bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of myristoylated methionine sulfoxide reductase A in the mouse protects the heart against ischemia‐reperfusion injury
Authors: Zhao Hang , Sun Junhui , Deschamps Anne M. , Kim Geumsoo , Murphy Elizabeth , Levine Rodney L.
Published: June 2021

Methionine sulfoxide reductase A (MsrA) has been reported to function as an antioxidant protein, either by scavenging reactive oxygen species via cyclic interconversion of the reduced and oxidized forms of methionine residues or by protecting critical protein residues from oxidation. However, the mechanisms and requirements for providing an effective oxidative defense in vivo are not known. In mammalian cells, ~75% of MsrA is located in the cytosol and is myristoylated while ~25% is in the mitochondria and not myristoylated. We created several strains of transgenic mice overexpressing MsrA directed to the cytosol or mitochondria or both. Quantitative Western blotting and activity measurements demonstrated a high level of functional MsrA in most tissues tested, with the highest in the heart. We tested whether the increased MsrA in heart protected against ischemia‐reperfusion injury in a Langendorff model. We found that MsrA overexpression in the cytosol but not in the mitochondria protected. We then examined whether myristoylation affected this protection. We created another transgenic mouse overexpressing MsrA only in the cytosol, but in this strain glycine at position 2 was mutated to alanine to prevent myristoylation. Analysis of heart tissue confirmed that the overexpressed MsrA was active but not myristoylated. However, this non‐myristoylated MsrA heart experienced no protection. These experimental results indicate that myristoylation of cytosolic MsrA is key to its ability to provide cardioprotection.

This research was supported by the Intramural Research Program of the NIH, NHLBI.
πGST increases peroxidase activity of Prdx6 in cultured cells
Authors: Zhou Suiping , Lien Yu‐Chin , Shuvaeva Tea , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Prdx6 is a non‐selenium GSH peroxidase that has an important role in ant‐oxidant defense. Previously, we have shown using in vitro assay and by liposome‐mediated delivery of the two proteins into the cells, that the regeneration of Prdx6's peroxidase activity requires πGST. Recently, we have shown that transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, increases the lipid peroxidase activity in the cells, presumably due to activation of endogenous Prdx6. Using the Duolink Proximity Ligation Assay we have shown that there is a dramatic increase in interaction between endogenous πGST and Prdx6 in mouse pulmonary microvascular endothelial cells upon their treatment with tert‐butyl hydroperoxide. Now, we have co‐transfected plasmids expressing πGST and Prdx6 into MCF7 cells. An increase in lipid peroxidase activity was obtained by transfecting Prdx6 and activity is significantly enhanced by co‐transfection with πGST. The functional interaction of Prdx6 with exogenous transfected πGST in MCF7 cells was also revealed by Duolink Proximity Ligation Assay. These cell culture studies are consistent with our previous finding that πGST is involved in regenerating the peroxidase activity of Prdx6.
Selective modulation of the redox state and thus the repressor function of SLUG by peroxiredoxin 5 at the BRCA2 gene silencer in human breast cancer cells
Authors: MISRA SMITA , MITTAL MUKUL K , CHAUDHURI GAUTAM
Published: June 2021

Human BRCA2 gene expression is inhibited by the transcriptional repressor protein SLUG at the G0/G1 phase of SLUG‐high breast cancer cells through the binding of SLUG to an E2‐box sequence in the BRCA2 gene. Interestingly, while SLUG maintains its repression of several of its target genes at the S/G2 phase of cell growth, it fails to repress BRCA2 gene at this growth phase. We discovered that, unlike other SLUG target gene promoters, BRCA2 gene silencer has the SLUG‐binding E2‐box at the close proximity of the binding site of the redox modulator protein peroxiredoxin 5 (PRDX5). Here, we provide evidence that in the S/G2 phase of cell growth, oxidized PRDX5 enters the nucleus at a higher rate and binds to BRCA2 silencer to rip off SLUG from its E2‐box through oxidation of its DNA binding zinc finger domains resulting in the de‐silencing of BRCA2 gene expression. This study presents a novel modality of PRDX5 in site‐specific oxidation of zinc‐finger transcription factors to regulate gene expression in a redox‐dependent manner. Supported by DOD‐CDMRP BCRP Grants W81XWH‐06‐1‐0466, W81XWH‐08‐1‐0446, BC086542, and Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC and 1U54RR026140‐01to SM.
Partial Purification and Characterization of Biliverdin Reductase from Mammalian Organs
Authors: Scavuzzo Raghela Marie , Fyalka Robert , Cartee William
Published: June 2021

The enzyme Biliverdin Reductase is utilized to aid in the breakdown of heme into bilirubin through the reduction of biliverdin. This is observed in both the yellow pigment in bruising and the jaundice coloring when liver failure occurs due to neurotoxicity. Prior studies have been restricted to rat and Guinea Pig organs. Throughout this experiment we examined enzyme activity of this enzyme in the bovine liver, kidney, spleen and brain then compared activity to that in mammalian species including porcine, bison, and rats. With a standard assay, the enzyme activity throughout the purification process was measured by observing the optimum of product (bilirubin) at 450 nm. The experimentation done on the fresh bovine liver was used as the standard for comparison due to previous experimentation done in this lab in the spring of 2010. Rats biliverdin activity was used to ensure results were comparable to previously designed experiments such as Kutty and Maines (J. Biol. Chem., 256, 3956–3963, 1981). This partial purification and comparative analysis allowed for a further understanding of the Biliverdin Reducatase throughout mammalians and its importance within various organs.
Protein kinase‐D1 overexpression in mice prevents lipid‐induced insulin resistance and cardiomyopathy by upregulation of glucose uptake
Authors: Dirkx Ellen , Schwenk Robert W. , Steinbusch Laura KM , Hoebers Nicole , Janssen Ben J , Brans Boudewijn , Basel‐Duby Rhonda , Olson Eric N , Bonen Arend , van Eys Guillaume JJM , Glatz Jan FC , Luiken Joost JFP
Published: June 2021

Disturbances in metabolic substrate uptake promote the development of cardiomyopathy and eventually lead to heart failure. Understanding the pathways that control substrate uptake could enable therapeutic approaches to prevent maladaptive cardiac remodeling.

