agaaaaacag ctacagatat


 


1441
gatcattctg tttcaaatga caaagccctg atggtgctaa ctgaagaacc attgctttac


 


1501
attcctccac ctccttgtca gcccctaatt aacacaacag agtctctcag gttaaatcat


 


1561
gaacttcgag gatgggttca tagacatgaa gtagaaagga ccaagtcaag aagaatgaca


 


1621
aataatcaac agaaaacccg tattcttcag ggtgctctgg aacagggctc aaattctcag


 


1681
ctgatggctg ttcaatacac agaaaccact agtagtatca gcaggaactc agggagtgag


 


1741
ctacaagtgt attatgcttc acccagaagt tatcaagact tttttgaagc catccgcaga


 


1801
aggggagaca cattttatgt tgtgtcattt cgaagggatc acctgctgtt accagctacc


 


1861
acccataaca agaccacaag accaaaaatg tcaattgtgt taccagcaat aaacataaat


 


1921
gagaatgtga tcaatgggca ggactacgaa gtgatgatgc agattgactg tcaggtgatg


 


1981
gacaccagga tcctccatat caaaagttcg tcagttcctc cttacctccg agatcagcag


 


2041
aggaatcaaa ccaacacctt ctttggctcc cctcccgcag ccacagaggc aacccacgtt


 


2101
gtcagcacca tccctgagtc attacaatag caccctgcag ctatgctgga aaactgagcg


 


2161
tgggaccctg ccagactgaa gagcaggtga gcaaaatgct gctttctgcc ttggtggcag


 


2221
gcagagaact gtctcgtact agaattcaag gaggaaagaa gaagaaataa aagaagctgc


 


2281
tccatttttc atcatctacc catctatttg gaaagcactg gaattcagat gcaagagaac


 


2341
aatgtttctt cagtggcaaa tgtagccctg catcctccag tgttacctgg tgtagatttt


 


2401
tttttctgta cctttctaaa cctctcttcc ctctgtgatg gttttgtgtt taaacagtca


 


2461
tcttctttta aataatatcc acctctcctt tttgccattt cacttattga ttcataaagt


 


2521
gaattttatt taaagctatg ccacacatgc atgttcaaat ggtttccact gattcgattt


 


2581
ttcattcatt taatgcaaac ccattctgga tattgtgctt atttgagaaa acacatttca


 


2641
aaaccagaaa agccaaaaac actccaaaaa caagcaaaac aatttggagc tttagataaa


 


2701
aggaaaaact cccagttggt aaagtttatc tttacttagg atttgtggct cacacctaaa


 


2761
caaagggggt cagggagtgg gtacaaattt gagaaaatag aagggtaagg gaagggccag


 


2821
tggtggggtt tggagagagg agatagctcc attaatacac atgtttaaaa gatggaaagt


 


2881
tcacgcctgt aatcccagca ctttgggagg ccgaggcggg tggatcacga ggtcaggaga


 


2941
tcaagaccat cccggctaaa acggtgaaac cccgtctcta ctaaaaatac aaaaaattag


 


3001
ccgggcgtag tgacgggcgc ctgtagtccc agctacttgg gaggctgagg caggagaatg


 


3061
gcgtgaaccc gggaggcgga gcttgcagtg agccgagatc ccgccactgc actccagcct


 


3121
gggcgacaga gcgagactcc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa gatggaaagt


 


3181
tcgatgtgac tgcagtatga gattaaagcc acaactattg tttattttgg ggactctagg


 


3241
ccaccaagta ttagcacaca tacttatgtt ttctctacta atctggtcca ggtcctcatg


 


3301
gaccacagga caaagctttc attttcattc attcttctat tgaaattata ccaaattcag


 


3361
ctgaggaata tggaagtaac tttagactta aacaagacaa aagttttttc actgaagaat


 


3421
tgacaagtat ttgctcctta aaacaacgca gattagtgaa cgtggattcc tgctgaggga


 


3481
gtgcatccca taatatggca ataattttca gtttctccaa cgaaaagata gtgaaggaat


 


3541
taaatctttt gtcctcccat ggttaaaaaa aaaaaaaaag ctgtgttcat ttttactgta


 


3601
ctatgcctct tttttcacca tagtagacaa ttatgtttca tttgatgaat tcatagaact


 


3661
ggatctcata cagcgatgtc ctctctaatg ttctaccttt cagtttctaa agtgagtctt


 


3721
cctccctctc ctacaaaact tttcaatttt ttgatgtaac tcatctacaa atactgtttc


 


3781
ttaccccagt tgacttgcct ttgtcagatt tcttcttgtt ccacactata gcaatcaatt


 


3841
tctcttcttc cttacaagaa agggaacgag aaattgtagc aacctctcaa ggattatatg


 


3901
cagctagtta gttttctgcc tgtgaaatta ggtctggctc ctaaataatt ttaaagaacc


 


3961
atcagcactt ctaactctct ggacaggtgc ctctttgtcc aagctagtta aatgctttcc


 


4021
aaggaaatca gttcaacttt tgtgagcggg gaaaagcagg gctttattgt tgtgttacct


 


4081
gggagtctgg agtttgaaaa gtgctaatta accttcctct ttttccacat tacaaacctt


 


4141
tttaagcagc gcagcactcc ccttagattt ggctatcctg ggtgattttc agacaagaac


 


4201
cattttctct ggggaccatt cttctgctgg gtgccaagga atataaggca aatgcccaga


 


4261
agaccttcag gtgactgggc agtcttatca tgggatattt cttctggccc tgccccttcc


 


4321
cattctgtaa tgtgaattag ccacaccaga ggctgtgacc atggctagta gacagtggca


 


4381
acatagtcat ccccaagatg ctaatcttct gctggaactg tcatacgtta tcatggtcaa


 


4441
tgtaaacctg gtttgtgtgg ggtgattata aatagagttt ccctcctctc tgtgacagaa


 


4501
tcacaggaga aggacccatc tcgtggcctt cttgttctta gcgcttcact tttacttcat


 


4561
ccctcgattc ccagcttttt ctatcatcat tttgccaact cctcagatgc aagactttgg


 


4621
ttatgtcata ctcaccaacg ttagtccctc tcttccaggt gaaaaggtgg gtagcggttg


 


4681
ggagggagtc tccactgaag agcaggaagg tggtagcagg gccggcagct ctgccacaga


 


4741
gctaggggtg cctgtaaggt gccgcctaga gcagcctggg agctttgcct tcttttgtct


 


4801
ctcactagcc cttctactct ttgtcattgc ctgttcttga gtggatcttt gaaatgaggg


 


4861
gacaggattc tcctaagggt agagtttcag gaaatgagtg aaaggcaatt gacaaatgca


 


4921
aagaagtagt cactttttaa attgctggca aagctataat taatccctag gcacaattgt


 


4981
agtttttatt ttaatgtttg tatgcacaag gccctttagg aaatgagaag ttgccatgcc


 


5041 
agattaattt tttttttttt ttttggtggg attgcctttt gggggttgca gccagaaatt



 


5101
gtgggtaatg tgtgtatttt tttatttatt aaattttaaa caggattgtg caagcttatg



 


5161
agacaattag ataaactcat ggaggaggca ggtcctcctg ttattagatg attttgtgct


 


5221
cttggggctg acaataatac actcttggga agtgatggta gagactgatg ggaatagtct


 


5281
ttctgcctgg ttgcaagtcc caaattttta agggttaatg gaagtaagtg gatgtttcct


 


5341
catgttaact actgaatcag atgttaggag cttgtccctt tggggttgac ttatgcccag


 


5401
cagtacaggg acacagcttc attagagtgt tagtgtaaac taactccaaa gttaggagtt


 


5461
aatgtgaaag gatcatcctt gaaacaaatc tgctgtttgc catgcttgta gtacagaaac


 


5521
ttcacatgga gttttgggtg ggatttgtgt tttcacaagt aaaaaatccc tcacgattat


 


5581
aaaactcaga gcatcatcta attttttttt ttaatgacta caagttccag cacaaaactg


 


5641
gcatttcttt gccatttctt gccagtaaga agttgacacg gaggtatttg aaagcaatgt


 


5701
tatgtgagtc attcttaagt gttccaagta agtttagaaa cagaaaagga acttgggatt


 


5761
caaattgatt tttcaaatca tttttaaaga gacatcatcc tgactaaatc ttagcctgaa


 


5821
ccttcctccc ctgtgtgtat tccccggtag tcaccgcagc gagatgctgg tgagactgcc


 


5881
gtggtggcat ttagcatcgt taaaactgga aaactctcaa gctctttgcc actttcctac


 


5941
tattttttga ttcttgccat tttaccaagc ttaggttgtg aaacttgaca gaaatgtatt


 


6001
acaggaaaaa cttataattg tatttgactt tctaacacat tgcaaagttt caaagtgact


 


6061
ttcactttca acaacatatt agaagtaacc acttttgctt tcacagcctg aagagttaga


 


6121
gcctgatctg atgccccctt tcactctgaa gtcatgggaa attttccagc catgaaagcc


 


6181
ctctttccac tgcatactga tgggctgact cagcttcctt cagccgactg agatcttttc


 


6241
atactattgg ctatttcata ccaattaacc tcttaaataa gattgtgaat tgccaaaatt


 


6301
gatagacact tattaccacc tgtggactcc atattcctta ccacaaatgt tattttcatc


 


6361
agtcctgagt cattttaact tacagaaatt aggattgttg ctgctaatat gaataccaat


 


6421
tataactttt agaaacaaga ataaagccta aaagagaatg aaatataaga aatgttcgtt


 


6481
cccaccccta ataacatttg gaagtgaata ttcccatttt cttccaccca cagggattgg


 


6541
gattgatttt taatttccta ggaaacaata ctagactacc caaaaagatg ttgccagaat


 


6601
ccaaaaggaa ctatgctcgt aaaagaaatg cagttttctc ctacctaaaa aaaagaaagt


 


6661
aaagtgtgtt ctgttcttat ctttttaatg actaagcttt aaacagttta ttttgggtaa


 


6721
gactagaact ttcggccatt tgttctaata tgtgtgttat tagatgcaat agaatttatg


 


6781
aaaagaagaa tgacaaaggt atctgattag aaaatttgat cttacgcatg aatccatgtc


 


6841
atggccagcc actgtcacat agtgggtgcc attctcaaca tattggtttg ctaactttaa


 


6901
gcattaggga tttagcacac taaaatactt ttaattatat taggtttggt aactaaggag


 


6961
taaataaatc ataatttatc atttgccaag gccaacaaac aacactattg tgctgtttgc


 


7021
tctcaatgaa gttgaataaa ccaggaggct tggcatatcc cctttatgtt aatcccagct


 


7081
agagattagt aggttgactt tcacagcaat tgtatattga tccattttaa ctcatccttg


 


7141
ccataatttc caggccagtc accaggacag aggagatgat ggggaaacag agctttagat


 


7201
gaaaactact atgcactact agccttagag gcactggttt cctgttacca ctttggcaag


 


7261
tatggatggt ctaagtccag tagggcttca tccatggagc cattagaact gaggggggag


 


7321
tgttagagat gccatttcac caggatcttt ttgctcaggt tgtacccatg ccaattgaag


 


7381
aacgtgttaa agatgaggag gagagatgta ccattctctc ccttaataat gatgttggtt


 


7441
tgcaaaacct aaagaaataa taacaacaga ctatttcata ctttcaagca agtctttata


 


7501
ctacctgtta tttctctaaa attcaaataa agaattttta aacttaaaaa aaaaaaaaaa


 


7561
aaa






The invention also provides for a nucleic acid encoding a XBP1 protein, or fragment thereof. XBP1 is also known as, “X-box binding protein 1” (“XBP1”), “XBP2”, “TREB5” and “XBP-1”. “XBP1” refers to all XBP1 orthologs, including, but not limited to, those found in mice and humans.
For example, the polypeptide sequence of human XBP1 is depicted in SEQ ID NO: 136. The nucleotide sequence of human XBP1 is shown in SEQ ID NO: 137. Sequence information related to XBP 1 isoform XBP 1 (U) is accessible in public databases by GENBANK Accession numbers NP 005071.2 (protein) and NM_005080.3 (nucleic acid).
SEQ ID NO: 136 is the human wild type amino acid sequence corresponding to XBP1 isoform XBP1(U) (residues 1-261):









1
mvvvaaapnp adgtpkvlll sgqpasaaga pagqalplmv paqrgaspea asgglpqark



 


61
rqrlthlspe ekalrrklkn rvaaqtardr kkarmseleq qvvdleeenq klllenqllr


 


121
ekthglvven qelrqrlgmd alvaeeeaea kgnevrpvag saesaalrlr aplqqvqaql


 


181
splqnispwi lavltlqiqs liscwafwtt wtqscssnal pqslpawrss qrstqkdpvp


 


241
yqppflcqwg rhqpswkplm n






SEQ ID NO: 137 is the human wild type nucleotide sequence corresponding to XBP1 isoform XBP1(U) (nucleotides 1-1820):









1
ggcgctgggc ggctgcggcg cgcggtgcgc ggtgcgtagt ctggagctat ggtggtggtg



 


61
gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc


 


121
gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga


 


181
ggggccagcc cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc


 


241
acgcacctga gccccgagga gaaggcgctg aggaggaaac tgaaaaacag agtagcagct


 


301
cagactgcca gagatcgaaa gaaggctcga atgagtgagc tggaacagca agtggtagat


 


361
ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat


 


421
ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct


 


481
gaagaggagg cggaagccaa ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc


 


541
gcagcactca gactacgtgc acctctgcag caggtgcagg cccagttgtc acccctccag


 


601
aacatctccc catggattct ggcggtattg actcttcaga ttcagagtct gatatcctgt


 


661
tgggcattct ggacaacttg gacccagtca tgttcttcaa atgcccttcc ccagagcctg


 


721
ccagcctgga ggagctccca gaggtctacc cagaaggacc cagttcctta ccagcctccc


 


781
tttctctgtc agtggggacg tcatcagcca agctggaagc cattaatgaa ctaattcgtt


 


841
ttgaccacat atataccaag cccctagtct tagagatacc ctctgagaca gagagccaag


 


901
ctaatgtggt agtgaaaatc gaggaagcac ctctcagccc ctcagagaat gatcaccctg


 


961 
aattcattgt ctcagtgaag gaagaacctg tagaagatga cctcgttccg gagctgggta



 


1021
tctcaaatct gctttcatcc agccactgcc caaagccatc ttcctgccta ctggatgctt



 


1081
acagtgactg tggatacggg ggttcccttt ccccattcag tgacatgtcc tctctgcttg


 


1141
gtgtaaacca ttcttgggag gacacttttg ccaatgaact ctttccccag ctgattagtg


 


1201
tctaaggaat gatccaatac tgttgccctt ttccttgact attacactgc ctggaggata


 


1261
gcagagaagc ctgtctgtac ttcattcaaa aagccaaaat agagagtata cagtcctaga


 


1321
gaattcctct atttgttcag atctcataga tgacccccag gtattgtctt ttgacatcca


 


1381
gcagtccaag gtattgagac atattactgg aagtaagaaa tattactata attgagaact


 


1441
acagctttta agattgtact tttatcttaa aagggtggta gttttcccta aaatacttat


 


1501
tatgtaaggg tcattagaca aatgtcttga agtagacatg gaatttatga atggttcttt


 


1561
atcatttctc ttcccccttt ttggcatcct ggcttgcctc cagttttagg tcctttagtt


 


1621
tgcttctgta agcaacggga acacctgctg agggggctct ttccctcatg tatacttcaa


 


1681
gtaagatcaa gaatcttttg tgaaattata gaaatttact atgtaaatgc ttgatggaat


 


1741
tttttcctgc tagtgtagct tctgaaaggt gctttctcca tttatttaaa actacccatg


 


1801
caattaaaag gtacaatgca






Sequence information related to XBP1 isoform XBP1(S) (also known as “spliced XBP 1” and “sXBP 1”) is accessible in public databases by GENBANK Accession numbers NP 001073007.1 (protein) and NM_001079539.1 (nucleic acid).
SEQ ID NO: 138 is the human wild type amino acid sequence corresponding to XBP1 isoform XBP1(S) (residues 1-376):









