bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and Purification of Soluble, Glycosylated T‐cell Receptors in Chinese Hamster Ovary Cells
Authors: Pearson Amanda Jo , Liedig Chance , Gallagher Elyssia S 
Published: June 2021

T‐cell receptors (TCRs) are glycoproteins that are expressed on the surface of T cells, which are a major component of the adaptive immune response. The glycans on the constant domain of this protein are involved in interactions with other proteins, such as galectin‐3, which mediates the immune response1. Additionally, altering the glycosylation of this protein has been associated with diseases such as Multiple Sclerosis1. Herein, we aim to express a soluble, glycosylated TCR in Chinese hamster ovary (CHO) suspension cells and purify it with the goal of characterizing the effects of glycosylation on TCR structure and function. To date, a soluble, unglycosylated TCR was expressed once, in 1990,2 but has not been replicated since. This project aims to express a glycosylated, soluble TCR with more current technology, which should result in more efficient expression.This project began with the design of a solubilized glycoprotein. We selected a starting sequence that has known binding affinity to ovalbumin and proceeded with modifications for optimized expression. The transmembrane sequence was removed after the last cysteine and replaced with a glycosylphosphatidylinositol (GPI) ‐ anchored sequence. This targets the protein to the endoplasmic reticulum (ER) and Golgi apparatus to ensure glycosylation. Following expression and glycosylation, the TCR should be localized to the surface of the cell, where the GPI sequence can be enzymatically cleaved using phosphatidylinositol‐phospholipase C (PI‐PLC), resulting in a soluble released protein. The α and β chains of the heterodimer were combined into a single plasmid using a 2A peptide. The use of 2A peptides instead of a more traditional technique, such as internal ribosome entry site (IRES), results in more efficient folding due to expression of stoichiometric ratios of both chains of the protein, as compared to a higher concentration of a single chain.The protein is transfected in CHO‐S cells and the expressed protein is harvested after 24 hours. The protein of interest is purified by affinity chromatography using a nickel column, followed by analysis with SDS‐PAGE. Imaging of the gels after surface cleavage with PI‐PLC and cell lysis confirmed that the protein was not localized to the plasma membrane as it was not recovered after enzymatic cleavage. However, the protein is purified from the inside of the cell after cell lysis. To determine the cellular localization of the protein we are using immunofluorescence using fluorescently labeled antibodies to target the His tag on the protein, as well as the ER, Golgi, and plasma membrane. Currently we are addressing the incorrect localization and analyzing methods to redirect the protein to the cell surface.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Alpha‐Arrestin Adaptor Protein Regulation of YOR1 in Yeast
Authors: Cheranda Nina Kiran , Wang Yuqi 
Published: June 2021

ATP‐binding cassette (ABC) proteins are membrane‐bound transporters that utilize ATP to pump various substrates across biological membranes. Serious pathological conditions can result when these transporters are defective, as exemplified by cystic fibrosis caused by a defective transporter CFTR. Yor1 is an ABC transporter in budding yeast, Saccharomyces cerevisiae. In this study, we investigated the potential regulation of Yor1 by alpha‐arrestins. Using immunoblotting techniques to check the effects of the overexpression of nine different alpha arrestin protein on Yor1 abundance in yeast cells, we found that upregulating the activity of specifically one alpha‐arrestin protein leads to a marked elevation of the level of Yor1. This observation is exciting because it provides a novel strategy for regulating and stabilizing Yor1, which could have valuable implications for the modulation of dysfunctional CFTR transporters in humans with cystic fibrosis.Support or Funding InformationKnoedler Undergraduate Fund from Saint Louis UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of βγ‐Mediated Membrane Localization on Growth Signaling by Overexpressed Gα13
Authors: Brown Katherine M. , White Nicholas F. , Tagliatela Alicia C. , Meigs Thomas E. 
Published: June 2021