We identified protein kinase‐D1 (PKD1) as a contraction‐activated kinase, which promotes GLUT4‐mediated glucose uptake, without affecting long chain fatty acids (LCFA) uptake. This corroborated with in‐vitro experimentation using PKD1 siRNA in HL‐1 cells and in‐vivo by microPET scan results showing a higher glucose uptake in the hearts of mice overexpressing constitutively active PKD1 (caPKD1). The increased glucose uptake in the heart of caPKD1 mice results in dilated cardiac hypertrophy and heart failure. In contrast, mice on a high fat diet with lipid‐overloaded hearts displayed concentric cardiac hypertrophy, accompanied by insulin resistance. However, hearts of caPKD1 mice on a high fat diet displayed neither dilated nor concentric hypertrophy. Further, these hearts did not show increased glucose uptake nor increased LCFA uptake, demonstrating the role of PKD1 to balance cardiac substrate utilization, which is crucial in preventing pathological cardiomyopathy. In conclusion, in the lipid‐overloaded heart, upregulation of PKD1 offers protection against the development of insulin resistance and hypertrophic remodeling.

Grant Funding Source: NWO (Dutch Organisation for scientific research)
Phosphoinositide‐3‐kinase pathway stringently controls cellular status of pyruvate kinase M2 to determine cancer metabolism
Authors: Iqbal Mohd Askandar , Bamezai Rameshwar NK
Published: June 2021

Growth factors signaling (GF) play a vital role in promoting anabolism in cancer cells, allowing them to grow and divide efficiently. However, it is not clear how GF contributes to this metabolic transformation. In this study we are reporting a mechanism that allows GF signaling to promote anabolism. As it has been shown recently that expression of pyruvate kinase M2 (PKM2) is central to cancer metabolism, we investigated the effect of inhibiting major GF transducers, Phosphoinositide 3‐kinase pathway (PI3K), Mammalian target of rapamycin (mTOR) and Mitogen activated protein kinase (MAPK) on cellular status of key enzyme PKM2. We observed down regulation of PKM2 expression and activity on mTOR and MAPK inhibition. Surprisingly, on PI3K inhibition ~25% increase in PKM2 activity was observed despite reduced expression of PKM2. The peculiar effect of PI3K inhibition on PKM2 activity was due to shift in crucial PKM2 Dimer/Tetramer (D/T) equilibrium in favor of highly active tetramer. We further investigated the implication of shifted D/T equilibrium on metabolism by LC‐MS, which interestingly showed substantial reduction in intracellular levels of anabolic markers, ribose‐5‐phosphate and lactate, indicating the suppression of cancer metabolism. The study depicts the unique potential of PI3K pathway in affecting cancer metabolism via PKM2 oligomeric state, hence widening the window of therapeutic interventions in cancer. Moreover, our results correlate with the recent reports that PI3K pathway is the most frequently mutated/dysregulated pathway in cancer. We acknowledge UGC, Govt. of India, for research funds.
Differential Expression of Cholecystokinin Receptors and Their Ligands in Normal and Neoplastic Pancreas
Authors: Ortega Emily E , Smith Jill P.
Published: June 2021

Background

The gastrointestinal peptide gastrin has been identified in fetal pancreas where it is thought to play a role in growth and differentiation. Gastrin has also been identified as a growth factor that stimulates pancreatic cancer through the CCK‐B and CCK‐C receptors by an autocrine mechanism.

Hypothesis

It is hypothesized that gastrin re‐expression is important to pancreatic carcinogenesis.

Methods

Endpoint RT‐PCR was performed on normal human pancreas mRNA to detect RNA levels of gastrin and CCK peptide. PCR products were visualized on an ethidium bromide stained gel after electrophoresis. Normal human pancreatic tissue samples were evaluated for gastrin and CCK by immunohistochemistry.

Results

The end‐point RT‐PCR showed significant levels of CCK‐B receptor and CCK peptide in normal pancreas samples. The gastrin end‐point RT‐PCR showed low levels in all of the normal pancreas samples. Gastrin immunoreactivity was not detected in normal pancreas, however, staining of the positive control was reactive; the negative control showed no staining.

