bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of BRCA2 gene expression through CpG methylation of its bi‐directional promoter induced by endogenous siRNAs
Authors: MISRA SMITA , KHEDKAR SHEETAL V , CHAUDHURI GAUTAM
Published: June 2021

Human BRCA2 gene expression is tightly regulated. It is not expressed at the G0/G1 phase of human breast cancer cells. Here we provide evidence that BRCA2 gene promoter, through its bidirectional activity, produces two partially overlapping transcripts (BRCA2 and ZAR2) in the G0/G1 breast cells potentially forming a 111 bp RNA duplex. Our data suggest that endogenous siRNAs formed from this RNA duplex suppressed BRCA2 gene expression through CpG methylation of the USF1/2‐binding site at the promoter by transcriptional gene silencing mechanisms. Involvement of DNA methylation in the siRNA‐induced inhibition of BRCA2 gene expression was evident by its dependence on folate and DNMT1. There was hypomethylation of CpG units at the BRCA2/ZAR2 promoter during folate deficiency or knockdown of DNMT1 activity. ChIP analysis revealed a decrease in the recruitment of HDAC1 and increased acetylations of histone H3 and H4 at the BRCA2 promoter DNA under these hypomethylation conditions. These data suggest that siRNA‐induced CpG methylation of BRCA2 promoter DNA regulates the expression of BRCA2 gene in human breast cells at the G0/G1 phase of cell cycle through chromatin remodeling. Supported by the DOD‐CDMRP IDEA Grant# W81XWH‐06‐1‐0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC.
Synthestic estrone derivatives as anticancer agents and as suppressors of hypoxia‐inducible factor‐1alpha in breast, prostate and glioma cancer cell lines
Authors: Alokail Majed Saleh
Published: June 2021

Hypoxia‐inducible factor‐1 alpha (HIF‐1á) is the main transcriptional factor activated by hypoxia and it plays a key role in reprogramming tumor growth. HIF‐1á inhibition represent an important target for antitumor intervention with pharmacologic agents and a possible role for HIF‐1á in the modulation of apoptosis. In order to study how HIF‐1á can modulate apoptosis, we analyzed in this study 63 of synthestic estrone (Peptidylestrone, Alkylamino and Alkylthioacetylamino) derivatives in MDA231, MCF7 breast cancer cell lines and PC3 prostate cancer cell lines, in addition to HIF‐1á transfected U251 human glioblastoma cell line. All cell lines were incubated at 16 hours under normoxia with or without CoCl2 (a chemical inducer of HIF‐1á). Our data have shown many of these derivatives significantly decreased HIF‐1á expression in the presence of CoCl2. These derivatives have also shown significant increase in apoptosis in all cell lines. These derivatives also suppress HIF‐1á gene expression in the presence of CoCl2 in all cancer cells in addition to HIF‐1á transfected U251 human glioblastoma cell line. In conclusion, our preliminary data suggested more experiments are needed to define if thease derivatives modulate the HIF‐1á role in cell death pathway via different transcription factors depending on the cellular context and to determine the exact mechanism of these derivatives in cancer cell apoptosis effects.
Functional characterization of novel BTB/POZ‐domain protein ZBTB8A
Authors: Kim Min‐Kyeong , Hur Man‐Wook
Published: June 2021

Human BTB/POZ proteins play important roles in the regulation of cell cycle, differentiation, and development. Some transcription factors with BTB/POZ domain have been identified as a key regulatory gene of cancer development. Previously, we found that BTB/POZ proteins such as FBI‐1, ZBTB2 and ZBTB5 regulate expression of the genes of p53 pathway important in the regulation of cell cycle. Recently, we isolated a cDNA encoding a new Krüppel‐like zinc finger BTB domain‐containing protein 8A (ZBTB8A). The ZBTB8A consists of 441 amino acids and contains two C2H2 type zinc fingers. Serial analysis of gene expression (SAGE) analysis showed that ZBTB8A is highly expressed in pancreatic cancer tissues. Over‐expression of ZBTB8A in HEK293A cells represses transcription of the genes of p53 pathway genes, which include ARF, MDM2, p53 and p21. In particular, ZBTB8A potently represses transcription of p21 gene through the distal p53 binding element. FACS and MTT assays show that ZBTB8A stimulates cell proliferation and cell cycle progression. These results suggest that ZBTB8A may be a potential proto‐oncogene.
ZBTB5 is a potential tumor suppressor in the absence of functional p53
Authors: Koh Dong‐In , Hur Man‐Wook
Published: June 2021

Previously, we isolated and characterized a novel POZ domain Krüppel like zinc finger transcription repressor, ZBTB5 (Zinc finger and BTB domain containing 5). SAGE analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Our data suggested that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto‐oncogene stimulating cell proliferation.