The G12/13 class of heterotrimeric G proteins is unique in its ability to stimulate cellular transformation via overexpression of the wildtype α subunit, Gα12 or Gα13, absent of activating mutations. We have studied the mechanism of wildtype Gα12/13 signaling to the transcriptional activator Serum Response Factor (SRF), a pathway implicated in multiple cancer types. When overexpressed in cultured human embryonic kidney cells, wildtype Gα13 showed robust stimulation of this transcriptional response. This signaling by Gα13 was blunted by overexpression of G protein β1 and γ2 subunits, suggesting that aberrant signaling by the overexpressed α subunit results from stoichiometric imbalance between the trimeric G protein subunits. Next, using an epitope‐tagged Gα13 to track its subcellular location, we discovered the overexpressed α subunit shifted from a membrane‐associated fraction to a soluble fraction, coincident with its sharp increase in SRF signaling. Overexpression of the β1γ2 dimer caused re‐localization of wildtype Gα13 to the membrane‐associated fraction, coincident with its diminished signaling. Interestingly, our preliminary data utilizing a non‐prenylated γ2 subunit suggest the ability of the βγ dimer to suppress signaling by overexpressed, wildtype Gα13 is independent of βγ association with the cell membrane. We currently are examining effects of this non‐membrane bound dimer on subcellular localization of wildtype Gα13. These findings add to our understanding of the mechanism utilized by overexpressed Gα13 to drive aberrant growth signaling.Support or Funding InformationWe acknowledge funding from the North Carolina Biotechnology Center, NC GlaxoSmithKline and CD Spangler Foundations, UNC Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The yeast Nem1‐Spo7 phosphatase complex, which dephosphorylates and regulates Pah1 phosphatidate phosphatase, is phosphorylated by protein kinase C
Authors: Dey Prabuddha , Mirheydari Mona M , Han Gil‐Soo , Carman George M 
Published: June 2021

Phosphatidic acid (PA) phosphatase, which catalyzes the conversion of PA to diacylglycerol, is a key regulatory enzyme in lipid metabolism; it plays a major role in controlling the balance between the synthesis of membrane phospholipids and triacylglycerol. Loss of PA phosphatase in yeast, mouse, and human results in a variety of cellular defects and lipid‐based diseases. The yeast PA phosphatase, Pah1, is regulated by its cellular location (cytosolic versus membrane), which is governed by its phosphorylation and dephosphorylation. The dephosphorylation is catalyzed by the Nem1 (catalytic subunit)‐Spo7 (regulatory subunit) phosphatase complex, and this modification facilitates Pah1 interaction with PA at the membrane for its reaction. Like Pah1, Nem1 and Spo7 are subject to phosphorylation, but the protein kinases involved are largely unknown. In this work, we examined the premise that both subunits are substrates for protein kinase C. Using purified reagents from yeast, we showed that protein kinase C phosphorylated Nem1 or Spo7 on multiple serine residues. Using Nem1 or Spo7 as a substrate, protein kinase C activity was dependent on time, the protein kinase, ATP, and substrate, as well as on phosphatidylserine and diacylglycerol. A multifaceted approach using synthetic peptide substrates, site‐specific mutagenesis, phosphopeptide mapping, and mass spectrometry lead to identification of Ser‐201 and Ser‐210 in Nem1 and Ser‐22 and Ser‐28 in Spo7 as the major sites phosphorylated by protein kinase C. Supported by NIH grant GM05679.Support or Funding InformationSupported by NIH grant GM05679.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The lipid acyltransferase LYCAT controls phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) signaling
Authors: Antonescu Costin N , Bone Leslie N , Khuffash Saba , Awadeh Yasmin , Genc Gizem E , Defferrari Marina , Botelho Roberto J 
Published: June 2021