Conclusions

Normal pancreas possesses the receptor CCK‐B and the peptide CCK. Gastrin is not present in normal pancreatic tissues, but is detected in neoplastic pancreatic samples. Therefore, the re‐expression of gastrin plays a pivotal role in the development of pancreatic cancer and could be a potential trigger to pancreatic carcinogenesis.
Leucine metabolism as a novel approach to improve T cell performance in managing cancer
Authors: Ananieva Elitsa A , Powell Jonathan D , Alme Angela , Patel Chirag , Hutson Susan M
Published: June 2021

T cells can recognize and eliminate tumors in the same way as they recognize and destroy infectious agents. There are a number of mechanisms by which tumors may evade or silence the immune response. Depriving T cells of amino acids mitigates T cell function and allows tumors to evade the immune response. We studied the impact of the branched‐chain amino acid, leucine, and its degrading enzyme, the cytosolic branched‐chain aminotransferase (BCATc) on T cell activation as a novel approach to improve T cell performance in managing cancer. Our results showed that leucine was required for T cell activation and proliferation and its depletion promoted T cell tolerance. BCATc transcripts were up‐regulated in tolerant T cells that are unable to provide a productive immune response. We observed a 15‐ fold increase in BCATc protein levels, a 13‐fold increase in BCATc activity and 6‐fold increase in leucine transamination in tolerant T cells. Moreover, BCATc was up‐regulated in prostate cancer‐infiltrating regulatory T cells known to suppress the anti‐tumor immune response. Our results indicate that leucine is a critical factor for T cell activation while BCATc is a new candidate for an immunosuppressive enzyme that may represent a novel means by which tumors evade the immune response. In as much as inhibitors of BCATc are available, our study may provide novel targets for cancer vaccines. Research support, NIH/NIDDK (R01DK081563).
Mast cell maturation accompanies neovascularization during skin tumor progression
Authors: Junior Devandir Antonio Souza , Oliver Constance , Jamur Maria Celia
Published: June 2021

In spite of evidence indicating an association between mast cells, tumor progression andangiogenesis, the role of these cells in neo‐vascularization and tumor progression remains obscure. This study aims to understand the involvement of mast cells in these processes. Tumors were induced by topical application of DMBA and TPA on BALB/c mice. Tumors were processed for routine histology, flow cytometry or biochemical analysis. Tumor progression was divided into phases I, II and III. At phase I tumors exhibited a large number of mast cells, which increased in phase II, but did not increase further. The expression of secretory granule components (mMCP‐4, mMCP‐5, mMCP‐6, mMCP‐7, carboxipeptidase A and histamine) and their activity was also altered during tumor progression. The activity of tryptase and chymase increased primarily in phases II and III, when there is no increase in the number of mast cells associated the tumor. An increase in new blood vessels, particularly in phase I, was observed. Whereas, in phases II and III there was no significant increase in new blood vessels, there was an enlargement of existing blood vessels. The pattern of expression of granule specific components even when there is no increase the number of the mast cells, suggests that mast cells found in tumors are in process of maturation mainly at phases II and III and this maturation correlates with neovascularization during skin tumor progression.
Dynamics of human hepatic cells metabolome during dengue virus infection
Authors: El‐Bacha Tatiana , Galina Antonio , Almeida Fabio C , Da Poian Andrea T
Published: June 2021

The metabolic resources crucial for viral replication are provided by host cells. The mechanisms by which viruses interact with cell metabolism, driving biosynthetic molecules for their own replication, however, are unknown. We employed nuclear magnetic resonance to study metabolic fluxes and identify metabolites in dengue virus (DenV) infection of human hepatic cells since liver dysfunction plays a central role in DenV pathogenesis. Metabolomics were done at 24h of infection. 13C spectra were acquired after cell incubation with D‐[U‐13C]glucose. Results showed that glycolytic (fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, dihydroxyacetone phosphate and glyceraldehyde‐3‐phosphate) and pentose phosphate pathway (PPP; 6‐phosphogluconate) intracellular intermediates were decreased in DenV infection and so was lactate accumulation in culture medium. Intracellular L‐glutamate and L‐alanine were increased upon DenV infection. The decrease in glycolytic and PPP intermediates indicates an enhancement of glucose utilization for biosynthetic purposes; Increased contents of L‐aminoacids indicates decreased oxidative metabolism suggesting impaired mitochondrial function. The use of metabolomics to evaluate carbon fluxes represents a key step towards the elucidation of the dynamics of DenV‐human hepatic cells metabolic interactions.

Supported by: CAPES, CNPq and FAPERJ.
MURINE MODEL FOR N‐ACETYLGLUTAMATE SYNTHASE (NAGS) DEFICIENCY
Authors: Mirzozoda Khulkar , Cabrera‐Luque Juan , Caldovic Ljubica , Morizono Hiroki , Tuchman Mendel
Published: June 2021

Ammonia is channeled into the urea cycle by means of carbamylphosphate synthetase (CPS1). This initial step of urea synthesis takes place in the mitochondria of the liver cell and requires N‐acetylglutamate (NAG). NAG is an essential cofactor and activator of CPS1 which is formed from AcCoA and glutamate by the enzyme N‐acetylglutamate synthase (NAGS). The lack of NAGS enzyme or its activity leads to severe neonatal hyperammonemia due to the inability of CPS1 to synthesize CP in the absence of NAG. NAGS deficiency is one of the more rare urea cycle disorders. In patients with NAGS deficiency, treatment with N‐carbamylglutamate (NCG) fully restores ureagenesis by activating CPS1. We have recently created a novel mouse model of NAGS deficiency using the VelociGene gene ablation method via the NIH‐funded KOMP resource. Following heterozygous mating, we have attempted to rescue homozygous NAGS deficient mice by providing NCG to the pregnant females in drinking water during pregnancy extending the life of homozygous pups for a period of 60 to 120 hours after birth, while untreated mice died within 24 hours after birth. We have subsequently been able to extend the lifespan of a homozygous NAGSko female for 12 days while a homozygous NAGSko male has survived pass weaning age using a combination of NCG and citrulline. This model recapitulates clinical observations in human and seems to be suitable model of the human disorder.
The Drosophila Estrogen‐Related Receptor Coordinates Carbohydrate Metabolism with Developmental Growth
Authors: Tennessen Jason M , Baker Keith D , Lam Geanette , Evans Janelle , Thummel Carl S
Published: June 2021