Interestingly, in the HCT116 p53 null cells lacking functional tumor suppressor p53, expression of ZBTB5 was greatly induced prior to induction of cell cycle arrest gene p21 when cells were expose to DNA damaging agent, etoposide. ZBTB5 potently activated transcription of p21 gene in the absence of p53. ZBTB5 and Sp1 synergistically activates transcription by acting on the p21 promoter lacking distal p53 binding elements both in the p53 null and p53 positive cells. Furthermore, ZBTB5 increased transcription of p21 in the presence of dominant negative mutant form of p53. Our data suggest that ZBTB5 has functional duality in the regulation of cell proliferation and can acts either as oncogene or tumor suppressor depending on presence or absence of functional p53.
Monitoring Cadmium Binding to the Yeast Transcription Factors Yap1 and Yap2
Authors: Stamm Chelsea E , West James D
Published: June 2021

The yeast transcription factors Yap1 and Yap2 regulate genes that promote defense against reactive molecules (e.g., hydrogen peroxide, organic electrophiles, and some transition metals). Regulation of Yap1 and Yap2 activity is controlled by alterations in subcellular localization. Under homeostatic conditions, Yap1 and Yap2 are predominantly cytosolic; however, in the presence of reactive molecules, Yap1 and Yap2 localize to the nucleus by interacting directly with these molecules. Here we describe an experimental approach to monitor transition metal binding to Yap1 and Yap2 through their cysteine‐rich regulatory domains. Specifically, we have conducted cellular experiments to monitor the localization of fluorescently tagged Yap1 and Yap2 in RKO human colorectal carcinoma cells and have observed nuclear accumulation with high concentrations of cadmium (5–10 mM). In addition, we are developing biochemical and biophysical methodologies to monitor direct transition metal binding by the regulatory domains of these proteins. These studies will be useful in determining the biochemical features that influence regulation of gene expression and cellular protection by these transcription factors.
Translational control of Sp1 against ischemia via a hydrogen peroxide‐activated IRES pathway
Authors: Hsu Tsung‐Yi , Yeh Shiu‐Hwa , Hung Jan‐Jong , Chang Wen‐Chang
Published: June 2021

The protein level of the transcription factor Sp1 (specific protein 1) is known to increase during hypoxia and oxidative stress. However, the exact mechanism underlying the increase of Sp1 and the physiological consequence thereafter remains to be fully clarified. We found in this study that, in rat primary cortical neurons, the oxygen‐glucose deprivation (OGD) causes an increase of H2O2 as well as Sp1 in the early phase of the treatment without, however, causing any change in either the level of mRNA or the stability of Sp1. We discovered hereby for the first time that there is a longer 5’‐UTR in the Sp1 mRNA that contains an IRES which is well‐known to render a fast and efficient translation of existing mRNAs. Importantly, by using the polysomal fragmentation and bicistronic luciferase assays, we found that H2O2 activates the IRES‐dependent translation. Accordingly, H2O2 or tempol, a superoxide dismutase‐mimetic agent, causes an increase of Sp1 in OGD‐treated neurons. The shRNA against Sp1 exacerbates the OGD‐induced apoptosis in primary neurons. In rat models of stroke, while the Sp1 level is increased in the cortex, blocking the Sp1 binding to DNA by mithramycin or pre‐silencing Sp1 with shRNAs leads to enhanced apoptosis and cortical injuries. Those results demonstrate that neurons can use H2O2 as a signaling molecule to activate the IRES‐dependent translation of Sp1 mRNA to increase Sp1 that defends against neuronal damage. Thus, appropriately maneuvering the H2O2 level or increasing Sp1 by targeting the IRES‐dependent translational pathway might represent efficient and novel avenue in combating neurodegenerative diseases.
Di‐Alkyl Aryl Phosphates are Irreversible Inhibitors of Butyrylcholinesterase; A Potential Therapeutic for the Treatment of Alzheimer's Disease
Authors: Acey Roger Albert , Brown Michael W. , Cage Felisha W. , Del Cid Joselyn , Nakayama Kensaku
Published: June 2021

The progression of Alzheimer's disease (AD) is characterized by an increase in brain butyrylcholinesterase (BuChE) activity. This results in diminished levels of acetylcholine and loss of cognitive function. Therapeutics currently used in the treatment of AD are competitive inhibitors of BuChE and acetylcholinesterase (AChE). Inhibition of enzyme activity leads to increased levels of acetylcholine and return of cognitive function. Here we present data on the potential use of irreversible inhibitors of BuChE for the treatment of AD. A series of di‐alkyl aryl phosphates were shown to be potent inhibitors of BuChE. In contrast, none of the compounds affect the activity of AChE, trypsin, hexokinase, or glucose‐6‐phosphate dehydrogenase. Enzyme inactivated by the organic phosphates undergoes varying degrees of spontaneous reactivation after 72 hrs of incubation in 0.10 M phosphate buffer, pH 7.5 at 37 oC. Incubation of rat brain extracts with di‐n‐butyl‐2‐chlorophenyl phosphate resulted in a 35% decrease in BuChE activity. The results suggest that this class of compounds is a potential therapeutic for the treatment of AD. Funded by the CSULB Mini Grant Program. Patent pending: Application Number PCT/US2007/083262.
CHARACTERIZATION OF HIV‐1 REVERSE TRANSCRIPTASE 3TC SPECIFICITY BY CONFORMATIONALLY SENSITIVE FLUORESCENCE REVEALS NEW INSIGHTS INTO THE KINETIC BASIS OF INHIBITOR DISCRIMINATION
Authors: Kellinger Matthew William , Johnson Kenneth Allen
Published: June 2021