1
mvvvaaapnp adgtpkvlll sgqpasaaga pagqalplmv paqrgaspea asgglpqark



 


61
rqrlthlspe ekalrrklkn rvaaqtardr kkarmseleq qvvdleeenq klllenqllr


 


121
ekthglvven qelrqrlgmd alvaeeeaea kgnevrpvag saesaagagp vvtppehlpm


 


181
dsggidssds esdillgild nldpvmffkc pspepaslee lpevypegps slpaslslsv


 


241
gtssakleai nelirfdhiy tkplvleips etesqanvvv kieeaplsps endhpefivs


 


301
vkeepveddl vpelgisnll ssshcpkpss clldaysdcg yggslspfsd mssllgvnhs


 


361
wedtfanelf pqlisv






SEQ ID NO: 139 is the human wild type nucleotide sequence corresponding to XBP1 isoform XBP1(S) (nucleotides 1-1810):









1
ggcgctgggc ggctgcggcg cgcggtgcgc ggtgcgtagt ctggagctat ggtggtggtg



 


61
gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc


 


121
gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga


 


181
ggggccagcc cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc


 


241
acgcacctga gccccgagga gaaggcgctg aggaggaaac tgaaaaacag agtagcagct


 


301
cagactgcca gagatcgaaa gaaggctcga atgagtgagc tggaacagca agtggtagat


 


361
ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat


 


421
ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct


 


481
gaagaggagg cggaagccaa ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc


 


541
gcagcaggtg caggcccagt tgtcacccct ccagaacatc tccccatgga ttctggcggt


 


601
attgactctt cagattcaga gtctgatatc ctgttgggca ttctggacaa cttggaccca


 


661
gtcatgttct tcaaatgccc ttccccagag cctgccagcc tggaggagct cccagaggtc


 


721
tacccagaag gacccagttc cttaccagcc tccctttctc tgtcagtggg gacgtcatca


 


781
gccaagctgg aagccattaa tgaactaatt cgttttgacc acatatatac caagccccta


 


841
gtcttagaga taccctctga gacagagagc caagctaatg tggtagtgaa aatcgaggaa


 


901
gcacctctca gcccctcaga gaatgatcac cctgaattca ttgtctcagt gaaggaagaa


 


961
cctgtagaag atgacctcgt tccggagctg ggtatctcaa atctgctttc atccagccac


 


1021
tgcccaaagc catcttcctg cctactggat gcttacagtg actgtggata cgggggttcc


 


1081
ctttccccat tcagtgacat gtcctctctg cttggtgtaa accattcttg ggaggacact


 


1141
tttgccaatg aactctttcc ccagctgatt agtgtctaag gaatgatcca atactgttgc


 


1201
ccttttcctt gactattaca ctgcctggag gatagcagag aagcctgtct gtacttcatt


 


1261
caaaaagcca aaatagagag tatacagtcc tagagaattc ctctatttgt tcagatctca


 


1321
tagatgaccc ccaggtattg tcttttgaca tccagcagtc caaggtattg agacatatta


 


1381
ctggaagtaa gaaatattac tataattgag aactacagct tttaagattg tacttttatc


 


1441
ttaaaagggt ggtagttttc cctaaaatac ttattatgta agggtcatta gacaaatgtc


 


1501
ttgaagtaga catggaattt atgaatggtt ctttatcatt tctcttcccc ctttttggca


 


1561
tcctggcttg cctccagttt taggtccttt agtttgcttc tgtaagcaac gggaacacct


 


1621
gctgaggggg ctctttccct catgtatact tcaagtaaga tcaagaatct tttgtgaaat


 


1681
tatagaaatt tactatgtaa atgcttgatg gaattttttc ctgctagtgt agcttctgaa


 


1741
aggtgctttc tccatttatt taaaactacc catgcaatta aaaggtacaa tgcaaaaaaa


 