Drosophila larval metabolism is tuned to promote exponential growth in response to environmental nutrients. We have discovered that the metabolic program required for larval growth is established by the Drosophila Estrogen‐Related Receptor (dERR), the sole Drosophila ortholog of the vertebrate ERR nuclear receptor subclass. dERR null mutants die as second instar larvae with abnormally low ATP levels, diminished triglyceride stores, and elevated levels of circulating sugars. These defects can be attributed to reduced expression of key genes in several metabolic pathways, including all of the genes that act in glycolysis. dERR appears to directly regulate of these pathways, as there are predicted dERR binding sites in nearly all of the misregulated genes examined. Intriguingly, the metabolic pathways induced by dERR at the onset of larval development are similar to those involved in the Warburg effect, by which cancer cells use glucose to support biomass production and proliferation. Our results demonstrate that the Warburg effect can be utilized in the context of normal developmental growth, indicate that dERR establishes the metabolic state that supports larval development, and implicate the ERR family as key regulators of the metabolic parameters that sustain cell proliferation and cancer progression. This work was supported by the NIH (1R01DK075607). JMT was supported by the NIDDK (F32DK083864).
Cloning and expression of loop 1 and loop 2 adiponectin receptor one mutants
Authors: Ose Allison Nicole , Jones Dana , Parker Chasta L
Published: June 2021

Adiponectin (Acrp 30) is a 30 kDa protein hormone that is released into the blood stream by adipocytes and is partly responsible for controlling fatty acid oxidation and glucose metabolism. Low levels of Acrp 30 can lead to the onset of obesity associated diseases such as type 2 diabetes. Adiponectin binds to a seven transmembrane receptor, Adiponectin Receptor 1 (Adipo R1) to influence cellular energy levels. Understanding how Acrp 30 binds to and influences cellular energy levels may lead to new targets for the treatment of obesity and diabetes. To begin this determination, the importance of loop 1 and loop 2 of AdipoR1 in the physiologically relevant binding of Acrp 30 will be assessed. Loop 1 and 2 mutants were produced using Overlap Extension PCR (OEPCR). The mutated sequence was based on the corresponding region within the protein PAQR 4. This protein is a member of the same family as AdipoR1, yet it does not bind Acrp 30. Once verified by sequencing, expression of the mutant proteins and Acrp 30 downstream signaling will be analyzed to determine if either loop region is critical in protein stability and/or downstream signaling events. If either region is determined to play a role in binding by analysis of downstream signaling, future researchers could develop new medical treatments that can be used to reduce not only the risks but also the incidence of obesity. Currently, Loop 1 and 2 of Adipo R1 have been produced and are undergoing sequence analysis to ensure that the desired mutation is in place. This work is supported by the Henson School of Science and Technology and Salisbury University.
Structural and sequence analyses of an infrared fluorescent tissue marker
Authors: Baker Anna W. , Salgado Daniel , Kennis John T. M. , Stojkovic Emina A.
Published: June 2021

Within the mammalian body infrared (IR) radiation is not scattered by tissues and fluids as much as visible light, making an IR fluorescent marker ideal for imaging applications in mammals. Recently, bacteriophytochromes (Bphs), red‐light sensing proteins from different bacterial strains, have been shown to contain significant levels of IR fluorescence. A Bph from Deinococcus radiodurans, after extensive mutagenesis, has been successfully expressed as IR fluorescent tissue marker in mice. Bph response to light depends on an organic cofactor, biliverdin (BV), which is abundant in the mammalian body. BV, upon the absorption of a photon, induces conformational changes in the surrounding Bph allowing it to reversibly photoconvert between spectrally distinct Pr (red light absorbing) and Pfr (far red light absorbing) states. Bph from Rhodopseudomonas palustris, RpBphP3 (P3), is unique among the Bphs in that its ground state is Pr and its excited state is Pnr (near red light absorbing). P3 and its mutant variants have been shown to emit significant IR fluorescence due to P3's unusual photoconversion properties. A single mutation D216A or Y272F in the BV binding region or C‐terminal truncation of the photosensory module of P3 increases IR fluorescence over the wild‐type protein. Using site‐directed mutagenesis we created mutant variants of P3 that contain combination of D216A or Y272F mutation with C‐terminal truncation of the photosensory module. We report that truncated P3 with D216A mutation displays higher levels of near IR fluorescence relative to wild type P3. Expression and visualization of IR fluorescent P3 mutants in mammalian cell lines followed by in vivo expression in mice are the subsequent goals of this research.

Supported by NIH EARDA pilot grant 5G11HD049644 – 04 to E.A.S..
Adhesion of lysozyme, albumin and transferrin to two types of FDA Group II contact lenses
Authors: Solanki Darshan , Chandrasekaran N. , Chodhry R. , Cuprillnilson S. , Desai S. , Liberman B. , Lee V.S. , Lin Y.J. , Nguyen K. , Roughi L. , Shah S. , Sharma V. , Thomas P. , Janoff A. , Keith E.O.
Published: June 2021

Tears contain ~60 different proteins that adhere to contact lenses, causing lens deterioration and ocular pathology. We examined the adhesion of three tear proteins to two different types of FDA Group II contact lenses (hilafilcon and omafilcon). Lenses were incubated in 2.0 mg/ml solutions of human lysozyme, albumin and transferrin for 1–4 days. Protein adhesion was determined by bicinchoninic acid assay. Lysozyme adhered to hilafilcon lenses in an up‐down pattern, with a maximum on day 3. Lysozyme adhesion to omafilcon lenses was high after 1 day and remained high on day 4. Albumin adesion to both types of lenses was high after 1 day, declined, and increased on day 4. Transferrin adhesion to both lenses was initally low, increasing to a maximum on day 3 and declining on day 4. These results are due to differences in lens material and tear protein structure. Hilafilcon, more negatively charged than Omafilcon, absorbed more lysozyme (+ charged at physiological pH). Omafilcon lenses are coated with phosphorylcholine, reducing adhesion by the hydrophobic domains of albumin. Human apo‐transferrin has few positive charges and thus bound more reluctantly to both materials.