HIV‐1 Reverse Transcriptase (HIV‐RT) nucleotide specificity is best quantified by analysis of the concentration dependence of the rate using single turnover rapid Quench‐Flow methods which provide a rate of polymerization (kpol) and an apparent dissociation constant (Kd) such that kpol/Kd = kcat/Km. Analysis of nucleoside analog RT inhibitors (NRTIs) has led to the surprising conclusion that most appear to bind more tightly than normal nucleotides. Using an enzyme linked fluorophore, we show that nucleotide binding is a two step process involving weak nucleotide ground state binding, followed by a conformational change from an “open” to “closed” state. These steps together define the true Kd for nucleotide binding at equilibrium. We show that contrary to previously reported findings, the dCTP analog 3TC binds 8‐fold more weakly to the enzyme than the correct nucleotide. The result is a specificity constant (kcat/Km) of 9.7μM−1s−1 for dCTP and 0.7μM−1s−1 for 3TC. The specificity constant for dCTP is determined solely by the rate of nucleotide binding (kcat/Km = K1k2 in the two‐step sequence), whereas the slower chemical reaction (k3) for 3TC incorporation allows the binding and isomerization to reach equilibrium so that kcat/Km = kpol/K1K2. This work provides mechanistic basis for discrimination of 3TC, and corrects how Km, Kd, and Kd,app must be assigned for NRTIs.
Polyisoprenylation potentiates the inhibitory and cell degenerative potency of sulfonyl fluorides towards polyisoprenylated methylated protein methyl esterase
Authors: Aguilar Byron , Amissah Felix , Duverna Randolph , Lamango Nazarius
Published: June 2021

Polyisoprenylated protein methyl transferase catalyses the esterification of polyisoprenylated proteins (PP) whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyses the ester bond. Significant changes in their enzymatic activity may alter PP function causing disease. Prior studies show that PMPMEase is Sus scrofa carboxylesterase, which hints at its susceptibility to serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF). PMSF was used as a prototypical compound to develop high affinity specific inhibitors of PMPMEase. Pseudo first‐order kinetics revealed an over 680‐fold increase in kobs/[I] (M−1s−1) values from PMSF (6), S‐trans,trans‐farnesyl (L‐28, 2000), to S‐trans‐geranylated (L‐23, 4100) 2‐thioethanesulfonyl fluorides. The compounds induced the degeneration of human neuroblastoma SH‐SY5Y cells with EC50 values of 49, 130 and >1000 μM for L‐28, L‐23 and PMSF, respectively. The increase in affinity with the polyisoprenyl derivatization corroborates the observed substrate specificity and the reported hydrophobicity of the PMPMEase active site. These results suggest that (1) PMPMEase is a key enzyme for PP metabolism, (2) regulation of its activity is essential for maintaining cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.
Human group III phospholipase A2 as a drug target: structural analysis and ligand binding studies
Authors: Hariprasad Gururao , Srinivasan Alagiri , Kumar Manoj , Kumar Prem , Kaur Punit , Singh Tej Pal
Published: June 2021

Group III phospholipase A2 is a known mediator of inflammation, atherosclerosis and cancer in mammals. It therefore makes the enzyme a potential drug target. The availability of the human group III phospholipase A2 (hIIIPLA2) amino acid sequence offers an opportunity to study its structural features by modeling. The monomeric hIIIPLA2 model is based on the 44% identity it has with the bee venom PLA2, the only known representative structure of this group. The overall structure comprises of three α‐helices, two sets of anti‐parallel β‐sheets and the calcium binding loop which is present at the N‐terminus of the enzyme. However, the unique structural features of hIIIPLA2 in comparison to the other well known group I/II PLA2s are: (1) The replacement of the ‘conserved’ tyrosine residue by phenylalanine at position 87 in the active site; (2) A decrease in the volume of the substrate binding hydrophobic channel and (3) presence of a C‐terminal extension which has a close proximity to the third helix. Docking studies of the enzyme with Non‐steroidal Anti‐Inflammatory Drugs (NSAIDs) gives a detailed insight into the participating residues of the enzyme and also the possible type of interactions it makes with the drug molecules. The NSAIDs have a binding affinity in micromolar to nanomolar range, thereby making them either potential lead molecules or potent drugs. This analysis paves the way for possible therapeutic applications in pathological states caused by this enzyme. This is the first report of structural and ligand binding studies of hIIIPLA2. The FAST TRACK grant given by Department of Science and Technology, Government of India and the Pool Officer fellowship given by Council of Scientific and Industrial Research, Government of India to GH is acknowledged.
Improved production of astaxanthin of the Paracoccus haeundaensis by mutagenesis
Authors: Kim Young Tae , Ji Keunho , Jeong Tae Hyug
Published: June 2021

Carotenoids are a widely distributed group of naturally occurring pigments and used extensively for natural colorants for food supplements and pharmaceutical applications. Astaxanthin has attracted commercial interest not only as a pigmentation source but also as a potent anti‐oxidative reagent that can delay aging and the onset of degenerative diseases. In addition its anti‐oxidative activity, astaxanthin has been shown to have biological functions as diverse as serving as a vitamin A precursor and modulating the immune system. We have previously reported the isolation of the Paracoccus haeundaensis, which produces astaxanthin. Cultures of Paracoccus haeundaensis were exposed to mutagens. The results showed that the survival rate decreased with the increase in UV exposure time and increase in EMS concentration. The hyper‐production mutant of astaxanthin was produced by UV irradiation (20 min exposure) with EMS treatment (0.4 M concentration). The isolated mutants exhibited an increase of 3.82 folds in astaxanthin production compared to untreated initial strain. This genetically stable mutant strain may be a suitable candidate for the industrial production of astaxanthin.
Lenses from Connexin50 Mutant Mice Exhibit the Unfolded Protein Response
Authors: Stull Jaime
Published: June 2021