1801
aaaaaaaaaa






A ILDR2, PERK, IRE1a, ATF6 or XBP1 molecule can also encompass ortholog genes, which are genes conserved among different biological species such as humans, dogs, cats, mice, and rats, that encode proteins (for example, homologs (including splice variants), mutants, and derivatives) having biologically equivalent functions as the human-derived protein. Orthologs of a ILDR2, PERK, IRE1a, ATF6 or XBP1 protein include any mammalian ortholog inclusive of the ortholog in humans and other primates, experimental mammals (such as mice, rats, hamsters and guinea pigs), mammals of commercial significance (such as horses, cows, camels, pigs and sheep), and also companion mammals (such as domestic animals, e.g., rabbits, ferrets, dogs, and cats). A ILDR2, PERK, IRE1a, ATF6 or XBP1 molecule can comprise a protein encoded by a nucleic acid sequence homologous to the human nucleic acid, wherein the nucleic acid is found in a different species and wherein that homolog encodes a protein similar to a ILDR2, PERK, IRE1a, ATF6 or XBP1 protein.
The invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (2001).
One skilled in the art can obtain ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.
The invention provides for ILDR2, PERK, IRE1a, ATF6 and XBP1 molecules that are encoded by nucleotide sequences. The ILDR2, PERK, IRE1a, ATF6 and XBP1 molecules can be a polypeptide encoded by a nucleic acid (including genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, a ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule can be encoded by a recombinant nucleic acid encoding a human ILDR2, PERK, IRE1a, ATF6 and XBP1 protein, or fragment thereof. The ILDR2, PERK, IRE1a, ATF6 and XBP1 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. The ILDR2, PERK, IRE1a, ATF6 and XBP1 molecules of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. A ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule of this invention can also encompasses variants of the human ILDR2, PERK, IRE1a, ATF6 and XBP1 proteins. The variants can comprise naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms.
In one embodiment, a fragment of a nucleic acid sequence that comprises a ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule can encompass any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 129, 131, 133, 135, 137, or 139. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of SEQ ID NO: 129, 131, 133, 135, 137, or 139. Fragments include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides.
A ILDR2, PERK, IRE1a, ATF6 and XBP1 molecule, can be a fragment of a ILDR2, PERK, IRE1a, ATF6 and XBP1 protein. For example, the ILDR2, PERK, IRE1a, ATF6 and XBP1 protein fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 22, 130, 132, 134, 136, or 138. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 80 consecutive amino acids, at least about 90 consecutive amino acids, at least about 100 consecutive amino acids, at least about 110 consecutive amino acids, or at least about 120 consecutive amino acids of SEQ ID NOS: 22, 130, 132, 134, 136, or 138. Fragments include all possible amino acid lengths between about 8 and 80 about amino acids, for example, lengths between about 10 and about 80 amino acids, between about 15 and about 80 amino acids, between about 20 and about 80 amino acids, between about 35 and about 80 amino acids, between about 40 and about 80 amino acids, between about 50 and about 80 amino acids, or between about 70 and about 80 amino acids.
Metabolic Disease
In one embodiment, the invention provides a method of treating a metabolic disease. In one embodiment, the metabolic disease is a fatty liver disease. In another embodiment, the metabolic disease is dyslipidemia. In another embodiment, the metabolic disease is metabolic syndrome. In another embodiment, the metabolic disease is a cardiovascular disease. In another embodiment, the metabolic disease is obesity. In another embodiment, the metabolic disease is a leptin disorder.
Metabolic syndrome is a name for a group of risk factors the occur together and increase the risk for coronary artery disease, stroke and type II diabetes. Risk factors for metabolic syndrome include, but are not limited to, extra weight around the midsection and upper parts of the body (“central obesity”), insulin resistance, aging, hormone changes, a genetic predisposition, lack of exercise, excess blood clotting, increased levels of inflammatory markers. A subject can be diagnosed with metabolic syndrome if they are diagnosed with three or more of the following conditions, including, but not limited to, blood pressure equal or higher than 130/85 mm/Hg, fasting blood sugar (glucose) equal to or higher than 100 mg/dL, large waist circumference of 40 inches or more for men and 35 inches or more for women, low HDL cholesterol of under 40 mg/dL for men and under 50 mg/dL for women, and triglycerides equal to or higher than 150 mg/dL.
Fatty liver disease involves large vacuoles of fat accumulation in liver cells. Fatty liver disease can be diagnosed by a variety of tests, including but not limited to liver function tests, liver biochemistry tests, serum alanine transaminase levels, serum aspartate transaminase levels, imaging studies, and histology.
In one embodiment, the fatty liver disease is hepatic steatosis. In another embodiment, the fatty liver disease is non-alcoholic steatohepatitis. In another embodiment, the fatty liver disease is non-alcoholic fatty liver disease. In another embodiment, the fatty liver disease is elevated liver cholesterol levels. In another embodiment, the fatty liver disease is elevated liver triglyceride levels. In another embodiment, the fatty liver disease is elevated liver fatty acid levels. In another embodiment, the fatty liver disease is elevated liver LDL-cholesterol levels. In another embodiment, the fatty liver disease is elevated liver VLDL cholesterol levels. In another embodiment, the fatty liver disease is elevated liver non-HDL cholesterol levels.
Dyslipidemia involves an abnormal amount of lipids in the blood. In one embodiment, the dyslipidemia is hyperlipidemia. In another embodiment, the dyslipidemia is mixed dyslipidemia. In another embodiment, the dyslipidemia is hypercholesterolemia. In another embodiment, the dyslipidemia is polygenic hypercholesterolemia. In another embodiment, the dyslipidemia is hypertriglyceridemia. In another embodiment, the dyslipidemia is hyperfattyacidemia. In another embodiment, the dyslipidemia is elevated ApoB. In another embodiment, the dyslipidemia is elevated cholesterol. In another embodiment, the dyslipidemia is elevated LDL-cholesterol. In another embodiment, the dyslipidemia is elevated VLDL-cholesterol. In another embodiment, the dyslipidemia is elevated non-HDL cholesterol.
In one embodiment, the cardiovascular disease is coronary heart disease. In another embodiment, the cardiovascular disease is acute coronary syndrome. In another embodiment, the cardiovascular disease is early onset coronary heart disease. In another embodiment, the cardiovascular disease is atherosclerosis.
In one embodiment, the leptin disorder is hyperleptinemia. In another embodiment, the leptin disorder is tissue leptin resistance.
In one embodiment, the treatment improves cardiovascular outcome. In another embodiment, the treatment results in slowed progression and/or amelioration of the metabolic disease. In one embodiment, in addition to a metabolic disease, the subject has type II diabetes or insulin resistance.
In one aspect the invention provides a method of decreasing lipid levels in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the lipid level is a cholesterol level. In another embodiment, the lipid level is a triglyceride level. In another embodiment, the lipid level is a ApoB level. In another embodiment, the lipid level is a LDL-cholesterol level. In another embodiment, the lipid level is a VLDL-cholesterol level. In another embodiment, the lipid level is a small LDL-particle level. In another embodiment, the lipid level is a small VLDL-particle level. In another embodiment, the lipid level is a non-HDL-cholesterol level. In another embodiment, the lipid level is a phospholipid level. In another embodiment, the lipid level is a or fatty acid level.
In one embodiment, the lipid level is the concentration in blood plasma. In another embodiment, the lipid level is the concentration in liver. In one embodiment the lipid level is decreased relative to a lipid level in the subject before administrating the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. Methods of measuring lipid levels in the blood and liver are known to one of skill in the art.
In one embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in liver tissue. In another embodiment, the expression of Ildr2 mRNA or ILDR2 protein is increased in hepatocytes.
In one aspect, the invention provides a method of increasing expression of Ildr2 mRNA or ILDR2 protein in a hepatocyte, the method comprising contacting the cell with an agent which increases expression of the Ildr2 mRNA or ILDR2 protein.
In one aspect, the invention provides an agent which increases expression of the Ildr2 mRNA or ILDR2 protein. In one embodiment, the agent is a nucleic acid which comprises a nucleic acid sequence encoding a ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or a ILDR2 functional fragment. In another embodiment, the agent is an ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or a ILDR2 functional fragment. In another embodiment, the agent is a peptide having SEQ ID NO: 2-9.
In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In another embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or any fragment thereof
In one embodiment, the subject has a reduced level of expression of Ildr2 mRNA or ILDR2 protein compared to the level of expression of Ildr2 mRNA or ILDR2 protein in a subject without a metabolic disease. In another embodiment, the level of expression is determined before administrating to the subject the therapeutically effective amount of an agent which increases expression of Ildr2 mRNA or ILDR2 protein. In one embodiment, the expression of ILDR2 protein in a subject is measured using an antibody. In another embodiment, the antibody specifically binds to a peptide having SEQ ID NOs: 2-9, or an ILDR2 protein, a ILDR2 polypeptide, a ILDR2 isoform, or any fragment thereof. Other methods of measuring the level of expression of Ildr2 mRNA or ILDR2 protein in a subject are known to one of skill in the art.
The invention provides methods to modify and measure expression of Ildr2 mRNA or ILDR2 protein in a subject. In one embodiment, the subject is a human or a non-human animal. Non-limiting examples of non-human animals include primates (such as monkeys), rodents, (such as mice, rats and rabbits), ovine species (such as sheep and goats), bovine species (such as cows), porcine species, equine species, feline species and canine species. In a particular embodiment, the subject is a human. The method can comprise detecting in a sample from the subject the expression of Ildr2 mRNA or ILDR2 protein. The expression of Ildr2 mRNA or ILDR2 protein in the sample can be measured through amplification, gene expression analysis, or a combination thereof.
In another embodiment, the method can comprise detecting the expression of Ildr2 mRNA or ILDR2 protein. RNA expression includes the presence of an RNA sequence, the presence of an RNA splicing or processing, or the presence of a quantity of RNA. These can be detected by various techniques known in the art, including by sequencing all or part of the Ildr2 RNA, or by selective hybridization or selective amplification of all or part of the RNA. In a further embodiment, the method can comprise detecting the presence of a Ildr2 polypeptide expression. Polypeptide expression includes the presence of a Ildr2 polypeptide sequence, or the presence of an decreased quantity Ildr2 polypeptide as compared to a sample from a subject without a metabolic disease. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies).
Various techniques known in the art can be used to detect or quantify DNA expression, RNA expression, or nucleic acid sequences, which include, but are not limited to, hybridization, sequencing, amplification, and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), oligonucleotide ligation, allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, denaturing HLPC, melting curve analysis, heteroduplex analysis, RNase protection, chemical or enzymatic mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Some other approaches are based on specific hybridization between nucleic acids from the subject and a probe specific for wild type gene or RNA. The probe can be in suspension or immobilized on a substrate. The probe can be labeled to facilitate detection of hybrids. Some of these approaches are suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand-specific for the polypeptide, for example, the use of a specific antibody.
Sequencing.
Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on the complete gene or on specific domains thereof, such as those known or suspected to carry deleterious mutations or other alterations.
Amplification.
Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. For example, nucleic acid primers useful for amplifying sequences from the gene or locus of Ildr2 are able to specifically hybridize with a portion of the gene locus that flanks a target region of the locus, wherein the target region is present in subjects having or are at risk of developing prostate cancer.
The invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a coding sequence (e.g., gene or RNA) of Ildr2 that is present in subjects having or at risk of developing prostate cancer. Primers of the invention are specific for sequences in a gene or RNA of Ildr2. By using such primers, the detection of an amplification product indicates the presence of the Ildr2 gene or the absence of such. Examples of primers of this invention can be single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, or about 8 to about 25 nucleotides in length. The sequence can be derived directly from the sequence of Ildr2. Perfect complementarity is useful, to ensure high specificity. However, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as described herein can be used in a method for detecting the presence of or a predisposition to prostate cancer in a subject.
Amplification methods include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560, 1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA 86:1173, 1989); and, self-sustained sequence replication (see, e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87:1874, 1990); Q Beta replicase amplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491, 1997), automated Q-beta replicase amplification assay (see, e.g., Burg, Mol. Cell. Probes 10:257-271, 1996) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger, Methods Enzymol. 152:307-316, 1987; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13:563-564, 1995. All the references stated above are incorporated by reference in their entireties.
Hybridization.
Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequences. A detection technique involves the use of a nucleic acid probe specific for wild type gene or RNA. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). For example, a sample from the subject can be contacted with a nucleic acid probe specific for wild type Ildr2. According to the invention, a probe can be a polynucleotide sequence which is complementary to and specifically hybridizes with a, or a target portion of a, Ildr2 gene or RNA. Useful probes are those that are complementary to the Ildr2 gene, RNA, or target portion thereof. Probes can comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance between 10 and 800, between 15 and 700, or between 20 and 500. Longer probes can be used as well. A useful probe of the invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a gene or RNA.
The sequence of the probes can be derived from the sequences of Ildr2 genes. Nucleotide substitutions can be performed, as well as chemical modifications of the probe. Such chemical modifications can be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Some examples of labels include, without limitation, radioactivity, fluorescence, luminescence, and enzymatic labeling.
A guide to nucleic acid hybridization is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 2001; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997; LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, PART I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.
Specific Ligand Binding.
As indicated herein, the presence of a Ildr2 gene locus or Ildr2 expression can also be detected. Different types of ligands can be used, such as specific antibodies. In one embodiment, the sample is contacted with an antibody specific for a Ildr2 and the formation of an immune complex is subsequently determined. Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays (IEMA).
These methods utilize a sample from a subject in order to assess the status of the Ildr2 gene locus. The sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, or tissue biopsies. The sample can be collected according to conventional techniques and used directly for diagnosis or stored. The sample can be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation. Also, the nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. In one embodiment, the sample is contacted with reagents, such as probes, primers, or ligands, in order to assess the presence of Ildr2. Contacting can be performed in any suitable device, such as a plate, tube, well, or glass. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers. The substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel. The contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.
Identifying a polypeptide, RNA or DNA of Ildr2 in the sample can be correlated to the presence, predisposition or stage of progression of metabolic disease. For example, an individual expressing reduced levels of Ildr2 may have an increased risk of developing a metabolic disease. The determination of the expression of Ildr2 in a subject also allows the design of appropriate therapeutic intervention, which is more effective and customized. Also, this determination at the pre-symptomatic level allows a preventive regimen to be applied.
The standard dose(s) of an agent which increases expression of Ildr2 mRNA or ILDR2 protein to be administered according to the methods described herein can vary, for example, depending upon the identity, size, and condition of the subject being treated and can further depend upon the route by which an agent according to the methods described herein, is to be administered, if applicable, and the effect which the practitioner desires the agent according to the invention to have upon the target of interest. These amounts can be readily determined by one of skill in the art. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a human.
Appropriate dosing regimens can also be determined by one of skill in the art without undue experimentation, in order to determine, for example, whether to administer the agent in one single dose or in multiple doses, and in the case of multiple doses, to determine an effective interval between doses.
In certain embodiments, an agent which increases expression of Ildr2 mRNA or ILDR2 protein administered according to the methods described herein can be administered alone, or in combination with other drugs, therapies, small molecules, biologically active or inert compounds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the agent. In one embodiment, the agent is administered with a lipid lowering therapy. In another embodiment the lipid lowring therapy is a therapeutic lifestyle change, a HMG-CoA reductase inhibitor, niacin, a fibrate, a cholesterol absorption inhibitor, a MTP inhibitor, or any combination thereof.
Therapy dose and duration will depend on a variety of factors, such as the disease type, patient age, therapeutic index of the drugs, patient weight, and tolerance of toxicity. The skilled clinician using standard pharmacological approaches can determine the dose of a particular therapeutic and duration of therapy for a particular patient in view of the above stated factors.
Methods of Administering
The invention provides methods for treating a metabolic disease in a subject. In one embodiment, the method can comprise administering to the subject an agent which increases expression of Ildr2 mRNA or ILDR2 protein, including, but not limited to an ILDR2 molecule (e.g, an ILDR2 polypeptide or an ILDR2 polynucleotide).
Various approaches can be carried out to increasing an ILDR2 gene expression level or activity and can be accomplished through gene or protein therapy
A eukaryotic expression vector can be introduced into cells in order to increase the expression or produce proteins (for example, ILDR2) encoded by nucleotide sequences of the vector. Cells can harbor an expression vector (for example, one that contains a gene encoding ILDR2) via introducing the expression vector into an appropriate host cell via methods known in the art. A nucleic acid encoding an ILDR2 molecule can be introduced into the cells of a subject. For example, the wild-type gene (or fragment thereof) can also be introduced into the cells of the subject in need thereof using a vector as described herein. The vector can be a viral vector or a plasmid. The gene can also be introduced as naked DNA. The gene can be provided so as to integrate into the genome of the recipient host cells, or to remain extra-chromosomal. Integration can occur randomly or at precisely defined sites, such as through homologous recombination. For example, a functional copy of an ILDR2 molecule can be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, or cationic lipid-mediated transfection. Gene therapy can be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional polypeptide.
A eukaryotic expression vector can be introduced into cells in order to decrease the expression of proteins (for example, ILDR2, PERK, IRE1a, ATF6, or XBP1). Cells can harbor an expression vector encoding an interfering RNA molecule (for example, one that encode a RNAi to ILDR2, PERK, IRE1a, ATF6, or XBP1) via introducing the expression vector into an appropriate host cell via methods known in the art.
Inhibition of RNA encoding a PERK, IRE1a, ATF6, or XBP1 molecule can effectively modulate the expression of the PERK, IRE1a, ATF6, or XBP1 gene from which the RNA is transcribed. Without being bound by theory, inhibition of RNA encoding a PERK, IRE1a, ATF6, or XBP1 molecule can effectively modulate the expression of ILDR2. An agent which increases expression of Ildr2 mRNA or ILDR2 protein can be selected from the group comprising: siRNA, interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; shRNAs; ribozymes; and antisense nucleic acid, which can be RNA, DNA, or artificial nucleic acid.
Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a PERK, IRE1a, ATF6, or XBP1 polypeptide can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al., (2006) Med. Sci. Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59; each herein incorporated by reference in its entirety).
siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. Antisense nucleotide sequences include, but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like. RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. The PERK, IRE1a, ATF6, or XBP1 modulating compound can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded. See for example Bass (2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914; each of which are herein incorporated by reference in its entirety.
siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector (for example, see U.S. Pat. No. 7,294,504; U.S. Pat. No. 7,148,342; and U.S. Pat. No. 7,422,896; the entire disclosures of which are herein incorporated by reference). Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. Patent Application Publication No. 2002/0173478 to Gewirtz, and in U.S. Patent Application Publication No. 2007/0072204 to Hannon et al., the entire disclosures of which are herein incorporated by reference.
An agent which increases expression of Ildr2 mRNA or ILDR2 protein can additionally be a short hairpin RNA (shRNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al., 2002, Genes Dev, 16:948-58; McCaffrey et al., 2002, Nature, 418:38-9; McManus et al., 2002, RNA, 8:842-50; Yu et al., 2002, Proc Natl Acad Sci USA, 99:6047-52; each herein incorporated by reference in its entirety. Such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
When a nucleic acid such as RNA or DNA is used that encodes a protein or peptide of the invention, it can be delivered into a cell in any of a variety of forms, including as naked plasmid or other DNA, formulated in liposomes, in an expression vector, which includes a viral vector (including RNA viruses and DNA viruses, including adenovirus, lentivirus, alphavirus, and adeno-associated virus), by biocompatible gels, via a pressure injection apparatus such as the PowderjectTM system using RNA or DNA, or by any other convenient means. Again, the amount of nucleic acid needed to sequester an Id protein in the cytoplasm can be readily determined by those of skill in the art, which also can vary with the delivery formulation and mode and whether the nucleic acid is DNA or RNA. For example, see Manjunath et al., (2009) Adv Drug Deliv Rev. 61(9):732-45; Singer and Verma, (2008) Curr Gene Ther. 8(6):483-8; and Lundberg et al., (2008) Curr Gene Ther. 8(6):461-73; each herein incorporated by reference in its entirety.
An agent which increases expression of Ildr2 mRNA or ILDR2 protein can also be a small molecule that binds to ILDR2, PERK, IRE1a, ATF6, or XBP and disrupts its function, or conversely, enhances its function. Suppression of ILDR2 could also be reduced by ER stress inhibitors, including, but not limited to TUDCA and 4PBA. Small molecules are a diverse group of synthetic and natural substances having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that modulate ILDR2, PERK, IRE1a, ATF6, or XBP can be identified via in silico screening or high-throughput (HTP) screening of combinatorial libraries.
An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art. For example, a retrovirus can be used to introduce a nucleotide sequence into cells (such as hepatocytes). In one embodiment, the retrovirus is an adenovirus. Other viral vectors known in the art can be used to introduce a nucleotide sequence, including, but not limited to a lentivirus, or an adeno-associated virus.
In one embodiment, a retrovirus can be used to introduce a nucleotide sequence into hepatocytes, in order to produce proteins encoded by said nucleotide sequences or to introduce RNAi to reduce protein levels (for example, ILDR2). A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences or to introduct RNAi to reduce protein levels (for example, ILDR2). Mammalian cells (such as hepatocytes) can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.
An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Other methods used to transfect cells can also include calcium phosphate precipitation, modified calcium phosphate precipitation, polybrene precipitation, microinjection liposome fusion, and receptor-mediated gene delivery.
A nucleic acid encoding a gene of interest or a functional part thereof can be introduced into the cells of a subject. For example, the wild-type gene (or a functional part thereof) can also be introduced into the cells of the subject in need thereof using a vector as described herein. The vector can be a viral vector or a plasmid. The gene can also be introduced as naked DNA. The gene can be provided so as to integrate into the genome of the recipient host cells, or to remain extra-chromosomal. Integration can occur randomly or at precisely defined sites, such as through homologous recombination. For example, a functional copy of the gene can be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, or cationic lipid-mediated transfection. Gene therapy can be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional polypeptide.
Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, and more specifically viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (see, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells can also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Biandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Non-limiting examples of in vivo gene transfer techniques include transfection with viral (e.g., retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
For reviews of gene therapy protocols and methods see Anderson et al., Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. Application Publication Nos. 2002/0077313 and 2002/00069, which are all hereby incorporated by reference in their entireties. For additional reviews of gene therapy technology, see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15; Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005; 99:193-260, all of which are hereby incorporated by reference in their entireties.
Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion. A replacement polypeptide can be synthesized according to known chemical techniques or can be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders. For example, a wild-type protein can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in the art. After the infusion, the exogenous protein can be taken up by tissues through non-specific or receptor-mediated mechanism.
Indications, dosage and methods of administration of the drugs of the present invention are known to one of skill in the art. In some embodiments, a drug of the present invention can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. Choice of the excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a drug of the present invention can also comprise, or be accompanied with, one or more other ingredients that facilitate the delivery or functional mobilization of the drugs of the present invention.
These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.
According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.
Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20th ed., 2000), the entire disclosure of which is herein incorporated by reference.
Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
Administration of a drug of the present invention is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.
The compositions of this invention can be formulated and administered to reduce the symptoms associated with a metabolic disease by any means that produce contact of the active ingredient with the agent's site of action in the body of a human or non-human subject. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20th ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers, such as PBS, Hank's solution, or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the agent which increases expression of Ildr2 mRNA or ILDR2 protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art
A composition of the invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the invention (e.g., that have a therapeutic effect) can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel-caps, caplets, or powders, that are adapted for sustained release are encompassed by the invention.
The dosage administered can be a therapeutically effective amount of the composition sufficient to result in treatment of a metabolic disease and can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.
In some embodiments, the effective amount of the administered an agent which increases expression of Ildr2 mRNA or ILDR2 protein is at least about 0.01 μg/kg body weight, at least about 0.025 μg/kg body weight, at least about 0.05 μg/kg body weight, at least about 0.075 μg/kg body weight, at least about 0.1 μg/kg body weight, at least about 0.25 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 0.75 μg/kg body weight, at least about 1 μg/kg body weight, at least about 5 μg/kg body weight, at least about 10 μg/kg body weight, at least about 25 μg/kg body weight, at least about 50 μg/kg body weight, at least about 75 μg/kg body weight, at least about 100 μg/kg body weight, at least about 150 μg/kg body weight, at least about 200 μg/kg body weight, at least about 250 μg/kg body weight, at least about 300 μg/kg body weight, at least about 350 μg/kg body weight, at least about 400 μg/kg body weight, at least about 450 μg/kg body weight, at least about 500 μg/kg body weight, at least about 550 μg/kg body weight, at least about 600 μg/kg body weight, at least about 650 μg/kg body weight, at least about 700 μg/kg body weight, at least about 750 μg/kg body weight, at least about 800 μg/kg body weight, at least about 850 μg/kg body weight, at least about 900 μg/kg body weight, at least about 950 μg/kg body weight, at least about 1000 μg/kg body weight, at least about 1500 μg/kg body weight, at least about 2000 μg/kg body weight, at least about 2500 μg/kg body weight, at least about 3000 μg/kg body weight, at least about 3500 μg/kg body weight, at least about 4000 μg/kg body weight, at least about 4500 μg/kg body weight, at least about 5000 μg/kg body weight, at least about 5500 μg/kg body weight, at least about 6000 μg/kg body weight, at least about 6500 μg/kg body weight, at least about 7000 μg/kg body weight, at least about 7500 μg/kg body weight, at least about 8000 μg/kg body weight, at least about 8500 μg/kg body weight, at least about 9000 μg/kg body weight, at least about 9500 μg/kg body weight, or at least about 10000 μg/kg body weight.
In one embodiment, an agent which increases expression of Ildr2 mRNA or ILDR2 protein is administered at least once daily. In another embodiment, an agent which increases expression of Ildr2 mRNA or ILDR2 protein is administered at least twice daily. In some embodiments, an agent which increases expression of Ildr2 mRNA or ILDR2 protein is administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 5 weeks, for at least 6 weeks, for at least 8 weeks, for at least 10 weeks, for at least 12 weeks, for at least 18 weeks, for at least 24 weeks, for at least 36 weeks, for at least 48 weeks, or for at least 60 weeks. In further embodiments, an agent which increases expression of Ildr2 mRNA or ILDR2 protein is administered in combination with a second therapeutic agent.
Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.
Administration of an agent which increases expression of Ildr2 mRNA or ILDR2 protein is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.
Methods of Detection
Embodiments of the invention provide for detecting expression of an ILDR2. In one embodiment, increased or reduced protein expression and/or activity can be detected. The detection can be performed at the level of the DNA, RNA, or polypeptide.
In some embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding an ILDR2 protein, or a reduction in an ILDR2 protein, or a combination thereof. In further embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding an ILDR2 protein, or a reduction in an ILDR2 protein, or a combination thereof.
Methods for detecting and quantifying ILDR2 molecules in biological samples are known the art. For example, protocols for detecting and measuring the expression of a polypeptide encoded by an ILDR2 molecule using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
In one embodiment, a biological sample comprises, a blood sample, serum, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including tissues obtained by biopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovial fluid), or from media (from cultured cells or cell lines). The methods of detecting or quantifying an ILDR2 molecule include, but are not limited to, amplification-based assays with (signal amplification) hybridization based assays and combination amplification-hybridization assays. For detecting and quantifying an ILDR2 molecule, an exemplary method is an immunoassay that utilizes an antibody or other binding agents that specifically bind to an ILDR2 protein or epitope of such, for example, Western blot or ELISA assays.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.
All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.
The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1
ILDR2: An Endoplasmic Reticulum Resident Molecule Mediating Hepatic Lipid Homeostasis
Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. Adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, were used to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting “relief” of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, the possible participation of ILDR2 in ER stress responses was investigated. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. Manipulation of lldr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time-course over which these studies were conducted, causal primacy cannot be assigned to either the effects on hepatic lipid homeostasis or ER stress responses.
In an earlier study [1] the differential diabetes susceptibilities of mouse strains C57BL/6J (B6) and DBA/2J (DBA) [2] segregating for the obesity mutation, Lepob, were used to identify a gene that encodes a predicted single-pass, trans-membrane molecule that, in B6. DBA congenic mice (segregating a DBA haplotype in a 1.8 Mb interval on Chr1), was associated with reduced b-cell replication rates accompanied by reduced b-cell mass, and persistent mild hypoinsulinemic hyperglycemia. This gene, formerly designated “Lisch-like”, has been renamed “immunoglobulin-like domain containing receptor 20” (Ildr2) [www.informatics.jax.org/mgihome/nomen/] to reflect the similarity of the conserved domain structure of the cognate protein to the two other members of this gene family: Ildr1 and Ildr3 (aka “LSR”—lipolysis stimulated receptor).
Despite their structural similarities, the three Ildr-genes exhibit widely divergent tissue-specific expression profiles, providing little evidence of significant overlap among their functions. The major isoforms of both ILDR1 and ILDR3 localize either to the plasma membrane (PM) or to the cytosol[3,4]. Although ILDR1 has been linked to neoplastic disease 2 [5] and non-syndromic deafness [6], how it functions is unknown. ILDR3, which was initially identified as a fatty acid-activated, liver-specific lipoprotein receptor [7], has since been characterized variously as a receptor for Clostridium toxin[8], as an hepatic receptor upregulated by leptin [9] and as a component of tri-cellular junctions in epithelial cells [10].
The Ildr2 gene is widely expressed, with 4 major isoforms that are differentially expressed in tissues relevant to the diabetic phenotype (hypothalamus, liver and islet b-cells). Expression levels of isoform 4, highest in liver, are reduced 20-fold in B6.DBA congenic animals and 30-fold in 10-week-old DBA mice versus B6 animals [1]. To assess the role of Ildr2 in the molecular physiology of normal, adult liver, adenoviruses containing overexpression or knockdown constructs was intravenously administered to study in vivo effects in liver and whole animal, and in transduced primary hepatocytes to study in vitro effects.
Described herein are results that ILDR2, in contrast to ILDR1 and ILDR3, is exclusively localized in the endoplasmic reticulum (ER), where it apparently participates in both lipoprotein physiology and the ER stress response, with consequences for hepatic lipid homeostasis.
Results
ILDR2 is Localized to the Endoplasmic Reticulum:
As previously described [1], the four major isoforms of ILDR2 (FIG. 1) contain an amino terminal immunoglobulin-like domain and long, carboxy tail. Isoforms 1, 2, and 4 also contain a single trans-membrane (TM) domain. Isoform 1 is full-length; isoform 2 lacks exon 6 (carboxy to the TM domain); isoform 4 lacks exon 4 (amino to the TM domain); isoform 3 lacks the TM domain and both flanking exons.
To determine the cellular location(s) of ILDR2, various isoforms were tagged at the C-termini with the green variant of the monomeric yellow fluorescent protein (mYFP), transiently transduced into mouse cells, and analyzed by confocal microscopy for co-localization with probes for the ER and PM (FIG. 2). Ildr2-isoform 2, predominantly expressed in the hypothalamus, was transduced into cells of the mouse hypothalamic neuronal cell line GT1-7 (FIG. 2A). Ildr2-isoform 4, the predominant isoform endogenously expressed in the liver, was transduced into cells of the mouse hepatoma cell line, Hepa1c1c7 (FIG. 2B). Both isoforms localized solely to the ER membrane, with no detectable fluorescence in the vicinity of the PM. Placement of the tagging peptide did not affect subcellular destination, since localization to the ER membrane was seen also in Hepa1c1c7 cells transduced with Ildr2-isoform 1 tagged at its N-terminus with the FLAG epitope (FIG. 2C). These results support the model depicted in FIG. 1, in which the hydrophobic, amino-terminal, immuno-globulin-like domain of isoforms 1, 2 and 4, extends into the ER lumen, and the hydrophilic carboxy-terminal tail, extends into the cytoplasm. No changes were observed in the subcellular distribution of C-terminal tagged isoform 4 in Hepa1c1c7 transfectants that were exposed to glucose, insulin, free fatty acids (FFA), and low-density lipoprotein (LDL). These results suggest that, unlike ILDR1 and ILDR3, whose final destination is the PM, ILDR2 is an integral ER trans-membrane molecule that likely does not further translocate within these cell types.
Functional analysis of Ildr2: The ER plays critical roles in protein and lipid synthesis, lipoprotein assembly and export, glucose and calcium homeostasis, and cellular responses to metabolic stress [11-15]. These protein functions affect liver [16], hypothalamus [17], and b-cells [18]. The metabolic phenotypes seen in the Ildr2 B6.DBA. congenic lines are consistent with effects on ER stress mechanisms [1]. Accessibility of the liver to in vivo and in vitro transcriptional manipulation using adenovirus vectors [19,20], led us to focus on the liver.
To examine the effects of short term changes in Ildr2 expression in liver on lipid and glucose homeostasis, 10-week-old chow-fed C57BL/6J (wild-type; WT) or B6.Cg-Lep/J (obese; OB) male mice were transduced with adenoviral expression vectors encoding shRNA (“ADKD”) that knockdown Ildr2, or with adenoviral constructs driven by the CMV promoter (“ADOX”) that overexpress Ildr2. To control for non-specific effects of adenoviral transduction on gene expression, mice were transduced with adenoviral expression vectors encoding shRNA that knockdown lacZ, or with adenoviral constructs driven by the CMV promoter that overexpress the green fluorescent protein (GFP). Expression levels in the hypothalamus and white adipose tissue were unaffected by transduction with either the ADKD or ADOX viral constructs (data not shown), confirming that their effects were restricted primarily to the liver. Knockdown efficiency exceeded 80% at 3 days post-transduction (p.t.) and 90% at 10 days p.t., while Ildr2 overexpression resulted in 2- to 4-fold increases in mRNA levels.
For studies of liver morphology, histology and chemistry, and for liver-specific gene expression analysis, animals were sacrificed at 3 days or 10 days p.t. To provide a general picture of the cellular/biochemical consequences of manipulations of expression of hepatic Ildr2, the livers were evaluated by visual inspection, light microscopy, chemical composition, and by quantitative expression of selected genes related to neutral lipid/cholesterol synthesis, lipid oxidation, glucose homeostasis, and ER stress. Mice were also evaluated by indirect calorimetry, ipGTT, and plasma lipid profiling. To identify very short term responses to changes in expression of Ildr2, mouse primary hepatocytes were transduced with the ADKD and ADOX constructs and analyzed responses at 24 hr p.t.
Ildr2 does not Cross-Regulate with Ildr1 or Ildr3:
Although the apparent lack of cellular colocalization of ILDR2 with other molecules of this family makes it unlikely that ILDR2 interacts directly with them, its functions could be mediated through secondary genetic effects. To test this possibility transcription levels among the ildr genes were analyzed in primary B6 mouse hepatocytes transduced with siRNAs specific to each gene (Table 1). Whereas siRNA specific to Ildr2 almost completely suppressed its own expression, it reduced expression of Ildr1 by only 3% and Ildr3 by 27%, with little effect on Ildr2 of knockdown of either Ildr1 or Ildr3. These results indicate that expression levels of Ildr genes do not significantly cross-regulate.