Supported by the Farquhar College of Arts and Sciences and the Health Professions Division, NSU.
Structure‐function relationships of pigment epithelium‐derived factor receptor (PEDF‐R): Identification of a PEDF binding region
Authors: Subramanian Preeti , Locatelli‐Hoops Silvia , Notari Luigi , Becerra S Patricia
Published: June 2021

PEDF‐R/ATGL is found in the retina and has high affinity for pigment epithelium‐derived factor (PEDF), a retina cell survival protein. PEDF‐R exhibits phospholipase A2 (PLA) activity, which is stimulated by PEDF. We aim at identifying the structural determinant for PEDF binding in PEDF‐R that relates to retina survival. Recombinant C‐terminal truncated PEDF‐R (M1‐L232) retained PLA activity, which increased with PEDF additions, as did full‐length PEDF‐R (M1‐L504). However, a single D166A alteration in PEDF‐R caused loss of PLA activity. Surface plasmon resonance (SPR) revealed that recombinant PEDF‐R, PEDF‐R[D166A] and L4 (L159‐M325) had similar PEDF affinity. Ligand blot, SPR and affinity chromatography demonstrated that synthetic peptides E5b (I193–L232), P1 (T210‐L249) and E5d (T210‐L232) specifically bound PEDF. Their effect on PEDF‐mediated survival activity was examined in retina R28 cells. PEDF‐R protein was detected in R28 membrane fractions and its PLA activity was stimulated by PEDF additions. PEDF mediated R28 cell survival, which was attenuated by E5b or P1 additions. Thus, the C‐terminal half of PEDF‐R is dispensable for PLA activity and PEDF binding. D166 is crucial for PLA activity. E5b contains structural determinants for binding PEDF. This novel site may form part of the heterodimer interface of PEDF‐R with the ligand, which is required for retina cell survival. Supported by NIH‐IRP.
Characterization of the Phytophthora sojae hypotaurocyamine kinase suggests early evolution of quaternary structure in the phosphagen kinase family
Authors: Van Houten Jason Michael , Palmer Allyson , Herring Kelsie , Snider Mark J , Fraga Dean
Published: June 2021

We investigated the substrate specificity and kinetic constants of a Phytophthora sojae hypotaurocyamine kinase (HTK) to better understand the evolution of the phosphagen kinase (PK) protein family. The substrate specificity and kinetic constants were determined using Isothermal Titration Calorimetry by titrating arginine, creatine, glycocyamine, or taurocyamine at varying concentrations in to a solution containing the HTK and MgATP. The protein had strong reactivity with taurocyamine and weak reactivity with glycocyamine, while it showed no reactivity with either arginine or creatine. The Km for taurocyamine and MgATP were found to be 0.59mM and 3.78mM respectively. The kcat was found to be 34.66s−1. Based on our phylogenetic analysis and the ability for the P. sojae HTK to react with glycocyamine, we hypothesized that the dimeric PKs evolved from an ancestral kinase that may have had some promiscuity in substrate specificity. Using protein sequences of known dimeric PKs, we tested this hypothesis by constructing ancestral sequences using the web‐based program ANCESCON. This project was made possible by the support of the Howard Hughes Medical Institute and the Henry J. Copeland Fund.
Pheromone Binding Protein: Structural & Mechanistic Insight into insect olfaction
Authors: Mazumder Suman , Katre Uma V , Mohanty Smita
Published: June 2021

In moths, pheromone binding proteins (PBPs) transport the hydrophobic pheromone molecules to the membrane‐bound receptors across the aqueous sensillar lymph. Here we show that the recombinant Antheraea polyphemus PBP1 (ApolPBP1) is expressed bound to the hydrophobic molecule(s) endogenous to Escherichia coli which keeps the protein in the bound conformation at high pH but switches to the free conformation at low pH. Thus delipidation is critical for bacterially expressed lipid‐binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the free form at all pH levels by solution NMR. Mutation of His70 and His95, located at one end of the binding pocket, to alanines and deletion of C‐terminus tail, on the other end of the pocket, drives the equilibrium toward the open conformation even at low pH for the ligand‐bound protein, eliminating the pH‐induced conformational switch which exists in wild type protein. Binding of 1‐aminoanthracene (AMA) by fluorescence spectroscopy and solution NMR studies revealed that, these proteins can bind ligand even at low pH in contrast to the wild type protein. C terminus truncated proteins showed reduced binding affinities towards the ligand at all pH indicates that apart from helping in the ligand releasing mechanism, C terminus might have a role in ligand binding mechanism and/or prevent the early escape of the ligand from the binding pocket.
Ct‐MGAM Activity Adapts to Various Botanical Food Starch Intakes by Alternative Splicing
Authors: Quezada‐Calvillo Roberto , Nichols Buford , Avery Stephen , Rocha Mireya , Hamaker Bruce
Published: June 2021