Cataract is the leading cause of blindness worldwide. Although the majority of cataracts are age‐related, they may also be caused by heritable mutations. In humans as well as mice, congenital cataract occurs in Connexin50 (Cx50) mutants although the cataract phenotypes are more severe than that observed in Cx50 null mice. We hypothesized that when cells attempt to make Cx50 from a mutated gene, the resulting protein does not fold properly, inducing a cell‐stress mechanism called the Unfolded Protein Response, UPR. We investigated this in mice harboring two different Cx50 mutations, Cx50S50P/S50P and Cx50G22R/G22R, that both exhibit similar lens phenotypes including microphthalmia, unorganized fiber cell structure, and cataract. We found a noticeable upregulation of the expression of the molecular folding chaperone BiP in mutant lenses after E15.5 in addition to an upregulation of XBP‐1 gene expression and unconventional splicing indicative of the activation of the UPR sensor IRE1. These data further support our prior work suggesting that UPR can contribute to lens phenotypes due to mutations in genes whose products must transit the secretory pathway. Supported by the Howard Hughes Medical Institute, Fight for Sight, and the National Eye Institute.
Interplay between Ca2+ Binding and Disulfide Bond Formation: Study of Lin‐12/Notch Repeats as a Model System
Authors: Ulu Didem Vardar , Choi Lauren , Seo Angie
Published: June 2021

Many multi‐domain proteins contain small protein domains that lack an extensive hydrophobic core or secondary structure. Their global folds are stabilized either by binding of a metal ion or the formation of disulfide bonds. This work focuses on a unique domain called the L in‐12/Notch repeat (LNR), first identified in Notch receptors and more recently, within functionally unrelated multi‐domain proteins, such as pregnancy associated plasma proteins and stealth proteins. Most LNRs are ~35 amino acids long and contain six cysteines defining a unique set of three disulfide bonds, as well as a group of acidic residues that coordinate a single Ca2+ believed to be essential for LNR folding. However, there are naturally occurring LNRs with only four of the conserved cysteines, as well as important deviations in the proposed Ca2+ binding residues. Here, we present our findings on the effect of the presence or absence of Ca2+, as well as various reduction potentials on the in vitro folding of several two‐ or three‐ disulfide bonded LNRs. Our results indicate that there is significance interdependence between Ca2+ binding and disulfide bond formation during the folding and the unfolding of the LNRs.
In Vivo, Non Invasive Evaluation of the Self Assembly of Unfolding Proteins in Transparent Lens Cells
Authors: Clark John I , Arnett Ernest , Clark Judy M , Greiling Teri M
Published: June 2021

With increasing human longevity, protein unfolding and aggregation characterize diseases that are among the greatest threats to aging humans and to the health care system. Progress on the development and screening of novel therapeutics is limited by the difficulties of studying the self assembly mechanisms of unfolding proteins in vivo. We report novel methods for overexpression of proteins responsible for aggregation diseases in transparent lens cells where protein unfolding and aggregation can be evaluated and quantified in living animals using inexpensive, noninvasive optical methods including slit lamp biomicroscopy, multiphoton microscopy and dynamic laser scattering spectroscopy (Seeberger et al(2004)J.of Biomed Optics.9:116–120;Greiling & Clark(2008)Seminars in Cell & Devel. Biol 19:94–99; Muchowski et al (2008)J Biol Chem 283:6330).Mouse lenses expressing exon 1 of the huntingtin protein with 72 polyglutamine (polyQ) repeats fused with enhanced green fluorescent protein (EGFP) were examined to determine the spatial distribution of the aggregating protein in vivo. The mobility and size of the huntingtin aggregates was measured using quasielastic light scattering (QLS) in the lens of a living mouse. The huntingtin aggregates labeled with EGFP were measured directly in the whole lens by 2 photon excitation microscopy and in frozen sections using fluorescence microscopy. The novel optical technology identified the earliest stages of protein unfolding and aggregation, when innovative therapies have the highest probability of success against the most prominent causes of neurodegeneration, cardiomyopathy and deteriorating visual function in the aging population. Supported by EY04542 from the NEI.
UNRAVELING A POSSIBLE CELLULAR MECHANISM BEHIND LEPTOMENINGEAL AMYLOIDOSIS USING AS MODEL A HIGHLY UNSTABLE TRANSTHYRETIN TETRAMER
Authors: Foguel Debora , Azevedo Estefania , Palhano Fernando , Lima Flávia
Published: June 2021

Transthyretin (TTR) is a 127‐residue homotetrameric β‐sheet‐rich protein that transports thyroxine in the blood and cerebrospinal fluid (CSF). In the present work we aimed to study A25T dissociation, aggregation and its role in leptomeningeal amyloidosis (LA). Using HPLC and native PAGE we monitored acrylodan‐labeled TTR aggregation in the plasma. The aggregates formed, when analyzed by AFM, Thioflavin T and Congo Red binding displayed amyloid structure. We solved the crystal structures of the wt and A25T and the comparative analysis shed light into the mechanism behind A25T highly amyloidogenicity: an expanded tetramer which is stabilized by a lower number of H‐bonds and hydrophobic interactions. Interestingly, in the presence of thyroxine and flufenamic acid (an anti‐inflammatory compound), two ligands of TTR, the structure of A25T was similar to that displayed by the wt protein. Since LA symptoms suggest a local inflammation, we questioned ourselves whether A25T aggregates can induce an inflammatory response in the CNS. Microglia primary cultures incubated for 48h with aggregates composed of A25T, underwent activation, phagocytosis (which could be blocked by cytochalasin D) and presented a significant nitric oxide and IL‐1 beta secretion. These results suggest that amyloid fibrils may have a role in LA pathogenesis, exacerbating tissue damage through inflammation.