TABLE 1







Relative expression of Ildr-family genes transduced with


Ildr-siRNAs. Genes of the Ildr-family do not significantly


cross-regulate. Data for effects of Ildr1 and Ildr3 siRNAs


were determined by qPCR. Data for effects of Ildr2 siRNA


are from microarray. Levels of mRNA are normalized to the


36B4 ribosomal housekeeping gene, expressed relative to


levels of each gene in control cells transduced with a


scrambled, non-specific siRNA sequence.











Ildr1 siRNA (n = 3)
Ildr2 siRNA (n = 10)
Ildr3 siRNA (n = 3)














Relative

Relative

Relative



Gene
Expression
P-value
Expression
P-value
Expression
P-value





Ildr1
0.14
1.2E−03
0.97
NS
ND
NA


Ildr2
0.97
NS
0.03
6.5E−06
1.28
3.3E−03


Ildr3
ND
n/a
0.73
8.0E−05
0.03
3.4E−03





n/a: not applicable;


ND: not done; NS: not significant.






Changes in Ildr2 Expression Affect Liver Morphology and Histology:
Control WT livers (lacZ) were normal in size and appearance in WT animals at 3 and 10 days p.t. but, as expected, were enlarged and grossly steatotic in OB animals (FIG. 3). The ADKD livers (WT and OB) were enlarged and grossly steatotic, whereas the ADOX WT livers were generally normal in appearance and size as were, remarkably, the ADOX OB livers.
Control WT livers were histologically normal with the exception of occasional mild lipid vesiculation and attendant monocytic infiltration, presumably due to adenovirus transduction per se. OB control livers showed extensive large vacuolization with minimal focal lobular lymphocytic infiltration[21]. Livers of ADKD WT mice at 3 days p.t. (FIG. 3A) showed mildly increased periportal vacuolization, modest mononuclear infiltration, occasional apoptosis and autophagy. By 10 days p.t. (FIG. 3B), histologic changes were striking: smaller lipid vesicles in the periportal region progressed to larger droplets at a distance from the portal tract, with ballooning of hepatocytes, autophagy, apoptosis and periportal monocytic inflammation. Some cells showed clumped pink intermediate filaments resembling human Mallory-Denk bodies in steatohepatitis, where they signify hepatocellular oxidative stress [22]. In the fed ADKD animals, increased apoptosis and inflammation were apparent in the context of a preponderance of large droplet fat vesicles. Lobular inflammation reminiscent of human non-alcoholic steatohepatitis was also seen. The livers of ADKD OB mice at 10 days p.t. (FIG. 3C) displayed extensive lipid deposition, with micro-vesiculation accompanied by severe monocytic infiltration, and areas of fibrosis in some animals.
In ADOX WT animals, phenotypic effects were generally modest. At 3 days p.t. (FIG. 3D), livers showed mild, small droplet steatosis, but by 10 days p.t. (FIG. 3E), there were areas of increased apoptosis with minimal lipid deposition or inflam-mation, consistent with a primary effect on ER stress-related responses. In fed animals, glycogen deposition was greatly increased. ADOX OB animals at 10 days p.t. (FIG. 3F) showed substantial reduction in the severity of steatosis (mostly medium and large droplet) with virtually no inflammation or apoptosis. These changes represented a striking “rescue” of the histology seen in the OB control and KD animals.
These gross effects and microscopic characteristics indicate the importance of Ildr2 in hepatic lipid homeostasis, with reduced expression causing lipid accumulation and overexpression acting to reduce this excess in OB livers. These effects generally increased in severity between 3 and 10 days p.t. Potential mechanisms for these effects and their molecular and physiological consequences were investigated.
Liver and Plasma Chemistry:
Hepatic triglyceride (TG) content was generally consistent with the histological effects of ADKD (Tables 2, 3, and 4) and ADOX (Tables 5, 6, and 7), whereas plasma TG, FFA, and glucose/insulin-related measurements were minimally affected. In contrast, hepatic and plasma cholesterol were greatly increased in refed 10 day ADKD animals. These phenotypes are described in more detail below.
At 3 days p.t. in ADKD WT animals (Table 2), body weight, liver weight and liver total cholesterol (TCH) content were unaffected, as were circulating concentrations of glucose, insulin, TG, TCH, and FFA. However, hepatic TG and glycogen content were significantly increased. Estimates of insulin resistance by HOMA2-IR and b-cell function by HOMA-2B % were unaffected, as were glucose excursions during IPGTT (see below). At 10 days p.t. in ADKD WT animals (Table 3), hepatic and circulating TCH and hepatic TG were increased while circulating TG was unchanged. Again, HOMA measurements were unaffected. In ADKD OB mice at 10 days p.t. (Table 4),—starting from higher baselines (as expected vs. WT animals) [21]—liver weight, TG and TCH content increased, and glycogen content decreased. Circulating concentrations of glucose, insulin, TG, TCH, and IPGTT were unaffected; circulating FFA concentrations were increased.
In ADOX WT animals at 3 days p.t. (Table 5), TG and TCH concentrations per unit wet weight of liver were higher (and glycogen lower) than in control mice. Plasma lipids were unaffected by ADOX. At 10 days p.t. in ADOX WT mice (Table 6), liver TG was lower, and glycogen content higher than in controls. Plasma TG and TCH trended higher in the ADOX animals. At 10 days p.t. in ADOX OB mice (Table 7), liver TG and TCH content were reduced without significant changes in circulating glucose, TG, TCH or FFA. Hepatic glycogen per gram wet weight was unaffected but, given the considerable reduction of TG, was probably decreased per unit liver nitrogen. Measure-ments of blood ALT and AST enzyme levels in these animals indicate that toxic effects of the adenoviral transductions on hepatocyte integrity were minimal.







TABLE 2







Liver and plasma chemistries of ADKD WT mice at 3 days p.t.


Mice were chow-fed, 10-week-old B6 (WT) males, intravenously


injected with ADKD vectors expressing RNAi for lacZ or Ildr2.


Measurements were taken at 3 days p.t. (following a 12-hr fast). 


ADKD WT 3 D


Fasted










Phenotype (n)
lacZ (10)
Ildr2 (10)
P-value





Body weight (g)
25.9 ± 0.5
25.7 ± 0.8
0.831


Liver weight (g)
 1.2 ± 0.0
 1.2 ± 0.0
0.772


Hepatic TG (mg/g Liver)
53.7 ± 5.1
69.2 ± 3.1
0.018


Hepatic TCH (mg/g Liver)
 6.7 ± 0.1
 7.2 ± 0.3
0.268


Hepatic glycogen (mg/g Liver)
 8.6 ± 2.0
20.2 ± 2.9
0.008


Plasma glucose (mg/dL)
182 ± 10
216 ± 16
0.096


Plasma insulin (μg/L)
 0.15 ± 0.04
 0.26 ± 0.06
0.190


Plasma TG (mg/dL)
156.8 ± 29.7
157.3 ± 20.0
0.989


Plasma TCH (mg/dL)
67.4 ± 1.4
70.8 ± 2.4
0.250


Plasma FFA (mEq/L)
 1.27 ± 0.03
 1.29 ± 0.02
0.564


Plasma ALT (mU/L)
134.3 ± 10.3
126.2 ± 7.8 
0.064


Plasma AST (mU/L)
125.5 ± 8.0 
126.8 ± 7.3 
0.707


HOMA2-IR
 0.59 ± 0.17
 1.11 ± 0.33
0.178


HOMA2-B (%)
14.3 ± 3.3
16.5 ± 2.8
0.598





n = number of animals in each study.


Data shown are mean 6 SEM;


P values were calculated by 2-tailed t test.


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase;


HOMA2-IR, homeostasis model assessment-estimated insulin resistance;


HOMA-2-B (%), homeostasis model assessment-b-cell function.













TABLE 3







Liver and plasma chemistries of ADKD WT mice at 10 days p.t. Mice were


chow-fed, 10-week-old B6 (WT) males, intravenously injected with ADKD vectors expressing


RNAi for lacZ or Ildr2. Measurements were taken at 10 days p.t. (following either a 24-hr fast


“Fasted” or following a 24-hr fast and 12-hr refeeding “Refed”). 


ADKD WT 10 D










Fasted
Refed
















P-


P-


Phenotype (n)
lacZ (5)
Ildr2 (6)
value
lacZ (5)
Ildr2 (6)
value
















Body weight (g)
25.1
27.5
0.084
27.5
27.5
0.983


Liver weight (g)
1.3
2.1
0.000
2.1
2.5
0.096


Hepatic TG (mg/g Liver)
46.4 ± 2.9 
88.6 ± 6.5 
0.001
18.6 ± 2.8 
76.9 ± 7.0 
0.000


Hepatic TCH (mg/g Liver) 
9.8 ± 0.6
18.0 ± 1.7 
0.005
7.6 ± 0.4
14.8 ± 1.4 
0.003


Hepatic glycogen (mg/g
1.9 ± 0.2
3.2 ± 0.5
0.039
58.0 ± 3.6 
37.1 ± 2.1 
0.007


Liver)








Plasma glucose (mg/dL)
81 ± 7 
86 ± 3 
0.619
187 ± 5 
153 ± 8 
0.009


Plasma insulin (μg/L)
0.26 ± 0.10 
0.25 ± 0.02
0.903
4.91 ± 0.75 
5.99 ± 1.68 
0.576


Plasma TG (mg/dL)
114.2 ± 19.0 
138.0 ± 17.0 
0.362
203.8 ± 22.9 
243.0 ± 27.0 
0.275


Plasma TCH (mg/dL)
131.7 ± 4.0  
254.0 ± 24.8 
0.003
135.5 ± 8.8 
313.3 ± 32.4 
0.001


Plasma FFA (mEq/L)
1.52 ± 0.06 
1.25 ± 0.13
0.093
1.05 ± 0.18
0.72 ± 0.03 
0.150


Plasma ALT (mU/L)
44.5 ± 6.2 
51.1 ± 9.3 
0.598
46.6 ± 3.0 
48.3 ± 6.0 
0.812


Plasma AST (mU/L)
46.5 ± 6.8 
48.8 ± 9.2 
0.852
49.6 ± 3.7 
47.5 ± 6.5 
0.797


HOMA2-IR
0.85 ± 0.35
0.80 ± 0.09 
0.901
n/a
n/a
n/a


HOMA2-B (%)
94.4 ± 22.2
91.0 ± 9.0 
0.989
n/a
n/a
n/a





n = number of animals in each study.


n/a: not applicable.