Maltase‐Glucoamylase (MGAM) is one of the intestinal mucosal α‐glucosidases responsible for the final digestion of starch to free glucose. In humans membrane‐bound MGAM is formed by two subunits: a N‐terminal (Nt‐MGAM) subunit with slow activity against αamylase pretreated α‐Limit Dextrins (α‐LDx); and a C‐terminal (Ct‐MGAM) subunit with fast activity against α‐LDx and substrate inhibition by the shortest glucans. In mammals, including humans, the 3′‐region of the MGAM gene coding the Ct subunit contains from 4 to 6 paralogous copies, each for a varied subunit. Although expression of mRNA from these copies has been observed, the biological significance of the variable 3′ Ct regions undocumented. Here we report 4 different mRNAs coding for Ct‐MGAM, each by alternative splicing. The recombinant expression of these 4 subunits produced active enzymes with differences in their kinetic properties of catalysis. These observations indicate that variable Ct‐MGAM subunits are the result of alternative splicing arising from paralogous duplications in the MGAM gene's 3′‐region. The resulting “splicoform” Ct‐MGSM enzymes may be a mechanism for efficient digestion of glucans with variable α‐LDx structures generated by αamylase from starches of different botanical origin. Supported by CNRC/ARS/USDA and CONACYT‐Mexico.
Transbilayer plasma membrane phospholipid asymmetry: Substrate specificity and protein‐protein interactions of the aminophospholipid flippase
Authors: Daleke David , Julien Michel , Paterson Jill , Pichika Rajeswari , Smriti Smriti , Zimmerman Michael
Published: June 2021

Phospholipids in the eukaryotic plasma membrane are distributed nonrandomly across the bilayer; the choline‐containing phospholipids are enriched in the outer, and the amine‐containing phospholipids localized to the inner, monolayer. This distribution is generated and maintained by inwardly‐directed phospholipid flippases, floppase proteins that pump lipids to the outer surface and scramblases that facilitate the bi‐directional movement of lipids in activated cells. Using synthetic analogs of PS in which each of the functional groups of the lipid have been modified or removed, the plasma membrane PS flippase demonstrates a high degree of structural and stereochemical specificity for PS that is distinct from other PS binding proteins, such as protein kinase C, clotting factors, and the macrophage PS receptor, but is shared by candidate flippases from erythrocytes and some members of a new subfamily (P4) of P‐type ATPases. Some of these lipid analogs are effective inhibitors of erythrocyte PS flippase activity, further defining the substrate recognition elements of the flippase. Recent evidence from other groups indicates that the activity of P4‐ATPases is regulated by the CDC50 family of proteins. Using activity‐inhibiting antibodies, our studies have also revealed that the erythrocyte flippase and scramblase may interact or be activities of the same protein.
Lipid droplets promote survival of nutrient‐deprived cells
Authors: Cabodevilla Ainara G , Nintou Eleni , Gubern Albert , Claro Enrique
Published: June 2021

Lipid droplets (LD) are intracellular organelles formed in most eukaryotic cells. They are a core of triacylglycerols (TAG) and choresteryl esters surrounded by a phospholipid monolayer with which a variety of proteins interact. Most of the present knowledge on LD biology is derived from experimental models where cells are loaded with external TAG and/or fatty acids. Little is known, however, about the physiological significance of LD formed under stress and in the absence of external lipids, conditions that lead eventually to cell death. Here we show that cells kept in isotonic buffer devoid of nutrients form LD whose TAG derive from membrane phospholipids in a process that depends on iPLA2 and cPLA2 activities. In this experimental model, inhibition of LD biogenesis accelerates cell death and increases the expression of markers of autophagy. And conversely, inhibition of autophagy increases the occurrence of LD. Taken together, our results show that LD biogenesis under stress has a cell protective role.

Supported by BFU2009‐0783 grant from the Spanish Ministry of Science.
Characterization of a Perilipin‐5 Splice Variant
Authors: DuBreuil Daniel M , Tansey John T
Published: June 2021

Perilipin‐5, the newest member of the perilipin family of lipid droplet proteins, has been found to be expressed in oxidative tissues, specifically heart, brown adipose tissue, and fasted liver. This work seeks to characterize a novel splice variant for perilipin‐5 we have termed Perilipin‐5B. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Highest levels of the protein were present in heart tissue from fed mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 7 and 8 and 8 and 9, either of which could give rise to a protein of approximately 35 kDa. Other members of the PAT family, such as Perilipin‐1, have multiple protein products arising from splice variants. The function of perilipin‐5B is yet unknown, but the interesting observation that it is found primarily in fed heart tissue suggests that it plays a role in the oxidation of the lipid droplet. Knowledge of this protein's function will give a more complete picture as to the overall mechanism of lipid storage and metabolism.
A novel anti‐aging drug extends longevity by remodeling neutral lipid metabolism
Authors: Titorenko Vladimir , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent
Published: June 2021

We use yeast as a model to understand a mechanism linking longevity and neutral lipid metabolism. Caloric restriction (CR) extends life span across species. We examined 1) the effect of CR and numerous mutations extending yeast life span on the spatiotemporal dynamics of the proteomes and lipidomes of organelles involved in neutral lipid metabolism, including the endoplasmic reticulum (ER), peroxisomes, and lipid bodies; and 2) the chronology of hallmark events of apoptotic and necrotic death pathways in various long‐lived yeast mutants impaired in neutral lipid metabolism. Our findings revealed that a calorie‐rich diet suppresses peroxisomal oxidation of fatty acids (FFA) that originate from neutral lipids synthesized in the ER and deposited within lipid bodies. The resulting age‐related accumulation of arrays of FFA within lipid bodies triggers a programmed necrotic cell death pathway. We designed a chemical genetic screen for small molecules that extend yeast longevity by altering neutral lipid metabolism. Our screen identified lithocholic acid (LCA) as one of such novel anti‐aging drugs. Using a combination of proteomic, lipidomic, metabolomic, and systems biological approaches, we established the mechanism underlying its life‐extending ability. We found that this bile acid remodels neutral lipid metabolism in the ER, lipid bodies, and peroxisomes, thereby postponing age‐related necrosis caused by the buildup of FFA. Our findings revealed two different ways for delaying aging by altering neutral lipid metabolism and thereby impairing the age‐related programmed necrotic death pathway. Supported by NSERC of Canada.
Role of phospholipase D in adrenal glomerulosa cell function
Authors: Seremwe Mutsa Patience , Bollag Wendy B
Published: June 2021