Support: CNPq and FAPERJ.
Characterization of the Tim17 associated protein, Tim50, in Trypanosoma brucei
Authors: Duncan Melanie R. , Jean Leolene , Sharma Shvetank , Singha Ujjal , Chaudhuri Minu
Published: June 2021

Trypanosoma brucei causes a fatal disease in humans and domestic animals known as African trypanosomaisis. T. brucei possesses a single, tubular mitochondrion with many unique characteristics. Similar to other eukaryotes, most of the mitochondrial proteins in T. brucei are encoded in the nucleus and must be transported into the mitochondrion. However, mitochondrial protein import machinery in T. brucei is poorly understood. In eukaryotes, proteins are imported into mitochondria post‐transcriptionally via receptor/translocase complexes located on mitochondrial outer and inner membranes known as TOM and TIMs. In T. brucei, only Tim17 has been characterized from the TIM complex. Recently we have identified Tim50 in this parasitic protozoan. T. brucei Tim50 (TbTim50) protein sequence showed 16–18% identity and 28–30% similarity with Tim50 from other eukaryotes. The putative Tim50 possesses an N‐terminal mitochondrial targeting sequence. Expression of the C‐terminal HA‐tagged TbTim50 in T. brucei followed by sub‐cellular fractionation and western blot analysis revealed that the expressed protein is enriched in the mitochondrial fraction. Anti‐HA antibody was able to co‐immunoprecipitate Tim17 from T. brucei mitochondrial lysate, leading us to conclude TbTim50 is associated with Tim17. This work was supported by NIH grants SC1GM081146 and 2T32HL007737‐16.
A Novel xLxxKN Targeting Motif of ATP Binding Cassette Class A Transporters
Authors: Mulugeta Surafel , Zhao Ming , Kotorashvili Adam , Shuman Henry , Beers Michael F
Published: June 2021

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps in cells of various organs of the body. Located within the N‐terminal domain of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define the role of this motif, the ABCA3 protein was employed as a model representing ABCA transporters. Transfection together with immunofluorescence and co‐localization studies using ABCA3 cDNA constructs were performed in A549 cells. Wild type ABCA3 was expressed in vesicles containing CD63 and surfactant protein C (SP‐C). In contrast, mutation of the ABCA3 LLLWKN motif to alanine produced an abnormal reticular expression which co‐localized with the ER marker calnexin. Moreover, scanning alanine mutagenesis of this motif as well as its flanking upstream and downstream residues demonstrated the importance of this motif for transporter's proper translocation to post‐Golgi vesicles. As proof of principle, substitution studies replacing the cytosolic targeting motif of SP‐C with LLLWKN showed vesicular targeting of SP‐C. These results suggest that post‐Golgi trafficking of ABCA3 is mediated by a LLLWKN motif located within the N‐terminus of the transporter. We propose a model whereby an N‐terminal signal sequence, xLxxKN is required for directing ABCA transporters to post‐Golgi vesicular compartments.
Characterization of clathrin‐independent endocytosis in epithelial cells
Authors: Petersen Shariska S , Donaldson Julie G
Published: June 2021

Major histocompatibility complex class I (MHCI) and more recently, membrane transport glycoprotein, CD98, were shown to be internalized via clathrin‐independent endocytosis (CIE) in HeLa cells. Following internalization, a fraction of vesicles containing MHCI fuse with early endosomes, defined by the presence of early endosomal autoantigen 1 (EEA1) and containing the transferrin receptor, a clathrin cargo. MHCI is then routed to late endosomes for degradation or to distinct tubular carriers that return CIE cargo back to the plasma membrane. In contrast to MHCI, CD98 bypasses the merge with early endosomes and directly enters into the tubular carriers. To study CIE in different epithelial cell types, surface antibody internalization assays were used to follow the trafficking of MHCI and CD98 in A431 and ARPE‐19 cells. Like HeLa cells, MHCI enters via CIE in vesicles that fuse with early endosomes and traffic to late endosomes for degradation. However, after endocytosis, CD98 reaches late endosomes although it bypasses the EEA1‐early endosome. Recycling of MHCI and CD98 is observed in both cell lines although tubular carriers seen in HeLa cells are absent. These observations confirm that CIE exists in different cell types but that itineraries for distinct cargo may vary.
Therapeutic Metabolomics Identifies Novel Treatments for Multiple Sclerosis and Chronic Pain
Authors: Siuzdak Gary
Published: June 2021

Quantitative global analysis of endogenous metabolites from cells, tissues, fluids or whole organisms ‐ metabolomics, is becoming an integral part of functional genomics efforts as well as a tool for finding diagnostic biomarkers. Where the genome and proteome is largely enabled by the predictable fragmentation pattern of peptides, metabolomics is complicated by the tremendous chemical diversity of metabolites. The experimental aim in our global metabolomics studies is to obtain a comprehensive quantitative, unbiased, view of the metabolome. We have explored multiple novel mass spectrometry platforms for metabolomics including both solution‐based approaches and surface‐based mass spectrometry, such as nanostructure‐initiator mass spectrometry (NIMS). These platforms will also be presented in the context of specific biochemical applications such as chronic pain, multiple sclerosis and tissue imaging.
The effect of the sn‐2 acyl chain of diacylglycerol on its interaction with C1 domains and its signaling role
Authors: Kurilova Svetlana , Yoon Youngdae , Lee Park Joo , Bhardwaj Nitin , Lu Hui , Cho Wonhwa
Published: June 2021