Data shown are mean 6 SEM;


P values were calculated by 2-tailed t test.


TG, triglycerides;


TCH, total cholesterol;


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase;


HOMA2-IR, homeostasis model assessment-estimated insulin resistance;


HOMA-2-B (%), homeostasis model assessment-b-cell function.


Other data showed B6 mice fasted for 12 hr and not treated with adenovirus had plasma ALT of 86.0 mU/L and AST of 94.6 mU/L.













TABLE 4







Liver and plasma chemistries of ADKD OB mice at 10 days p.t. Mice were


chow-fed, 10-week-old B6.V-Lepob/J (OB) males, intravenously injected with ADKD vectors


expressing RNAi for lacZ or Ildr2. Measurements were taken at 10 days p.t. (following either a


24-hr fast “Fasted” or following a 24-hr fast and 12-hr refeeding “Refed”). 


ADKD OB 10 D










Fasted
Refed













Phenotype (n)
lacZ (3)
Ildr2 (4)
P-value
lacZ (3)
Ildr2 (4) 
P-value





Body weight (g)
46.7 ± 0.4 
46.8 ± 0.9 
0.961
46.4 ± 3.1 
47.3 ± 1.9 
0.845


Liver weight (g)
4.2 ± 0.5
5.8 ± 0.3
0.012
3.4 ± 0.1
6.4 ± 0.7
0.047


Hepatic TG (mg/g Liver)
77.5 ± 4.0 
113.7 ± 6.8 
0.037
77.0 ± 1.0 
118.0 ± 7.3 
0.041


Hepatic TCH (mg/g Liver)
10.6 ± 1.5 
16.5 ± 3.4 
0.189
8.9 ± 0.7
16.9 ± 3.6 
0.203


Hepatic glycogen (mg/g Liver)
43.0 ± 0.4 
14.6 ± 3.8 
0.004
44.2 ± 1.9 
5.8 ± 1.3
0.001


Plasma glucose (mg/dL)
175 ± 29 
116 ± 20 
0.215
379 ± 79 
214 ± 28 
0.233


Plasma insulin (μg/L)
18.1 ± 10.1
27.2 ± 19.5
0.707
77.8 ± 33.1
103.5 ± 5.3  
0.576


Plasma TG (mg/dL)
97.6 ± 28.0 
133.5 ± 16.1 
0.395
121.0 ± 24.5 
195.4 ± 24.2 
0.135


Plasma TCH (mg/dL)
110.3 ± 2.5 
104.0 ± 2.5 
0.173
112.6 ± 9.7 
133.7 ± 8.5 
0.231


Plasma FFA (mEq/L)
1.23 ± 0.01
1.71 ± 0.08
0.012
1.31 ± 0.07
2.44 ± 0.16
0.013


Plasma ALT (mU/L)
93.2 ± 4.0 
107.2 ± 4.1 
0.006
105.9 ± 9.6 
126.8 ± 25.3 
0.274


Plasma AST (mU/L)
88.9 ± 2.3 
110.0 ± 11.3  
0.036
93.4 ± 10.6
115.8 ± 37.1 
0.421





n = number of animals in each study.


Data shown are mean 6 SEM;


P values were calculated by 2-tailed t test.


TG, triglycerides;


TCH, total cholesterol;


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase.













TABLE 5







Liver and plasma chemistries of ADOX WT mice at 3 days p.t.


Mice were chow-fed, 10-week-old B6 (WT) males, intravenously


injected with ADOX vectors expressing GFP or Ildr2. Measure-


ments were taken at 3 days p.t. (following a 12-hr fast).


ADOX WT 3 D


Fasted










Phenotype (n)
GFP (9)
Ildr2 (9)
P-value





Body weight (g)
25.1 ± 0.7
24.3 ± 0.6
0.439


Liver weight (g)
 1.2 ± 0.0
 1.0 ± 0.0
0.004


Hepatic TG (mg/g Liver)
 93.0 ± 11.9
124.3 ± 9.6 
0.073


Hepatic TCH (mg/g Liver)
10.7 ± 1.2
15.6 ± 1.6
0.038


Hepatic glycogen (mg/g Liver)
14.6 ± 2.2
 6.2 ± 2.1
0.009


Plasma glucose (mg/dL)
159 ± 6 
166 ± 13
0.637


Plasma insulin (μg/L)
 0.15 ± 0.04
 0.21 ± 0.04
0.316


Plasma TG (mg/dL)
127.3 ± 21.9
153.3 ± 14.2
0.338


Plasma TCH (mg/dL)
65.3 ± 0.9
72.1 ± 2.8
0.052


Plasma FFA (mEq/L)
 1.28 ± 0.03
 1.35 ± 0.09
0.502


Plasma ALT (mU/L)
 79.5 ± 15.1
 99.4 ± 13.1
0.008


Plasma AST (mU/L)
 78.0 ± 20.5
 93.3 ± 13.0
0.089


HOMA2-IR
 0.56 ± 0.16
 0.79 ± 0.15
0.347


HOMA2-B (%)
17.7 ± 3.8
23.5 ± 4.7
0.302





n = number of animals in each study.


Data shown are mean ± SEM;


P values were calculated by 2-tailed t test.


TG, triglycerides;


TCH, total cholesterol;


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase;


HOMA2-IR, homeostasis model assessment-estimated insulin resistance;


HOMA-2-B (%), homeostasis model assessment-β-cell function.













TABLE 6







Liver and plasma chemistries of ADOX WT mice at 10 days p.t. Mice were


chow-fed, 10-week-old B6 (WT) males, intravenously injected with ADOX vectors expressing


GFP or Ildr2. Measurements were taken at 10 days p.t. (following either a 24-hr fast “Fasted” or


following a 24-hr fast and 12-hr refeeding “Refed”). 


ADOX WT 10 D










Fasted
Refed













Phenotype (n)
GFP (5)
Ildr2 (6)
P-value
GFP (5)
Ildr2 (6)
P-value
















Body weight (g)
24.1
23.2
0.607
27.5
26.0
0.112


Liver weight (g)
1.0
1.3
0.011
1.7
2.2
0.030


Hepatic TG (mg/g Liver)
64.9 ± 6.9 
33.0 ± 4.7 
0.004
31.3 ± 5.5 
20.0 ± 5.6 
0.191


Hepatic TCH (mg/g Liver)
11.4 ± 0.8 
7.4 ± 0.6
0.005
6.8 ± 0.7
5.4 ± 0.9
0.272


Hepatic glycogen (mg/g Liver)
1.1 ± 0.3
4.0 ± 0.9
0.033
63.0 ± 1.3 
71.2 ± 0.4 
0.048


Plasma glucose (mg/dL)
105 ± 4 
92 ± 4 
0.056
211 ± 4 
155 ± 3 
0.001


Plasma insulin (μg/L)
0.34 ± 0.09
0.34 ± 0.07
0.988
5.67 ± 0.09
4.95 ± 0.07
0.560


Plasma TG (mg/dL)
57.6 ± 2.5 
 98 ± 9.6
0.005
192.8 ± 23.0 
246.1 ± 22.4 
0.117


Plasma TCH (mg/dL)
114.6 ± 8.9 
123.9 ± 11.6 
0.563
125.7 ± 2.3 
145.7 ± 1.2 
0.005


Plasma FFA (mEq/L)
1.25 ± 0.16
1.70 ± 0.19
0.148
0.65 ± 0.18
0.82 ± 0.10
0.470


Plasma ALT (mU/L)
66.9 ± 5.1 
32.1 ± 0.3 
0.021
72.2 ± 6.3 
41.1 ± 1.9 
0.042


Plasma AST (mU/L)
65.6 ± 3.7 
31.4 ± 0.5 
0.012
68.0 ± 6.2 
41.1 ± 2.7 
0.028


HOMA2-IR
1.36 ± 0.25
1.10 ± 0.19
0.435
n/a
n/a
n/a


HOMA2-B (%)
86.0 ± 14.8
100.0 ± 18.8  
0.570
n/a
n/a
n/a





n = number of animals in each study.


n/a: not applicable.


Data shown are mean ± SEM;


P values were calculated by 2-tailed t test.


TG, triglycerides;


TCH, total cholesterol;


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase;


HOMA2-IR, homeostasis model assessment-estimated insulin resistance;


HOMA-2-B (%), homeostasis model assessment-β-cell function.













TABLE 7







Liver and plasma chemistries of ADOX OB mice at 10 days p.t. Mice were


chow-fed, 10-week-old B6.V-Lepob/J (OB) males, intravenously injected with ADOX vectors


expressing GFP or Ildr2. Measurements were taken at 10 days p.t. (following either a 24-hr fast


“Fasted” or following a 24 hr fast and 12 hr refeeding “Refed”). 


ADOX OB 10 D










Fasted
Refed
















P-


P-


Phenotype (n)
GFP (4)
Ildr2 (4)
value
GFP (4)
Ildr2 (4)
value





Body weight (g)
45.1 ± 1.4 
48.2 ± 0.6 
0.120
46.9 ± 1.1 
43.7 ± 0.8 
0.066


Liver weight (g)
3.4 ± 0.2
3.9 ± 0.3
0.321
4.6 ± 0.3
3.4 ± 0.1
0.042


Hepatic TG (mg/g Liver)
49.5 ± 5.7 
27.1 ± 3.0 
0.019
56.4 ± 4.4 
32.3 ± 4.0 
0.007


Hepatic TCH (mg/g Liver)
7.7 ± 0.6
5.6 ± 0.2
0.038
6.4 ± 0.2
 5.1 ± 0.29
0.015


Hepatic glycogen (mg/g
50.4 ± 4.3 
53.5 ± 3.7 
0.606
 62.8 ± 101.6
 61.3 ± 103.6
0.689


Liver)








Plasma glucose (mg/dL)
151 ± 10 
174 ± 24 
0.420
224 ± 29 
263 ± 20 
0.349


Plasma insulin (μg/L)
13.6 ± 4.4 
24.5 ± 2.5 
0.168
73.9 ± 19.1
89.3 ± 4.0 
0.489


Plasma TG (mg/dL)
94.3 ± 27.2
129.8 ± 43.4 
0.538
137.6 ± 18.3 
135.4 ± 11.7 
0.925


Plasma TCH (mg/dL)
134.0 ± 3.5 
129.0 ± 3.9 
0.383
143.1 ± 4.8 
130.9 ± 2.6 
0.086


Plasma FFA (mEq/L)
1.66 ± 0.09
1.46 ± 0.05
0.119
2.25 ± 0.29
1.76 ± 0.12
0.206


Plasma ALT (mU/L)
115.1 ± 18.4  
109.7 ± 15.7 
0.668
108.0 ± 5.4 
100.1 ± 15.3 
0.386


Plasma AST (mU/L)
106.1 ± 8.8 
101.7 ± 13.8 
0.611
102.7 ± 12.7  
98.7 ± 17.0
0.718





n = number of animals in each study.


Data shown are mean ± SEM;


P values were calculated by 2-tailed t test.


TG, triglycerides;


TCH, total cholesterol;


FFA, free fatty acids;


ALT, alanine aminotransferase;


AST, aspartate aminotransferase.













TABLE 8







Triglyceride and cholesterol content of ADKD and ADOX hepatocytes.


Hepatocytes from 5, 10-week-old B6 mice were extracted, pooled and plated into individual


wells and exposed for 24 hr to either the ADOX or ADKD (or empty vector control) virus in


triplicate. Cells were lysed and triglyceride and total cholesterol were determined.










ADKD
ADOX














lacZ
ILDR2
P-value
GFP
Ildr2
P-value





Triglyceride
103.1 ± 2.3 
119.4 ± 3.4 
0.019
105.5 ± 3.9 
92.7 ± 1.3 
0.056


(mg/g








protein)








Cholesterol
7.58 ± 0.37
8.45 ± 0.40
0.083
8.43 ± 0.96
6.62 ± 0.45
0.081


(mg/g








protein)
















TABLE 9







Area under the curve calculations for calorimetry. 










WILD-TYPE
OB/OB












ADKD
ADOX
ADKD
ADOX















AUC
lacZ
Ildr2
GFP
Ildr2
lacZ
Ildr2
GFP
Ildr2





24-hr 
43.2 ± 0.6
40.8 ± 0.6*
42.1 ± 0.6
42.0 ± 0.4
42.0 ± 0.8
40.9 ± 1.4 
39.5 ± 0.8
40.3 ± 1.3


Day
20.1 ± 0.5
19.2 ± 0.5
19.6 ± 0.4 
19.7 ± 0.2
19.8 ± 0.4
20.0 ± 0.6
19.1 ± 0.3
19.5 ± 0.6


Night
23.1 ± 0.3
21.6 ± 0.3**
22.4 ± 0.2
22.3 ± 0.3
22.3 ± 0.6
20.9 ± 0.8
20.5 ± 0.5 
20.8 ± 0.7





*p < 0.05;