Objective

To study the role of agonist‐induced phospholipase D (PLD) activation in modulating aldosterone secretion in human adrenocortical carcinoma cells (HAC15).

Methods

HAC15 cells were treated with elevated extracellular potassium (K+) and angiotensin II (AngII) in the presence and absence of 5‐fluoro‐2‐indolyl des‐chlorohalopemide (FIPI), a selective PLD inhibitor. To verify that FIPI was not cytotoxic, we used 22(R)‐hydroxycholesterol [22(R)OH‐cholesterol], a cholesterol analog that can bypass signal transduction pathways and be directly used for aldosterone production. Supernatants were collected and assayed for aldosterone. [3H]Oleate‐prelabeled HAC15 cells were stimulated with AngII in the presence of ethanol, and lipids extracted and assayed for the PLD product, phosphatidylethanol.

Results

FIPI inhibited agonist‐induced aldosterone secretion in a manner unrelated to cytotoxicity, as shown with 22(R)OH‐cholesterol. Furthermore, AngII stimulated PLD activity in HAC15 cells.

Conclusion

Aldosterone secretion in HAC15 cells in response to secretagogues was inhibited by FIPI, suggesting that PLD plays a role not only in AngII‐ but also in K+‐induced aldosterone secretion. Experiments are in progress to determine the time course and dose dependence of the PLD response to AngII and lastly, to define its mechanism of action. Funded by NIH award#HL70046 and a VA Merit award.
Mass‐Spec Identification of Human Genetic Disease
Authors: Stiles Ashlee R , McDonald Jeffrey G , Russell David W
Published: June 2021

Oxysterols are metabolites of cholesterol that play important roles in the brain, immune system, and liver. Mutations in four of seven genes that produce oxysterols cause learning defects, immunoglobulin abnormalities, and liver failure. To identify mutations in the remaining three genes, a mass spectrometry (MS)‐based method was developed to quantify sterols and oxysterols in small volumes (~200 μl) of human serum. Every procedural step was optimized, including organic solvents used to extract total lipids from serum, partial purification of sterols and oxysterols by aminopropyl silica chromatography, derivatization prior to gas chromatography‐MS, and resolution by ultra‐HPLC prior to quantification via isotope‐dilution electrospray or atmospheric pressure ionization MS. To test the method, levels of 60 sterols and oxysterols were determined in sera from 200 volunteers from the Cooper Aerobics Clinic in North Dallas. The power of the approach to detect mutations was illustrated by identification of an undiagnosed case of sitosterolemia (abnormal accumulation of plant sterols). Sterol and oxysterol levels are now being measured in ~3,500 individuals from the Dallas Heart Study, a cohort whose genomic DNAs and clinical histories are maintained on campus. Oxysterol biosynthetic and catabolic genes will be sequenced in individuals with oxysterol levels in the lowest and highest 5th percentiles and the consequences of mutations identified determined from clinical histories.
Elucidation of the cytosolic phospholipase A2‐α‐‐ceramide‐1‐phosphate binding site
Authors: Olmstead Deborah Nicole , Ward Katherine E , Sudhahar Christopher G , Stahelin Robert V
Published: June 2021

Cytosolic phospholipase A2‐alpha (cPLA2α) has been a protein of interest for over 16 years because of its presence in inflammatory diseases, including asthma and rheumatoid arthritis. cPLA2α is composed of two domains, a catalytic domain that generates arachidonic acid by cleaving phospholipids, and a C2 domain responsible for anchoring the protein to the lipid membrane. Ceramide‐1‐phosphate (C1P) is a sphingolipid found in inflamed tissue that previous research demonstrates to significantly upregulate the activity of cPLA2α. cPLA2α's C2 domain contains a debated binding site for C1P, and the goal of this study is to culture 15N‐tagged cPLA2α C2 domain for structural investigation by NMR. In addition, binding studies utilizing large unilamellar vesicles (LUV) and increasing concentrations of C1P will help us construct a binding curve, thus better understand C2 binding to C1P. Preliminary results of the NMR study and LUV assay support our hypothesis that C1P and cPLA2α interact. This interaction is likely significant in inflammatory disease processes and is potentially a novel drug target. Funding provided by American Heart Association SDG0735350N.
Food intake and dietary selection during buffet feeding in women classified by 6‐n‐propylthiouracil (PROP) taster phenotype ‐ A preliminary report
Authors: Shafaie Yasmine , Koelliker Yvonne , Hoffman Daniel
Published: June 2021