Diacylglycerol (DAG) is a signaling lipid that interacts with the C1 domain in various target proteins, including protein kinases C, and Ras guanyl nucleotide‐releasing proteins, thereby recruiting them to the plasma membrane. Although these proteins can all bind DAG, some are known to target other intracellular membranes. To understand the basis of this differential membrane targeting, we explored the possibility that a difference in the acyl chain composition of DAG in the plasma membrane and other membranes may affect the C1‐DAG interaction. To test this notion, we measured the binding of the various C1 domains to DAGs with different sn‐2 acyl groups by fluorescence correlation spectroscopy (FCS). The C1 domains were expressed as an enhanced green fluorescence protein‐fusion protein to enhance protein stability and allow FCS measurements. FCS measurements as well as the molecular dynamics simulation show that the sn‐2 acyl group of DAG has significant and differential effects on the membrane binding properties of C1 domains, which is consistent with the differential subcellular localization of the C1 domains.

Supported by NIH GM76581
Phosphatidylserine binding is important for plasma membrane targeting of 3′‐phosphoinositide‐dependent kinase‐1 pleckstrin homology domain
Authors: Lucas Nathan Thomas , Kim Hee‐Yong , Cho Wonhwa
Published: June 2021

3′‐Phosphoinositide dependent kinase (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide‐3‐kinase activation. An increase in phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol‐3,4‐bisphosphate (PtdIns(3,4)P2) is thought to recruit PDK1 and its substrates to the 3′‐phosphoinositide‐rich regions of the plasma membrane for subsequent phosphorylation. PDK1 contains a C‐terminal catalytic domain and an N‐terminal pleckstrin homology (PH) domain that was reported to have high affinity for PtdIns(3,4,5)P3 and PtdIns(3,4)P2. To better understand how lipids regulate the subcellular localization and activation of PDK1, we rigorously investigated the membrane binding properties of PDK1 PH domain by surface plasmon resonance analysis. The results show that the PDK1 PH domain has high affinity for not only PtdIns(3,4,5)P3, PtdIns(3,4)P2 and phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2) but also for phosphatidylserine (PS). We identified the separate binding sites for phosphoinositides and PS through molecular modeling and mutagenesis and analyzed the differential effects of phosphoinositides and PS in membrane recruitment of PDK1.

Supported by a NIH grant GM68849.
Unique lipid‐protein interactions observered in molecular dynamics simulations of rhodopsin in a polyunsaturated membrane
Authors: Feller Scott Edward
Published: June 2021

The structure and dynamics of polyunsaturated lipids, specifically those containing docosahexaenoic and docosapentanoic acid, will be described and compared with saturated and mono‐unsaturated lipids as well as with experimental measurements utilizing NMR and x‐ray diffraction. Both simulation and experiment suggest the polyunsaturated fatty acid chains possess an extreme flexibility. The role of the torsional energy profile for rotation about single bonds between vinyl groups will be emphasized as the key factor in these observations. A mechanism by which this flexibility allows unique interactions with integral membrane proteins will be demonstrated.
Regulation of mitochondrial glycerol 3‐phosphate acyltransferase by phosphorylation‐dephosphorylation mechanism in 3T3‐L1 cells
Authors: Zhang Man , Pandey Atul , Haldar Dipak
Published: June 2021

The posttranslational regulation of mitochondrial glycerol 3‐phosphate acyltransferase (mtGPAT) by phosphorylation‐dephosphorylation was investigated. All of the kinase sites were identified on the cDNA of the enzyme. The effects of four kinases, protein kinase C (PKC), protein kinase A (PKA), tyrosine kinase (TK) and casein Kinase II (CKII) were investigated either by treatment of isolated mitochondria with a kinase, or by treating intact cells with an activator or inhibitor of the kinase. Out of these kinases, PKC gave the highest stimulation (approximately 50%) of the mtGPAT with mitochondria isolated from 3T3‐L1 cells. MtGPAT was identified by Western Blot, or when the mitochondria or whole cells were incubated with γ‐32P‐ATP, by autoradiography. PMA (0.1 μM), a stimulant for PKC, gave approximately 50% stimulation whereas AG490, AG1478 or Genistein (approximately 35 μM each), inhibitors of TK, caused approximately 50% inhibition of the mtGPAT when 3T3‐L1 cells were treated with these agents. Substantial inhibition of mtGPAT was also observed when the cells were treated with H‐7 (10 μM) or Gö6976 (10 μM). The data are in keeping with the view that the regulation of mtGPAT occurs not only at the level of transcription (Guha et al., Arch. Biochem. Biophys. 490 (2009) 85–95), but also by phosphorylation‐dephosphorylation mechanism. Supported by NIH grant GM‐57643.
Unfolded protein response interferes IGF‐1 signaling of capillary endothelial cell survival
Authors: Baksi Krishna , Banerjee Aditi , Couto Javier A , Banerjee Dipak K
Published: June 2021