**p < 0.01; AUC, area under the curve.






Analysis of In Vivo Lipoprotein Production and Clearance:
The increase in hepatic TG and TCH in the ADKD mice could reflect: 1. increased assembly/reduced secretion of lipoproteins; 2. increased synthesis/decreased oxidation of TG; 3. increased synthesis/decreased disposal of cholesterol. Detergents such as Triton WR1339 block clearance of TG in circulating lipoproteins by inhibiting lipoprotein lipase (LPL)-mediated lipolysis of circulating TG-rich lipoproteins [23]. When LPL is inhibited, changes in circulating concentrations of lipoprotein species reflect hepatic secretion rates of very low-density lipoprotein (VLDL). Concentrations of plasma TG were measured following LPL inhibition with Triton WR1339 in 10-week-old chow fed ADKD and ADOX WT mice at 7 days p.t. (FIG. 4). Area under the curve (AUC) analysis of hepatic lipoprotein secretion shows no significant difference in either ADKD (FIG. 4A) or ADOX mice (FIG. 4B). These results suggest that in ADKD mice, the increased hepatic lipids did not stimulate increased VLDL secretion and that, consequently, the increased plasma lipids, notably TCH, reflected reduced hepatic lipoprotein clearance. Likewise, the decreased hepatic lipids in ADOX mice were not due to increased VLDL secretion. Increased hepatic lipid synthesis in ADKD mice was not coupled to secretion or decreased hepatic fatty acid oxidation. In contrast, ADOX mice could have had decreased hepatic lipid synthesis or increased fatty acid oxidation. Finally, it is interesting to note that in these animals, glucose tolerance was normal despite severe hepatic steatosis [24].
Steady-State Lipoprotein Analysis:
Based upon the striking changes in hepatic lipid content without evidence of change in lipoprotein export, qualitative and quantitative changes in circulating lipoproteins in response to transient manipulations of Ildr2 expression were of interest. Particularly, determining if the dramatic increase in TCH in the ADKD mice represented increased TCH in VLDL or LDL, or decreased TCH in high-density lipoprotein (HDL) was of interest. In analysis by fast protein liquid chromatography (FPLC) of fasted plasma obtained prior to the Triton study (FIG. 5), VLDL cholesterol (fractions 12-16) and IDL/LDL cholesterol (fractions 17-23) were clearly higher, and HDL cholesterol (fractions 24-30) was  ̃20% lower in the ADKD animals (FIG. 5A, 5C). In ADOX animals (FIG. 5B, 5D), VLDL was similarly increased, LDL was not altered, and HDL was also slightly reduced. These results are consistent with the data reported in Tables 2, 3, 4, 5, 6, and 7 in which ADKD animals had higher absolute circulating TG than ADOX animals and where WT fasted and refed ADKD mice exhibited increases in plasma TCH and TG vs. controls, whereas WT ADOX mice exhibited more moderate changes.
Hepatic Gene-Expression Signatures:
To assess possible molecular bases for these changes in liver histology and lipid/glycogen chemistry with the remarkably minimal effects of these changes on systemic lipid and insulin homeostasis, hepatic expression of genes related to acylglyceride, cholesterol and glucose homeostasis and ER-resident molecules mediating responses to metabolic stress were examined (FIG. 6).
In ADKD WT animals, at 3 days p.t. (FIG. 6A), increases in transcript levels of genes involved in acylglyceride synthesis were consistent with increased TG content; however, at 10 days p.t. (FIG. 6B), transcripts of genes related to acylglyceride synthesis were reduced (where Fas expression was especially decreased in the fed animals), as were genes related to cholesterol homeostasis and FFA catabolism. Unlike the livers at 3 days p.t., those at 10 days displayed a general suppression of transcripts of genes mediating both synthesis and oxidation of hepatic lipids. The general suppression of transcripts of genes mediating both synthesis and oxidation of hepatic lipids between days 3 and 10 suggests that the accumulation of hepatic lipids due to effects of inhibition of Ildr2 transcription resulted in secondary suppression of the expression of these genes.
In ADKD OB animals at 10 days p.t. (FIG. 6C), Ildr2 knockdown greatly reduced the expression levels of all transcripts examined compared to the control (lacZ) animals. These effects were comparable, though more extensive and proportionately greater, than in the corresponding studies of WT animals, possibly reflecting, in part, the consequences of pre-existing hepatic steatosis.
In livers of ADOX WT mice at 3 days p.t. (FIG. 6D), as in the ADKD livers, in the context of an increase in TCH content (and a trend towards increased TG), transcript levels of genes mediating the synthesis of these molecules were generally reduced, although transcript levels of some fatty acid oxidation genes were slightly increased. However, at 10 days p.t. (FIG. 6E), transcript levels of genes related to glucose metabolism and acylglyceride synthesis were increased.
In livers of ADOX OB animals at 10 days p.t. (FIG. 6F), the very high levels of lipid accumulation due to the obesity of these animals were reduced by over-expression of Ildr2. In association with this reduction, levels of transcripts related to neutral lipid synthesis and cholesterol are increased, suggesting “relief” of the secondary suppression imposed by lipid accumulation as mentioned above [25].
ER Stress Pathways:
The apparent fixed location of ILDR2 in the ER (see FIG. 2) raises the possibility that the protean effects of hypomorphism for this gene might be related to a role in the mediation of ER stress responses [12]. Such a role would not be inconsistent with an independent effect on lipoprotein metabolism [26]. Accordingly, transcription rates of canonical members of the ER stress response pathways [27] were examined.
In ADKD WT animals at 3 days p.t., expression of ER stress effectors Perk, Atf6, and Ire1 was slightly reduced, while expression of transducers Atf4 and Chop was increased. In general, Ildr2 over-expression was associated with increased expression of ER stress pathway genes, while Ildr2 knockdown was associated with decreased expression. Effects were greater at 10 days than at 3 days p.t.
Increases in hepatocyte lipids activate ER stress pathways [28]-[30], and activation of ER stress pathways increases hepatic lipid deposition[31]-[33]. Given these reciprocal interactions, and the relatively extended time course over which these studies were conducted, it is not possible to assign causal primacy to either the effects on lipid synthesis or ER stress responses. The data are also consistent with the possibility that ILDR2 has primary effects on both processes. Experiments conducted in isolated hepatocytes (see FIG. 7) demonstrate that in vitro knockdown of Ildr2 modestly increases ER stress-related transcripts over a 48-hr period. Longer term, these responses may be exhausted [34], [35]. The apparent paradox of increased ER stress responses in both ADKD and ADOX hepatocytes may be due to the former's reflecting the response to increased cellular lipids, and the latter to direct interactions of the ILDR2 molecule with elements of the ER stress pathways. The increase in ER stress molecules is presumably partially protective [36]-[38].
Short-Term Effects of Ildr2 Expression on Lipid Metabolism and ER Stress Pathways in Hepatocytes:
ER stress can affect lipid metabolism and vice versa [31], [39], [40], and molecules such as XBP1 can independently affect both pathways [26]. In an effort to disarticulate—by shortening the experimental time course—possible contributions of ILDR2 to ER stress response mechanisms, C57BL/6J mouse primary hepatocytes were transduced for 24 hr with Ildr2 ADKD and ADOX adenoviral vectors and examined cellular lipid content (Table 8) and expression of genes of lipid biochemical and UPR/ER stress pathways (FIG. 7).
In the ADKD cells, TG and TCH content were increased, consistent with the changes seen in the in vivo studies. Only slight changes were seen in the transcripts analyzed, with no indication of primacy of Ildr2 knockdown effects on either lipid synthesis or ER stress genes (FIG. 7A). In the ADOX cells, TG and TCH content were reduced in the context of large, reciprocal changes in both lipogenesis, where Ldlr and Srebp1c decreased, and ER stress effectors, where Atf6 and Ire1 increased (FIG. 7B). The comparable magnitude of these changes makes it difficult to assign primacy, but is consistent with a role for Ildr2 in both pathways. If these in vitro data at 24 hr are viewed in the context of the 3 and 10 day in vivo studies, it is apparent that there are strong temporal effects of responses of the ER stress pathways, and that the responses are influenced by intercurrent processes, probably lipid deposition per se.
Effects of Feeding Status, Diet, and Ob Genotype on Ildr2 Expression:
Hepatic lipid homeostasis is strongly affected by fasting and refeeding and by diet [41]. To study their effects on Ildr2, levels of Ildr2 expression were compared in livers of WT mice fed either chow or a high-fat diet (FIG. 8). These results show that feeding status (the difference between fasted and refed mice) had little effect on Ildr2 expression, whereas obesity achieved by feeding a high-fat diet, increased Ildr2 levels by 3.6 fold (FIG. 8A). To determine if this effect was leptin-dependent, Ildr2 expression in livers of OB (leptin-deficient) mice was also analyzed. These mice showed a 3.7 fold increase in Ildr2 expression compared to age-matched WT controls (FIG. 8B). The large increases in ildr2 expression, caused by leptin deficiency and high-fat feeding are presumably secondary—at some level—to the deposition of TG in the hepatocytes under both circumstances. Given the possible dual roles of Ildr2, this effect could reflect a role of Ildr2 in ER stress responses.
Calorimetry:
To determine if there were differences in energy expenditure, physical activity, or metabolic substrate use in the mice in any of the models used (ADKD and ADOX in both WT and OB), indirect calorimetry (72-hr) was performed on chow-fed, 10-week-old WT and OB males, 4 to 5 days p.t. (FIG. 9; Table 9).
There were no differences in rates or patterns of 24-hour energy expenditure in WT mice between knockdown and control. However, in WT (FIG. 9A) and OB (FIG. 9C) ADKD mice, the nocturnal respiratory exchange ratio (RER) was 7% lower vs. controls, indicating that, at night, the ADKD mice preferentially oxidize fat to a greater extent than the WT mice. In WT (FIG. 9B) and OB (FIG. 9D) ADOX animals, the RER was not significantly different between the two groups at any time, although the OB mice had a slightly higher RER during the dark period. These data are consistent with hepatic lipid content influencing systemic fuel oxidation: higher fat content increasing fatty acid oxidation, resulting in a lower RER.
ipGTT (Intraperitoneal Glucose Tolerance Tests):
To assess systemic effects of changes in hepatic lipid and glycogen content on peripheral glucose homeostasis, ipGTT was performed on chow-fed, 10-week-old WT and OB males, 7 days p.t. with Ildr2 ADKD and ADOX constructs (FIG. 10). Surprisingly, no differences in systemic glucose tolerance were detected in ADKD or ADOX animals versus either their respective controls, or each other. Consistent with these findings, HOMA IR values based on data obtained at the time of sacrifice of ADKD and ADOX mice (at 10 days p.t.) were not significantly different (see Table 2). Thus, large changes in hepatic lipid content were not accompanied by changes in systemic glucose/insulin homeostasis. This finding has implications for the possible mechanism(s) underlying the effects of Ildr2 on hepatic lipid synthesis and handling.
Discussion.
Based upon manipulation of levels of expression of Ildr2 in liver and isolated hepatocytes using ADKD and ADOX constructs, the results described herein show ILDR2 is an ER membrane protein that participates in cellular lipid synthesis and responses to ER stress. The most salient phenotype in the ADKD mice is TG accumulation, accompanied by increased hepatic and plasma cholesterol and a mix of micro- and macro-vesicular lipid droplets in periportal hepatocytes. Overexpression of Ildr2 in ob/ob mice substantially rescued their hepatic steatosis, as Ildr2 over-expressing mice had significantly decreased hepatic TG and TCH and reduced periportal vacuolar deposition.
Despite the excess lipid in the livers at 10 days p.t., transcript levels for major lipogenic and fat-oxidative genes were reduced in ADKD mice and up-regulated in ADOX mice. At 3 days p.t., several of these genes showed changes in expression in the opposite direction, suggesting that down-regulation in ADKD mice at 10 days p.t. may have been a response to excess lipid accumulation. These changes in transcriptional profiles are likely secondary to the respective increased/decreased lipid content of the hepatocytes. Also relevant in considering the molecular pathogenesis of the steatosis in the ADKD animals is the absence of major changes of circulating TG or cholesterol in these animals, their apparently normal rates of hepatic TG secretion, and the absence of significant changes in glucose or insulin homeostasis. In ADKD mice, reduced hepatic VLDL/IDL/LDL clearance and increased circulating IDL/LDL cholesterol suggests a reduction in hepatic LDL receptor-mediated clearance of those lipoproteins, consistent with reduced expression of hepatic LDL receptor [42], [43].
Lipid accumulation in the liver is commonly associated with liver and/or systemic insulin resistance and resultant hyperglycemia. Indeed, hepatic steatosis is commonly implicated as a causative factor in these phenotypes that are aspects of the metabolic syndrome [44]-[46]. However, in the ADKD animals, large changes in hepatic lipid content were not accompanied by changes in systemic glucose/insulin homeostasis [47]. Decreased lipid droplet turnover and/or enhanced traffic of newly synthesized TG from the ER to the cytoplasmic droplets might be related to the apparent absence of effect of the increased lipid deposition on glucose/insulin homeostasis. Since neither insulin resistance nor hyperglycemia was present in the ADKD mice (similar in this regard to the phenotype of mice hypomorphic for hepatic Atgl) [24], [48], [49], other mechanisms relating Ildr2 to hepatic steatosis were investigated.
Localization of ILDR2 in the ER membrane, up-regulation of ER stress markers in the livers of ADOX mice and down-regulation in ADKD mice, along with the emerging relationship between hepatic lipid accumulation and ER stress in several metabolic disorders, including obesity, hepatic steatosis and type II diabetes [16], [31], [50], suggest that ILDR2 might have a role in cellular ER stress responses.
Three known pathways provide mechanisms whereby Ildr2 regulation of hepatocyte lipid metabolism and ER stress could be achieved:
1. ILDR2 has a primary role in ER function, where ER stress produced by Ildr2 knockdown leads to lipid accumulation. Overexpression of the ER stress chaperone BIP (GRP78) in ob/ob mice (as with Ildr2) reverses hepatic steatosis [51], and hypomorphic expression of UPR modulators Atf6, Ire1α, Chop, and Crebh in mouse models cause hepatic dyslipidemia [32], [33], [52], [53].
2. ILDR2 has a primary role in lipid metabolism, where Ildr2 knockdown leads to lipid accumulation, which causes ER stress. Excess intracellular fatty acids induce ER stress in the liver via pathways affecting ER membrane integrity and calcium homeostasis [40], [54], increasing Chop expression[55], inducing PERK signaling [28] and stimulating CREBh-induced inflammation[56], [57]. Additionally, fatty acid-binding protein-4 (aP2) has a primary role in lipid metabolism and mitigates ER stress in macrophages [39].
3. ILDR2 is independently involved in both ER function and lipid metabolism, as has been suggested for the ER stress-related gene, Xbp1, a key transcription factor and effector of the UPR which is spliced by IRE1a in response to ER stress. Xbp1 KO mice show reduced hepatic TG secretion and decreased fatty acid oxidation, along with down-regulation of key hepatic lipogenic genes [26]. Without being bound by theory, ILDR2 may be a transcription factor, by acting on downstream signaling targets, including transcription factors, it could independently affect the UPR and lipid metabolism.
The broad down-regulation of lipid metabolism and ER stress genes in the ADKD mice at 10 days p.t. is consistent with studies of pharmacologically-induced ER stress, in which expression of genes involved in lipid metabolism and ER stress initially increases and then declines. This pattern has been observed both in vitro and in vivo for spliced Xbp1, Chop, Bip, lipogenic transcription factors, lipid droplet proteins, and TG synthesis genes [34]. If Ildr2 knockdown induces ER stress in these mice, then by 10 days the decline of previously up-regulated ER stress and metabolic genes may be observed.
Since it remains uncertain, which gene expression effects are primary in the pathogenesis of the hepatic steatosis, and which may be responses to the steatosis per se, the data obtained in the 3 and 10 day adenovirus transductions should be cautiously interpreted. Absence of a clear pattern in the differential responses of the canonical UPR pathways seen in the studies reported here suggests that these variable responses may reflect differences in the timing of the responses of specific molecules and pathways coupled with secondary effects of the accumulation of hepatic lipids [58].
Ildr2-mediated effects on lipid homeostasis and ER stress responses could account for both the hepatic steatosis observed in the ADKD animals reported here, and the reduced β-cell mass and accompanying glucose intolerance in the Chr1 B6.DBA ob/ob congenic animals [1]. Perk-null mice develop ER stress specifically in the β-cell, with morphological abnormalities within the pancreatic ER leading to loss of β-cells, and hypoinsulinemic hyperglycemia [59]. Deregulation of lipid metabolism in a β-cell line impaired insulin secretion[60].
In a recent study, ILDR2, along with ILDR1 and ILDR3, was localized to tricellular junctions on the PM of mouse epithelial cells—specifically fibroblasts, mammary and retinal epithelia, and choroid plexus [61]. Without being bound by theory, these molecules can mediate macromolecular access through these “tight junctions”. However, no specific effort was made to visualize ILDR2 or other ILDR molecules in the ER. As has been described for the thyrotropin-releasing hormone receptor, which is localized to the PM in pituitary cells but to the ER and Golgi in non-pituitary cells [62], ILDR2 may localize primarily to the PM in epithelial cells, and to the ER in other cell types (e.g. hepatocytes, neurons, β-cells). Alternatively, subcellular distribution of members of this family of proteins may reflect cell type-specific splicing patterns, as reported for BAT3 [63].
Materials and Methods.
Animal Care: Mice were housed in a vivarium maintained on a 12 hr-12 hr light-dark cycle, with ad libitum access to 5058 Purina PicoLab Mouse Diet 20 (9% fat) and water, unless otherwise stated. All mice were sacrificed at the same time-of-day (1000-1200 hr).
Strains: Male 9-week-old C57BL/6J (B6) (Stock number 000664) and B6.V-Lepob/J (ob/ob) (Stock number 000632) mice were obtained from Jackson Laboratories (Bar Harbor, Me.) and allowed to adjust to conditions in our local colony for 1 week prior to starting experiments. Mice used to study the effects of feeding status and diet (FIG. 8A) were fed high-fat chow (60% kcals from fat) at Jackson Laboratories from 6 weeks of age until purchase at 18 weeks of age. Mice were fed ad libitum high-fat chow (Research Diets D12492i) for 4 additional weeks.
Metabolic Parameters: Body mass and composition: Weight was measured with a Vicon Vic-212 integrating laboratory scale (Acculab). Fat and lean mass were measured with a Minispec TD-NMR Analyzer (Bruker Optics), calibrated using mouse carcasses [64].
Serum: Blood was collected at sacrifice. Plasma was analyzed for glucose using an Autokit Glucose (Wako), for TG using an L-Type TG M Color A (Wako), for TCH using Cholesterol E (Wako), for FFA using HR Series NEFA-HR(2) Color Reagent B (Wako) and insulin using an Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem). Glucose in living mice was measured with a FreeStyle Lite portable glucose meter (Abbott) using 3 μl blood from a capillary tail bleed. IPGTT was performed in the morning after overnight fast. Blood for fasting glucose analysis was collected by tail bleed. Mice were injected with 2 mg/g of glucose using a 50 mg/ml solution in autoclaved water. Blood was collected at 5, 15, 30, 60, and 120 minutes and glucose was measured with the Autokit Glucose.
Plasma Lipid Profile and Triton Experiment: 250 μl of pooled plasma from 6 mice fasted for 5 hr was used for FPLC analysis using 2 Sepharose 6 Fast Flow columns in series (Amersham Biosciences). The buffer contained 100 mM Tris and 0.04% NaN3, pH 7.5; a flow rate of 0.7 ml/min was used. TCH and TG levels of FPLC fractions were measured using Wako enzymatic kits. To block clearance of nascent lipoproteins, these mice were then injected with Triton WR1339 (0.5 mg/g body weight; Sigma-Aldrich) via tail vein. Blood samples were collected at 0, 30, 60, and 120 min post-injection. Initial plasma samples were used for TG quantification.
Liver Glycogen: Liver fragments (0.1-0.2 g) were digested in 1 ml of 30% KOH at 95° C. for 30 min; 0.2 ml of 2% Na2SO4 and 3.2 ml of 70% ethanol were added and the mixture was centrifuged for 30 min at 6800 RCF. Pellets (containing glycogen) were washed with 70% ethanol and resuspended in 0.5 ml of 0.2 M acetate buffer; 0.1 ml of the solution was incubated for 30 min at 55° C. with 5 μl of amyloglucosidase (Sigma) and then incubated 5 min at 37° C. with Autokit Glucose (Wako Diagnostics, Richmond, Va.). Glycogen content was expressed as mg of glucose/g of wet liver.
Liver Lipids: Whole lipids were extracted by Bligh-Dyer extraction[65]. In brief, 1.5 ml of chloroform:methanol (12, v/v) with 0.4 ml of PBS was added to liver pieces (0.1-0.2 g) in a screw-capped glass test tube and mixed vigorously for 1 min. Vigorous mixing followed successive additions of 0.5 ml chloroform and 0.5 ml H2O. The mixture was centrifuged at 1800 RCF for 5 min and the lower (organic) phase, containing whole lipids, was collected and stored at −20° C. until assay. TCH and TG were determined with a colorimetric kit (Wako; Cholesterol E 439-17501; L Type TG 461-08892 and 461-09092).
HOMA-IR: HOMA2-IR (homeostasis model assessment-estimated insulin resistance) and HOMA-2-B % (homeostasis model assessment-β-cell function) were calculated using the HOMA calculator, www.dtu.ox.ac.uk/homacalculator/index.php, based on the nonlinear updated HOMA2 model [66], which takes account of variations in hepatic and peripheral glucose resistance, increases in the insulin secretion curve for plasma glucose concentrations >10 mmol/L (180 mg/dL) and the contribution of circulating proinsulin.
Energy Expenditure: Energy expenditure was measured with a LabMaster-CaloSys-calorimetry System (TSE Systems, Bad Homburg, Germany). 02 and CO2 measurements were taken every 26 min during a 72 hr period from 32, 10-week-old male mice (8 ADOX, 8 ADOX control, 8 ADKD, and 8 ADKD control). Mice were injected with the adenovirus on day 0 and placed in calorimeters from days 5-7. Because of stress related to transfer to the calorimetry chambers, only measurements taken within the last 48 hr were used to calculate total 24-hr energy expenditure (TEE; in kcal/24-hr) and respiratory exchange rate (RER=VCO2/VO2). Resting energy expenditure (REE in kcal/24-hr) was defined as the1-hr period of lowest energy expenditure. This coincided with the 1 hr of lowest total ambulatory activity (generally early afternoon), during the 48-hr period; this value was extrapolated to 24 hr. Non-resting energy expenditure (NREE) was calculated as the difference between TEE and REE (NREE=TEE−REE). Physical activity was measured by an infrared beam system integrated with the LabMaster system. Total activity (beam breaks) in X, Y, and Z axes was recorded every 26 min. The system is designed to differentiate between fine motor movement (defined as a single X or Y axis beam break), ambulatory movement (defined as the simultaneous breaking of two adjacent X or Y beams), and rearing, defined as the breaking of the Z axis infrared beam. Lights were off at night from 1900 to 0700 hr.
Adenovirus Studies: Adenoviral expression vectors: Adenoviruses were prepared and amplified with the ViraPower Adenoviral Expression System (Invitrogen). Viral titers were determined by plaque-forming assays on HEK 293A cells. PCR-amplified, full-length Ildr2-cDNA was subcloned into the pENTR/D-TOPO vector using the pENTR Directional TOPO Cloning Kit (Invitrogen). After verifying the sequence, inserts were transferred into the pAd/CMV/V5-DEST vector by the Gateway system using LR Clonase II Enzyme Mix. Sequences corresponding to the shRNAs for Ildr2 and lacZ were cloned into pBlock-it (Invitrogen). The sequence of the shRNA for Ildr2 was: 5′-cac cGT TCA AAT CCT ACT GCC Aga cgt gtg ctg tcc gtC TGG CAG TAG GAT TTG AAC-3′ (SEQ ID NO: 147), where the 5′ uppercase 18-nucleotide sequence corresponds to the coding strand in exon 2 for the amino acid sequence FKSYCQ (SEQ ID NO: 148).
Virus purification: To obtain virus particles, plasmids were linearized by Pac I digestion and transduced into HEK 293A cells with LIPOFECTAMINE 2000 using Opti-MEM medium. The transduced HEK 293A cells were incubated at 37° C. in a 6 cm dish until the cells started to die (about 10 days). The cells and supernatant were harvested in a 50 ml tube and subjected to 3 freeze-thaw cycles to lyse the cells. The suspension was centrifuged at 1800 RCF for 15 min to eliminate cellular debris. The supernatant was collected and used to transduce a new 10 cm dish of HEK 293A cells. This process (grow, lyse, centrifuge, transduce a larger number of cells) was repeated until 20, 15-cm dishes, were incubated simultaneously. The cells and supernatant were collected and spun at low speed (200 RCF) for 2 min. The cells and 5 ml of supernatant were then subjected to 3 freeze-thaw cycles to lyse cells, followed by centrifugation at 1800 RCF for 15 min. A CsCl step gradient was set up with a lower layer of 4 ml of 1.4 g/ml CsCl and an upper layer of 3 ml of 1.2 g/ml CsCl. 5 ml of supernatant was over-layered and ultracentrifuged at 65,000 RCF for 90 min at 4° C. The 1.2 g/ml cesium chloride layer, containing virus, was extracted and dialyzed vs. 10 mM Tris/HCL at pH 8.0. Viral concentration was determined by OD260 assay.
Injection: Recombinant viruses were administered via tail vein injection and mice were sacrificed 3 days or 10 days p.t.
Real-Time qPCR: RNA was extracted with TRIZOL acid-phenol reagent (Invitrogen) and purified with on-column DNase digestion using RNEASY Mini Kit (Qiagen). RNA integrity was verified by visual inspection of ethidium bromide stained electrophoresis gels and by OD260 nm/OD280 nm>1.9 and OD260 nm/OD230 nm>2.0. First strand cDNA synthesis was performed using 1 μg of total RNA each and the Sprint RT Complete-Random Hexamer kit (Clontech) according to the manufacturer's instructions. Reverse transcription (RT) followed by PCR was used to analyze mRNA abundance in response to treatments. Primers for genes were designed to produce an amplification product which spanned at least one exon using the Universal Probe Library Assay Design Center (www.universalprobelibrary.com, Roche Applied Sciences); primers were synthesized by Invitrogen. qPCR analysis was performed on a LIGHTCYCLER 480 (Roche) using the LIGHTCYCLER 480 SYBR Green I Mastermix (Roche).
Primers for PCR: Primers used for the PCR amplification of full-length Ildr2-cDNA. Ildr2 Forward: caccATGGATAGGGTCGTGTTGGG (SEQ ID NO: 59). Ildr2 Reverse: TCAGACTACAAGGGACATCCTGGTTGGAAAGTCACC (SEQ ID NO: 60). The first TCA in the reverse is the stop codon. The ATG in the forward is the start codon. Primers used in expression analysis are shown in Table 10.