Taste blindness to PROP is a common phenotype that has been associated with increased adiposity in women, and might be linked to increased energy and fat intake. Since exposure to a variety of energy‐dense foods is known to promote excess energy intake, we investigated if PROP non‐taster (NT) women would consume more fat and/or energy in a buffet setting than medium taster (MT) or super‐taster (ST) women. Subjects were non‐diet restrained, lean (BMI= 21.8 ± 0.8), women (26.8 ± 1.5 yr). To date, 14 NT, 20 MT and 16 ST participated. Subjects ate lunch and dinner in the laboratory for 3 consecutive control (CONT) days and 3 consecutive buffet (BUF) days; there was a 4‐d washout between conditions. During CONT they consumed ad‐libitum, fixed‐item meals; during BUF they selected from a variety of foods. As expected, all groups consumed more energy during BUF than CONT (p<0.0001). Compared to NT, ST consumed more protein (% en) across all days of the study (p<0.0002), as well as more fruit and non‐caloric beverages and less sweet‐fat snacks (p<0.05–0.003). Energy intake was marginally higher in NT compared to ST. These data suggest that NT women consume more energy‐ dense foods than ST which might contribute to group differences in energy balance over time. Supported by the American Heart Association.
Low vs. high glycemic load diets reduce insulin‐like growth factors and inflammatory factors in overweight persons in a controlled feeding study
Authors: Neuhouser Marian L Stone , Pollak Michael N , Coronado Gloria , Schwarz Yvonne , Lampe Johanna W
Published: June 2021

Our objective was to investigate whether associations of obesity with chronic disease risk biomarkers are mediated by dietary patterns that cause unfavorable metabolic profiles. Our randomized, cross‐over, controlled feeding study tested the effect of low‐ and high‐glycemic load (GL) experimental diets on disease risk biomarkers [glucose, insulin, IGF1, IGFBP3, leptin, adiponectin, interleukin‐6 and C‐reactive protein (CRP)]. Normal weight (n=40) and overweight/obese (n=42) men and women completed two 28‐day feeding periods – low GL and high GL. All meals were prepared in a metabolic kitchen and were isocaloric; both arms had identical macronutrients but differed in GL. Fasting blood was drawn before and after each feeding period. Linear mixed models tested the intervention effect on the biomarkers; models were adjusted for baseline biomarker concentrations, diet sequence, feeding period, age, sex and body fat mass. Compared to the high‐GL diet, the low‐GL diet significantly reduced IGF1 (p=0.04) and the IGF1:IGFBP3 ratio (p=0.01). Results were more pronounced among overweight/obese participants. When stratified by body fat mass, CRP was significantly lower for the low‐ vs. the high GL diet for those with high body fat (p=0.01). A dietary change emphasizing low GL foods may improve the metabolic and inflammatory profile of overweight and obese persons. Supported by NIH U54 CA116847, NIH R03 CA132158.
Extracellular matrix (ECM) protein lumican regulates innate immune signals at the host cell surface and promotes bacterial phagocytosis
Authors: Chakravarti Shukti , Lee Seakwoo
Published: June 2021

Here we show that lumican aids phagocytosis of gram− bacteria through cell surface interactions with integrins and CD14, an adaptor protein for LPS signaling. Lum−/− peritoneal macrophages (MF) were tested for response to LPS (induction of TNF‐α), and phagocytosis of gfp‐E. coli in the presence or absence of wild type (wt) and mutant recombinant lumican (rLum). Lum−/− MF showed poor induction of TNF‐α, and impaired phagocytosis. Pretreatment of Lum−/− MF with wt rLum rescued LPS response and phagocytosis completely, while mutated rLumY20A rescued only 30% of wild type functions. Phagocytic functions in Lum−/−Cd14−/− MF could not be rescued with wt rLum, indicating complete CD14 dependence. CD14‐antibody blockade reduced phagocytosis in Lum+/+ MF to Lum−/− levels, with no further decrease in Lum−/− MF. CD18 blockade reduced phagocytosis in Lum+/+ and Lum−/− MF, indicating some CD18 phagocytic functions to be lumican‐independent. Lumican, β2 integrin (CD18) and CD14 co‐localize on the surface and phagosomes of Lum+/+ MF. Surface Plasmon resonance indicated specific binding between CD14 and rLum (KA=2.15 ×106 M−1) but weaker affinity for rLumY20A (KA= 3.50 × 104 M−1). These studies identify a novel role for lumican in innate immunity and bacterial phagocytosis, in a CD14 and CD18 dependent manner. An N‐terminal tyrosine in lumican is critical for binding CD14, LPS response and phagocytosis.

Supported by NEI11654 to SC
RNAi screening reveals new players in the defense against RNA viruses
Authors: Xu Jie , Sabin Leah , Zhou Rui , Hannon Greg , Cherry Sara
Published: June 2021

Virus‐host interactions are a delicate interplay of opposing forces: the virus subverts cellular machinery to promote its replication while the host mounts an immune response to eliminate the infection. Innate immunity is the first and most ancient line of defense, yet little is known about the cellular factors that restrict viral infection. Our lab uses RNAi screening in cells of Drosophila melanogaster, a model organism that only has an innate immune system, to identify novel antiviral factors. Of particular interest to us are pan‐antiviral factors that restrict arthropod‐borne viruses, for which there is significant morbidity and mortality worldwide due to the lack of vaccines or therapeutics. Using this approach, we previously identified Ars2, which restricts human arthropod‐borne viruses via the antiviral RNA silencing pathway. To find additional antiviral factors, we screened a panel of genes involved in various aspects of RNA metabolism against 4 RNA viruses, including 2 arthropod‐borne pathogens. We focused on genes whose depletion by RNAi leads to increased infection by at least 3 of the 4 viruses tested. We found that factors involved in various steps of RNA biogenesis and metabolism had antiviral effects. We are now characterizing the antiviral activity of these factors and their role in small RNA silencing.

Funded by RO1AI074951 to SC.