Angiogenesis is crucial for breast tumor growth and metastasis. Regulated pattern of endothelial cell survival and death play a central role in the timely evolution and regression of angiogenic responses. We have shown that dolichol‐linked oligosaccharide (i.e., LLO) biosynthesis and turnover are required for angiogenesis. Investigating the efficacy of a LLO biosynthetic inhibitor tunicamycin for anti‐angiogenetic breast cancer therapy indicates cell cycle arrest in G1 and induction of apoptosis by unfolded protein response (upr) signaling. This paralleled upregulation of c‐Jun, c‐Myc and down regulation of c‐fos. Upregulated p53 expression indicated failure of cell survival. IGF‐1 (20ng/ml) addition exhibited no change in the cell status as confirmed by down regulation of PI3KR, pAkt and p53(S14). This is supported by down regulation of phospho Akt (473)/Akt (308), GSK3 beta, phosphocaspase‐9, phospho‐Bad and phospho mTOR expression. Changes in NFkB phosphorylation (S14) however remained silent. QRT‐PCR confirmed upregulation of GRP‐78, Bcl‐2, caspase‐3, p53 expression and down regulation of PI3KR following IGF‐1 challenges in tunicamycin treated cells. IGF‐1 signaling triggers glucose uptake for energy metabolism but ATP level could not be raised when tunicamycin was present. This strongly suggested that tunicamycin down regulates the PI3‐K/AKt signaling of cell survival and could help developing glycotherapeutic for treating breast cancer. Supported by NIH/NCRR/RCMI G12‐RR03035 (KB); NIH U54‐CA096297 and Komen for the Cure BCTR06582 (DKB).
A novel microdissection model for probing cellular interactions. V. Free Mannan
Authors: Coyle‐Thompson Catherine , Abramian Sarineh , Akinlolu Adelaja , Allen Jenieke , Askari Tina , Dreyfuss Justin , Frederick Robert , Jarvis Krystal , Makhoul Elias , Manukyan Mariam , Mkrtchian Mary , Nazaryan Ani , Ollawa Patrick , Portillo Georgina , Qubrosi Mirey , Ramos Luz , Vasquez Eric , Oppenheimer Steven
Published: June 2021

The adhesion between the NIH model sea urchin embryo roof of the blastocoel and tip of the archenteron is a cellular interaction that has interested investigators for over a century, yet its molecular basis is unknown. Here, 102 blastocoel roofs and archenterons were microdissected out of 102, 54 hr Lytechinus pictus embryos that adhered to each other when placed together. 25 mg per ml free mannan was added to the adhered roofs and archenterons. They remained adhered and then were pulled apart and placed together again in the free mannan solution. All 102 roofs and archenterons adhered once again, the mannan possibly bridging mannan‐binding receptors on both components. As reported in Part III of these studies, mannan conjugated agarose beads also adhered to the dissected roofs and archenterons and here this work was extended to 65, 54 hr L.pictus embryos. Part I of these studies indicated that alpha methyl mannose blocked the cellular interaction. Part II of these studies showed that mannose‐binding lectins could block the interaction. Taken together, this set of studies suggests that mannose‐containing glycans and mannose‐binding receptors may be involved in the cellular interaction between the archenteron and blastocoel roof. This microdissection method, that removes the components of adhesive events from intact embryos, is novel and likely to lead to a better understanding of the molecular basis of cellular interactions (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology FIRE course funding).
Use of post‐fertilization aggregates of sea urchin embryos from denuded eggs to study self‐adhesive properties of the hyaline layer: glycosidases
Authors: Jarvis Krystal , Idoni Brian , Dreyfuss Justin , Coyle‐Thompson Catherine , Carroll Edward , Oppenheimer Steven , Hutchins‐Carroll Virginia
Published: June 2021

Vitelline envelope free sea urchin eggs do not form fertilization envelopes; however, the coat free embryos form aggregates (or clumps) as their exposed hyaline layers develop (Epel, Exp Cell Res 61(1):69–70, 1970). Here we describe a study using that property to probe hyaline layer interactions. De‐jellied Lytechinus pictus eggs were treated with 0.02 M dithiothreitol, pH 9.1, to remove vitelline envelopes, fertilized, washed 3X with artificial sea water (ASW), incubated at 5°C for 45 min and then incubated for 75 min at 17°C with no additions or with 40–9800 units/ml ASW of independently characterized α‐amylase, β‐amylase or β‐glucosidase. In 12 replicate experiments using eggs from 48 female sea urchins, counting at least 31,400 single embryos and clumps for all experiments, only β‐glucosidase caused disaggregation of clumps (p < 0.0001 compared with controls). Beta amylase caused single embryos to adhere to each other forming clumps (p < 0.03 compared with controls) and α‐amylase had no significant effect on the embryos (p > 0.19 compared with controls). The results suggest that the self‐aggregating property of coat free, fertilized embryos is a simple and effective system to probe hyaline layer‐hyaline layer interactions and that β‐glucosidic linked polysaccharides may be involved in these interactions (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust and CSU Northridge FIRE course funding).
Heparan and chondroitin sulfate modifications in signaling pathways regulating articular cartilage homeostasis
Authors: Randell Rachel Leigh , Wittmeyer Richard , Selva Erica M
Published: June 2021