TABLE 10







Primers used for expression analysis. PCR primers used in experiments 


described in Table 1, FIG. 6, and FIG. 7.













SEQ 

SEQ 


Gene
Forward
ID NO:
Reverse
ID NO:














Ildr1
TCATTGTCCTGCATTGG
61
CAACAGCGGGTAGGACA
62



CTGA

GCA



 


Ildr2
ACAGGGCTCGACGGTT
63
ACACCCACTCCAACACC
64



AC

AGC



 


Ildr3
TCACCATCACAGGAAAT
65
GCTTCTGAGGTCCTGCC
66



GCTGAC

AAGG



 


Pepck
TGTCATCCGCAAGCTGA
67
TTCGATCCTGGCCACAT
68



AGA

CTC



 


Gck
TCCCTGTAAGGCACGAA
69
ATTGCCACCACATCCAT
70



GACAT

CTCA



 


Glut2
GGAACCTTGGCTTTCAC 
71
GGAACACCCAAAACATG
72



TGTCTT

TCGAT



 


Gpat
GGCTACGTCCGAGTGG
73
AACATCATTCGGTCTTGA 
74



ATTTT

AGGAA



 


Mgat1
CTGGTTCTGTTTCCCGT
75
GGTGAATGTTCCTGGGT
76



TGT

GAG



 


Dgat1
CCTCAGCCTTCTTCCAT
77
ACTGGGGCATCGTAGTT
78



GAG

GAG



 


Dgat2
TCCAGCTGGTGAAGACA
79
GATGCCTCCAGACATCA
80



CAC

GGT



 


FAS
ATCCTGGAACGAGAACA
81
AGAGACGTGTCACTCCT
82



CGATCT

GGACTT



 


ACC1
GGGCACAGACCGTGGT
83
CAGGATCAGCTGGGATA
84



AGTT

CTGAGT



 


Pparg2
TTCCACTATGGAGTTCA
85
TCCGGCAGTTAAGATCA
86



TGCTTGT

CACCTA



 


Srebp1c
CGGCGCGGAAGCTGT
87
TGCAATCCATGGCTCCGT
88


 


Srebp2
CTGCAGCCTCAAGTGCA
89
CAGTGTGCCATTGGCTG
90



AAG

TCT



 


Ldlr
TGGAGGATGAGAACCG
91
GCACTGAAAATGGCTTC
92



GCT

GTTTA



 


Apob
TCACCCCCGGGATCAAG
93
TCCAAGGACACAGAGGG 
94





CTTT



 


Ppara
CCTCAGGGTACCACTAC
95
GCCGAATAGTTCGCCGAA
96



GGAGT





 


Cptla
CCTGGGCATGATTGCAA
97
GGACGCCACTCACGATG
98



AG

TT



 


Acox1
CGATCCAGACTTCCAAC
99
CCATGGTGGCACTCTTC
100



ATGAG

TTAACA



 


Mcad
TGCTTTTGATAGAACCA
101
CTTGGTGCTCCACTAGC
102



GACCTACAGT

AGCTT



 


Ucp2
GACCTCATCAAAGATAC
103
ATCTCGTCTTGACCACAT
104



TCTCCTGAA

CAACAG



 


Rxra
GGCAAACATGGGGCTG
105
GCTTGTCTGCTGCTTGA
106



AAC

CAGAT



 


Fxra
TGGGCTCCGAATCCTCT
107
TGGTCCTCAAATAAGATC
108



TAGA

CTTGG



 


Fxrb
GGGCTTAGAAAATCCAA
109
CGTCCGGCACAAATCCTG
110



TTCAGATTA





 


Perk
CCTTGGTTTCATCTAGC
111
ATCCAGGGAGGGGATGAT
112



CTCA





 


Atf6
GGACGAGGTGGTGTCA
113
GACAGCTCTTCGCTTTG
114



GAG

GAC



 


Ire1
TGAAACACCCCTTCTTC 
115
CCTCCTTTTCTATTCGGT
116



TGG

CACTT



 


Atf4
ATGATGGCTTGGCCAGT
117
CCATTTTCTCCAACATCC
118



G

AATC



 


Xbp1
TGACGAGGTTCCAGAG
119
TGCAGAGGTGCACATAG
120



GTG

TCTG



 


Chop
TCCCTGCCTTTCACCTT
121
GCCCTGGCTCCTCTGTC
122



G

A



 