Degradation of articular cartilage leads to osteoarthritis (OA) in humans. Articular cartilage homeostasis depends on signaling events modulated by extracellular matrix glycoproteins. We tested the hypothesis that disrupting heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans would alter signaling pathways relevant to cartilage homeostasis. Mosaic mutant and wild‐type Drosophila wing discs were used to visualize the effects of mutations in two genes: O‐xylosyltransferase (oxt), required for linking HS and CS to core proteins, and sulfateless (sfl), essential for decorating HS with negatively charged sulfate groups. We performed immunostaining of signaling ligands and pathway activation targets, including: Decapentaplegic, the homolog of human Bone Morphogenic Protein involved in anabolic cartilage processes; Wingless, the homolog of human Wnts implicated in cartilage repair; and Hedgehog and its receptor Patched. We demonstrate the importance of HS and CS for proper activation and movement of signaling molecules. Surprisingly, sfl appears to be more disruptive than oxt on signaling. Ongoing experiments examine the role of the core proteoglycan Dally‐like and look for signals that might upregulate cartilage deposition. These results will help to identify targets for the development of novel OA therapeutics.
Identification of Potential Partners in ENaC Assembly and Modulation
Authors: Ramirez Erika L , Ybanez Raquel V , Booth Rachell E
Published: June 2021

Epithelial sodium channels (ENaC) are voltage independent Na+ channels situated within the apical membrane of epithelia. Their function is especially critical in the distal portion of nephron where Na+ and water must be reabsorbed in order to maintain appropriate blood pressure levels. Anomalous ENaC activity can lead to disorders such as Liddle's syndrome, which is due to overactive ENaC resulting in hypertension, or pseudohypoaldosteronism type I, which is caused by decreased ENaC activity resulting in hypotension. Understanding assembly and modulation of ENaC are essential to understanding its physiological role. We have previously reported the development of a quickly screen for monitoring ENaC activity, which takes advantage of ENaC's ability to transport Na+ into yeast cells and cause growth inhibition (i.e salt sensitivity phenotype). These results can easily be visually compared by using a survival pronging dilution assay. In an effort to examine critical partners in ENaC assembly and modulation, we have screened a series of Saccaromyces Genome Deletion Project strains. We report the identification of four genes required for proper ENaC function in yeast: SUR4, ERV14, EMP24, and ERV25 as well as ULA, which showed significant reduction in growth suggesting an overly active ENaC or an increased stability of otherwise normal channels. Funding provided by Research Corporation and the Welch Foundation.
Translational Research, Technology Transfer and Small Business Grants – Alternate Funding Resources
Authors: Opperman Lynne A.
Published: June 2021

Many institutions highly value technology development and commercialization, and list these activities as criteria for consideration for faculty tenure and promotion. Funding for such research can be through regular R‐type grants (R01, R21 and R03). However, if the technology requires proof‐of‐principal testing (for example demonstrating functional prototype devices, specific siRNAs, new drug molecules) partnering with the business community might enable access to small business innovation research (SBIR) and small business technology transfer research (STTR) grants. Specific examples of university/small business partnerships and technologies will be described, and tips for finding/creating partnerships and preparing these proposals will be covered.
Research Career Development Awards: Which one is right for me?
Authors: Lysakowski Anna
Published: June 2021

After successfully completing the Ph.D. dissertation and perhaps one postdoctoral training period, how does one get established in a research career? This talk will describe, contrast and compare the various Research Career Development Awards available through NIH. We will cover "F" awards, "K" awards and early career stage "R" awards. The audience should then have a clearer understanding of the various funding mechanisms.
Mass Spectrometry and its contribution to hybrid structure determination
Authors: Robinson Carol
Published: June 2021

Standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organization of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. We have combined two existing methodologies to tackle these challenges: affinity purification and nanoflow ESI‐MS. We use methods designed to maintain non‐covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. Structural models will be presented for oligomeric protein complexes with different degrees of structural information including the human U1snRNP and eIF3 complexes. These models will then be examined within the context of their function.

Recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross‐section. Using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross–sections.

Very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase. This enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. I will demonstrate some of the advantages of this approach by presenting recent insights into the structures of intact V‐type ATP synthases.
Effect of Angiotensin II on Oxidative Stress in Female Borderline Hypertensive Rats
Authors: Zimmerman Margaret Ann , Sullivan Jennifer
Published: June 2021

Oxidative stress is a contributing factor to cardiovascular tissue injury. Angiotensin II (Ang II) produces oxidative stress in male animals, but it's effect in females in unknown. The focus of this study was to determine the effect of chronic Ang II on oxidative stress levels in the kidneys of female Borderline Hypertensive Rats (BHR). Following two weeks of Ang II infusion (200ng/kg/min), BHR mean arterial pressure rose from 111±3 to 135±7 mmHg. Protein levels in the urine, which corresponds to kidney tissue damage, increased by 17±3 mg/day in Ang II‐treated BHRs. Whole body oxidative stress was assessed through observations of H2O2 levels in urine. Ang II treated animals showed a 72±29 mg/ml increase in H2O2 in urine as compared to control animals. Protein expression of NOX4, gp91, p22, p67, CuSOD, MnSOD, ECSOD and catalase was determined by western blot. Ang‐II treated BHRs renal tissue showed a decrease in MnSOD and catalase, and no significant differences in NOX4, gp91, p22, p67, CuSOD, and ECSOD protein levels. We found that chronic Ang II treatment resulted in hypertension, kidney tissue damage and elevation in whole body oxidative stress. Research was supported by funds from grant number 1 RO1 HL093271‐O1A1.