Bip
CTGAGGCGTATTTGGGA
123
TCATGACATTCAGTCCA
124



AAG

GCAA









Construction of Tag Protein Fusions. N-terminal 3×FLAG Ildr2 fusion construct. The Ildr2 open reading frame from exon 2 was subcloned into p3×FLAG-CMV-8 (N-terminal FLAG with PPT LS, Sigma-Aldrich # E4151-20UG). Ildr2 was amplified using a forward primer on the sequence coding for the first amino acids of exon 2 with a HindIII site (5′ ATT TAC AAG CTT CAG GTC ACA GTG CCT GAC AAG AAG AAG GT3′ (SEQ ID NO 125), and a reverse primer with an in-frame stop codon and EcoR1 restriction site at the end of Ildr2 last exon (5′-CAT GCA GAA TTC TCA GAC TAC AAG GGA CAT CCT G-3′ SEQ ID NO: 126). The destination vector and the PCR amplified Ildr2 sequence were digested with HindIII and EcoR1 (NEBiolabs) in NEBuffer EcoR1 and BSA at 37° C. for 60 min, purified and ligated.
C-terminal-tagged ILDR2 mYFP construct. The ildr2 open reading frame from exon 1 was subcloned into pmEYFP-N1 (Clontech #6006-1). Ildr2 was amplified using a forward primer on exon 1-including Kozak sequence—with a site for the restriction enzyme NheI (5′-ATC TTG CTA GCG GTA ATG GAT AGG GTC GTG TTG G-3′ SEQ ID NO: 127), and a reverse primer that bypass the stop codon and an EcoRI restriction site (5′-CAT GCA GAA TTC GGA CTA CAA GGG ACA TCC TG-3′ SEQ ID NO: 128). The destination vector and the PCR-amplified Ildr2 sequence were digested with Nhe and EcoRI (NEBiolabs) in NEBuffer EcoRI+BSA at 37° C. for 60 min, purified and ligated.
Isolation of Hepatocytes: Hepatocytes were pooled from 5, 10-week-old C57BL/6J mice. The mice were anaesthetized with cocktail containing ketamine (100 mg/kg) and xylazine (20 mg/kg) and then laparotomized to expose the liver and the portal vein. A 25G winged needle attached to a 50 ml syringe filled with 37° C. EGTA-Hanks solution (Hanks Balanced Salt Solution, Gibco; EGTA final concentration 0.5 mM) was inserted into the portal vein and clipped in place with a clamp at the root of the mesentery and the needle. After cutting the inferior vena cava, the liver was perfused with 30 ml EGTA-Hanks Using a fresh syringe, the liver was then perfused with 20 ml of a solution containing collagenase (5 mM CaCl2 in Hanks with 1 mg/ml of collagenase type II Gibco #17101-015) being careful not to inject bubbles. Liver was excised and placed in a 10 cm sterile dish on ice with 2-3 ml of collagenase solution and minced with scissors to remove visible blood clots. Minced livers were pooled and incubated for 5-10 min at 37° C. and homogenized by pipetting up and down 20-30 times. Then 20 ml of RT Hanks balanced salt solution was added to the dish and mixed. The suspension was filtered through sterile gauze into a 50 ml conical tube, spun 1 min at 200 RCF at RT and aspirated to remove supernatant. Cells were resuspended in 20 ml Gibco HG medium (with 10% FBS, 1% Penicillin Streptomycin, 10 nm DEX, 100 nm insulin, and 0.1% Fungizone) and pipetted up and down 5 times. The suspension was filtered through a 100 um Nylon cell strainer (BD Falcon REF352360) and collected in 50 ml conical tube. The filtrate was centrifuged again at 200 RCF for 1 min at RT and aspirated to remove the supernatant. Cells were resuspended in 25 ml of Gibco HG medium and gently pipetted. Cells were counted with an Invitrogen Countess using trypan blue staining and then distributed at 3×106 cells per 10 cm plate and incubated overnight at 37° C. in a humidified 5% CO2 incubator before administering virus.
Cell Line Studies: Cell microscopy, image acquisition: Cell cultures were prepared and maintained according to standard cell culture procedures. Hepa1c1c7 and GT1-7 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum using BD Falcon T75 cell culture flasks. For transient transfection, cells were harvested by Trypsin/EDTA digestion, seeded on coverslips (1×105 per coverslip) and incubated for 24 h in a cell culture incubator at 37° C. and 5% CO2. Transfection of plasmid DNA for GFP-tagged ILDR2 was performed with LIPOFECTAMINE 2000 according to the manufacturer's instructions. In brief, cells were incubated with 500 ng plasmid DNA and 1.25 μl LIPOFECTAMINE 2000 in OptiMEM (24-well plate format) over night before being analyzed. Transfection was up-scaled accordingly if other plate formats were used.
Confocal images: Transduced cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature (rt) for 15 min, washed again with PBS, permeabilized with 0.1% Triton-X 100 (2 min at rt) if necessary and incubated with blocking buffer (5% normal goat serum and 0.05% Tween 20 in PBS) for 30 min. For immunostaining, cells were incubated overnight at 4° C. with the corresponding specific antibody diluted in blocking buffer, washed with PBS, incubated for 1 hr at rt with 2 μg/ml Alexa Fluor 546 goat anti-mouse secondary antibody diluted in blocking buffer washed with PBS and mounted in ProLong Gold antifade reagent with DAPI as a nuclear staining marker. Samples were analyzed with a Confocal Laser Scanning Microscope (TCS SP2, Leica, Germany). When fluorescent proteins were used, GFP was excited at 488 nm and fluorescence was detected at 500-540 nm. YFP was excited at 514 nm and fluorescence was detected at 520-560 nm. DsRed was excited at 557 nm and fluorescence was detected at 592 nm. Alexa Fluor 546 was excited with the 543 nm line of the helium laser and fluorescence was detected at 555-700 nm. Cells were imaged with a 63.0×/1.25 HCX PL APO objective lens. Images were processed with Adobe Photoshop software. Images shown are stacks of several confocal sections.
siRNA reverse transfection: RNA interference-mediated gene knockdown was achieved using custom Stealth RNAiTM siRNA designed using the BLOCK-iTTM RNAi Designer software (Invitrogen), and its correspondent controls. 10 nM RNAi duplex was diluted in OptiMEM® I Reduced Serum Medium (Invitrogen) and 5 ul of LIPOFECTAMINE RNAiMAX (Invitrogen) was added to each well containing the diluted RNAi. After mixing, the complexes were incubated for 20 min at room temperature. After incubation, approximately 0.15-0.25×106 cells per well in complete growth medium without antibiotics were added to the wells containing the siRNA-lipid complexes. Cells were then cultured in normal growth medium processed for RNA or protein extraction as indicated by experimental procedure.
Western blotting: Cells were lysed in M-PER Mammalian Protein Extraction reagent (Thermo Scientific) with protease (Calbiochem) and phosphatase inhibitor (Thermo Scientific). Equivalent amounts of protein (100 to 400 μg) from each sample were diluted in 1×LDS sample buffer (Invitrogen) containing 100 mM DTT and incubated for 10 min at 70° C. After denaturing, the mixture was cooled at rt for at least 15 min and the protein was resolved by SDS-PAGE. The gel was transferred onto polyvinylidene fluoride (PVDF) membrane and blocked by incubation for 1 hr at rt in a solution 5% bovine serum albumin fraction V (BSA) in Tris-buffered saline with Tween (TBST) at pH 7.4, followed by overnight incubation at 4° C. with primary antibody in 5% BSA/TBST. Following 3 15-min washes with TBST, membranes were incubated for 1 hr at rt with the corresponding secondary antibody and washed 3 more times in TBST. Membranes were developed using a chemiluminescence assay system and proteins were visualized using Kodak exposure film. Membranes were stripped using RestoreTM PLUS Western blot stripping buffer (Thermo Scientific) with vigorous agitation for 10 min at rt, followed by 3 TBST washes.
Statistical Analysis: Two tailed T tests were performed using Microsoft Excel.
Example 2
Generation of ILDR2 Conditional Knockout Mouse
A ILDR2 conditional knock-out mouse was generated that was designed to delete exon 1 of Ildr2. A neo cassette was inserted 1.6 kb upstream of exon 1 of Ildr2. The addition of the neo cassette alone does not affect expression of ILDR2, this neo gene is in the opposite orientation to the Ildr2 gene (FIG. 48). This mouse was mated with an albumin ere mouse that expresses ere specifically in the liver. The addition of the ere excises the region between the two loxP sites; this removes the neo cassette and the first exon of Ildr2, knocking it out in the tissues where the ere is expressed. Quantification of Ildr2 expression in this mouse by qPCR, shows it has no Ildr2 expression in the liver.
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Example 3
Effects of ER Stress on ILDR2
Mice (WT) were transduced with adenoviral expression vectors encoding shRNA (“ADKD”). Ten day ADKD mice had hepatic steatosis and increased circulating plasma lipids. In analysis of plasma lipoprotein fractions, VLDL-TC and VLDL-TG were higher in the ADKD mice. This suggests that ILDR2 affects a change in the TG and TC content of VLDL particles.
ILDR2 binds to apoE. KYLYYVE (SEQ ID NO: 149), encoded by ILDR2 exon 7, is a critical binding site of apoE. The effects of deletion of the apoE binding sequence in mouse primary hepatocytes was investigated. A KYLYYVE (SEQ ID NO: 149) deletion adenoviral construct was used. This construct contains the sequence of ILDR2 with the sequence encoding KYLYYVE (SEQ ID NO: 149) deleted from exon 7. ILDR2 overexpression significantly decreased TG content in primary mouse hepatocytes consistent with in vivo findings. However, overexpression of the KVLYYVE-deleted allele (“KVLYYVE” disclosed as SEQ ID NO: 150) did not markedly decrease TG content. This suggests that ILDR2 has a modulating role in the VLDL secretion cascade and may have a function in the assembly of VLDL mediated by apoE.
ER stress decreases ILDR2 expression and reduced ILDR2 levels may contribute to ER stress-dependent hepatic steatosis. Hepatic VLDLR expression is increased in response to ER stress. Increased VLDLR facilitates uptake of VLDL in liver and hepatic lipids increase. VLDLR acts as a receptor for apoE-containing lipoproteins. ApoE is an important constituent of TG-rich lipoproteins and is essential for lipoprotein uptake at LDL and VLDL receptors. ApoE-deficient mice accumulate hepatic TG and decrease rates of VLDL-TG production compared with wild-type mice. Overexpression of apoE in the liver of apoE-deficient mice increases secretion of VLDL-TG. Increased apoE expression in rat hepatoma cells increases VLDL-TG secretion. ApoE may play a role in facilitating hepatic secretion of VLDL-TG and may have a function in the assembly and/or secretion of VLDL by the liver. ILDR2 may interact with apoE in these processes, with functional impairment when ILDR2 is insufficient.
ER stress causes an increase in VLDLR which increases uptake of VLDL and accumulation of hepatic lipids. At the same time, decreased ILDR2 by ER stress impairs VLDL assembly mediated by apoE and reduces VLDL secretion, causing hepatic lipid content to be markedly increased. Reduced apoE content of circulating VLDL could reduce the efficiency of hepatic VLDL uptake, leading to an equilibrium at higher circulating concentrations of TG and cholesterol. Deficiency in ILDR2 function may, therefore, impair VLDL export from the liver, increase VLDLR at the hepatocyte surface and thereby lead to cholesterol and TG accumulation in the liver. ER stress, by reducing levels of ILDR2, could promote this process (FIGS. 49A-E).
Interaction of ER Stress-Related Proteins with ILDR2
To assess whether ER stress-related proteins interact with ILDR2, co-immunoprecipitation was performed. The results show that ILDR2 strongly binds to PERK and Bip (FIG. 50). As shown in FIG. 50, PERK binds to ILDR2 and cleaves ILDR2. Cleaved ILDR2 releases two fragments. Full length ILDR2 and cleavage of ILDR2 were decreased by co-transfection with IRE1a. IRE1a, which controls the splicing of Xbp1 mRNA, may affect the degradation of ILDR2. ILDR2 binds to Bip, a major chaperone protein that protects cells from ER stress, suggesting that ILDR2 may play a role in ER stress maintenance
Interaction of PERK with ILDR2
To assess whether ILDR2 is cleaved by PERK, N-terminus tagged ILDR2 was constructed and co-transfected with PERK (FIGS. 51A-B). ILDR2 fragments were assessed by western blot. N-terminus-tagged ILDR2 was also decreased by PERK, but cleaved ILDR2 was not detected, suggesting that N-terminal ILDR2 was cleaved by PERK. The C-terminal cleaved ILDR2 fragment may be too small to be detected in this assay. PERK-K618A which is a dominant negative mutant vector did not cleave ILDR2.
IRE1a Degrades ILDR2 mRNA
When ILDR2 and IRE1 (an ER stress mediator) were co-transfected, ILDR2 protein levels were decreased (FIG. 52). To assess whether ILDR2 protein levels are decreased by IRE1a, 293 cells were co-transfected with ILDR2 vector and an IRE1a dominant-negative isoform. ILDR2 protein levels were decreased by IRE1a but not the K599A dominant negative. It ispossible that IRE1a degrades Ildr2 mRNA because IRE1a also splices Xbp1 mRNA. To assess whether IRE1a degrades Ildr2 mRNA, mRNA levels of Ildr2 were measured by real-time PCR (FIG. 53). Ildr2 mRNA levels were markedly decreased by IRE1a but not IRE1a K599A, suggesting that IRE1a degrades Ildr2 mRNA. To assess whether endogenous Ildr2 mRNA is degraded by IRE1a, Hepa1c1c cells were transfected with either IRE1a or K599A. Endogenous Ildr2 mRNA was also degraded by IRE1a in time dependent manner (FIG. 54).
ILDR2 Protein Levels were Decreased by Co-Transfection with IRE1a
IRE1a, which controls the splicing of Xbp1 mRNA, may affect with degradation of ILDR2. To assess whether IRE1a affects ILDR2 splicing and/or degradation, an Ildr2 mRNA degradation assay was performed. Both IRE1a-WT and IRE1a-K599A were expressed in 0 h. To assess Ildr2 mRNA degradation, Actinomycin D was added in Hepa1c1c7 cells. Xbp1 was spliced by IRE1a in time dependent and spliced Xbp1 increased. Ildr2 mRNA transcription was decreased by IRE1a but not dominant negative K599A (FIG. 55).
To assess whether ILDR2 expression levels were affected by tunicamycin which induces ER stress, Hepa1c1c cells were treated with tunicamycin. ILDR2 expression levels were decreased by ER stress. ER stress response proteins were increased (FIGS. 56A-B).
Effects of ATF6 and XBP1 on ILDR22 Expression
The ILDR2 promoter has an ER stress response element (ERSE)-like, suggesting that ATF6 and XBP1 may bind Ildr2 promoter and regulate transcription levels. To assess if ATF6 and XBP1 suppressed Ildr2 transcript, a luciferase assay was performed. Hepa1c1c cells were co-tranfected with the ILDR2 promoter and either ATF6 or XBP1. Both ATF6 and spliced XBP1 (sXBP1) decreased ILDR2 transcription activity in a dose dependent manner (FIG. 57)
To assess if ATF6 and XBP1 suppressed endogenous ILDR2 transcript levels, Hepa1c1c7 cells were transfected with either ATF6 or spliced XBP1 (sXBP1) expression vectors, When transfected with either ATF6 or sXBP1, ILDR2 mRNA levels decreased, demonstrating that ATF6 and XBP1 regulate Ildr2 transcript levels (FIG. 58).
The results described herein demonstrate that ILDR2 is suppressed by ER stress (FIG. 59). Firstly, ILDR2 binds PERK which cleaves ILDR2 to an inactive form. Secondly, transcription factors, ATF6 and XBP1 are increased by ER stress and downregulate ILDR22 transcripts by binding to its promoter region. In addition, IRE1a activated by ER stress degrades Ildr2 mRNA. Suppression of ILDR2 could be reduced by ER stress inhibitors, including, but not limited to TUDCA and 4PBA. Increasing levels of cellular ILDR2 could mitigate the effects of ER stress by offsetting the decreases imposed by ER
The in vivo effects of tunicamycin on liver ILDR2 (lowering), and the protective effect of overexpressing ILDR2 in liver exposed to tunicamycin were investigated. Tunicamycin (ER stress-inducer) administration (72 hrs) decreased hepatic Ildr2 expression by 45% and was associated with hepatic steatosis (tripling of TG, 50% increase in cholesterol) (FIGS. 60A-D). Conversely, over expression of Ildr2 in the livers of animals exposed to tunicamycin mitigated the steatotic effects (FIGS. 61A-D).

## Claims

1. A method of treating hepatic steatosis in a subject, the method comprising administering to the subject a therapeutically effective amount of an adenoviral expression vector comprising a nucleic acid encoding full-length ILDR2, and wherein the subject has metabolic syndrome, type II diabetes, insulin resistance, or is obese, or any combination thereof.

2. A method of decreasing hepatic triglyceride level or hepatic total cholesterol level in a subject, the method comprising administering to the subject a therapeutically effective amount of an adenoviral expression vector comprising a nucleic acid encoding full-length ILDR2.

3. The method of claim 2, wherein the hepatic triglyceride level or hepatic total cholesterol level is decreased relative to a hepatic triglyceride level or hepatic total cholesterol level in the subject before administrating the therapeutically effective amount of an adenoviral expression vector comprising a nucleic acid encoding full-length ILDR2.

4. The method of claim 1 or 2, wherein the expression of Ildr2 mRNA or ILDR2 protein is increased in liver tissue.

5. The method of claim 1 or 2, wherein the expression of Ildr2 mRNA or ILDR2 protein is increased in hepatocytes.

6. The method of claim 1 or 2, wherein the subject is administered an additional therapy.

7. The method of claim 6, wherein the additional therapy is a lipid lowering therapy.

8. The method of claim 7, wherein the lipid lowering therapy is a therapeutic lifestyle change, a HMG-CoA reductase inhibitor, niacin, a fibrate, a cholesterol absorption inhibitor, a MTP inhibitor, or any combination thereof.

9. The method of claim 1 or 2, wherein expression of ILDR2 protein in the subject is measured using an antibody.

10. The method of claim 9, wherein the antibody specifically binds to a peptide selected from the group consisting of SEQ ID NOs: 2-9, an ILDR2 protein, or an ILDR2 isoform.

11. The method of claim 1, wherein the subject has a reduced level of expression of Ildr2 mRNA or ILDR2 protein compared to the level of expression of Ildr2 mRNA or ILDR2 protein in a subject without hepatic steatosis.

12. The method of claim 9, wherein the level of expression is determined before administrating to the subject the therapeutically effective amount of an adenoviral expression vector comprising a nucleic acid encoding full-length ILDR2.

13. The method of claim 11, wherein the expression of ILDR2 protein in the subject is measured using an antibody.

14. The method of claim 13, wherein the antibody specifically binds to a peptide selected from the group consisting of SEQ ID NOs: 2-9, an ILDR2 protein, or an ILDR2 isoform.

15. A method of decreasing liver fat deposits in a subject, the method comprising administering to the subject a therapeutically effective amount of an adenoviral expression vector comprising a nucleic acid encoding full-length ILDR2.