Phosphatidylinositol‐3,4,5‐trisphospate (PIP3) is a potent signaling lipid that exerts key control over cell growth, survival, adhesion and migration. PIP3 signaling is often disrupted in various cancers, making understanding the regulation of PIP3 signaling an important priority. Much has been learned from the study of phosphorylation of the inositol headgroup of phosphoinositides, such as the phosphorylation of phosphatidylinositol‐4,5‐bisphospate (PIP2) to produce PIP3, as well as negative regulation of PIP3 by phosphoinositide phosphatases such as PTEN. However, much less is known about another key dimension of control of phosphoinositide action, the regulation of the fatty acyl profile of these lipids. Indeed, phosphatidylinositol and phosphoinositides exhibit remarkable selectivity of acyl chains (>50% harboring 1‐stearoyl, 2‐arachidonoyl), an acyl profile quite distinct from other phospholipids. This suggests that control of fatty acyl profile of phosphoinositides may be an important determinant of the function of these lipids that has to date remained largely unexplored. We recently uncovered that the acyltransferase LYCAT is a key regulator of the acyl profile of specific phosphoinositides such as PIP2, thus exerting control over membrane traffic phenomena dependent on these phosphoinositides (Bone LN et al 2017 Mol Biol Cell. 28:161–172). We now examine how LYCAT controls PIP3 signaling and PIP3‐dependent control of cell physiology, including the activation of Akt and control of actin dynamics. We find that LYCAT perturbation impairs the activation of Akt and the activation of a number of Akt substrates, which in turn impacts cell growth and survival. Furthermore, LYCAT perturbation elicits dramatic changes in actin filament morphology, cell migration and cancer cell invasion. These results indicate that control of phosphoinositide acyl chain profile is an important novel dimension of regulation of PIP3 signaling, impacting proliferative, growth and migration signaling, and as such may be a novel dimension to control of cancer cell growth and progression.Support or Funding InformationThis work was supported by an Ontario Early Researcher Award, a Canada Research Chair Award, and a Natural Sciences and Engineering Research Council Grant to R.J.B, and a Project Grant and New Investigator Salary Award from the Canadian Institutes of Health Research to C.N.A.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Regulatory Mechanism for Nuclear Lipid Droplet Biogenesis
Authors: Lee Jonghwa , Salsman Jayme , Dellaire Graham , Ridgway Neale 
Published: June 2021

Lipid droplets (LDs) are metabolic energy reservoirs that store and release fatty acids in response to nutrient and hormonal signals. Cytosolic LDs (cLDs) are thought to form in the endoplasmic reticulum (ER) by a process that requires a coordinated synthesis of triglycerides (TG) and phosphatidylcholine (PC), the primary phospholipid component of the surface monolayer that stabilizes the neutral lipid core. Nuclear LDs (nLDs) are present in many eukaryotic cells, suggesting a related mechanism for nLD assembly that involves de novo phospholipid and TG synthesis on the inner nuclear membrane (INM). The rate‐limiting enzyme for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα), is activated by translocation to the INM and nLDs. As well, nLDs and CCTα associate with promyelocytic leukemia nuclear bodies (PML‐NBs). PML‐NBs are dynamic sub‐nuclear structures involved in regulating several important biological processes, including gene expression and oxidative stress response. The storage of TG in nLD/PML‐NB/CCTα complexes could buffer the nucleus against fatty acid overload and detect and respond to lipid stress via PML‐NB‐associated signaling pathways. In this study, we investigated how the nLD/PML‐NB/CCTα complex is formed during oleate treatment of human U2OS osteosarcoma cells and Caco2 colorectal adenocarcinoma cells. Super‐Resolution Radial Fluctuation (SRRF) imaging revealed that PML proteins and CCTα co‐localized in a bead‐like ring around the nLD. Quantitative fluorescence microscopy revealed that PML‐NBs and CCTα were localized on larger nLDs in both U2OS and Caco2 cells. Knocking out PML expression by CRISPR/Cas9 in U2OS cells (PML‐KO) resulted in fewer and smaller nLDs, and the remaining nLDs had a 40% reduction in associated CCTα. The size of CCTα‐positive nLDs in control and PML‐KO cells was comparable. In contrast, CRISPR/Cas9 knockout of CCTα in Caco2 cells (CCTα‐KO) increased the size and decreased the number of LDs, resulting in increased association of PML‐NBs with large nLDs. Collectively, these data suggest that PML‐NBs and CCTα are required for regulating the size and number of nLDs in response to exogenous oleate. Fatty acids promote the formation of large nLDs that interact with PML‐NBs. This interaction recruits CCTα to potentially stimulate CCTα activity and PC biosynthesis required for the surface monolayer.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of a novel small‐molecule activator that corrects G6PD deficiency
Authors: HWANG SUNHEE 
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. However, no medications are available to treat G6PD deficiency directly; thus, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity and/or stability of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation and characterization of a myeloid cell‐specific Cyp19a1 knockout mouse model
Authors: Han Weiguo , kovalchuk Nataliia , Ding Liang , Liu Pengfei , Yang Weizhu , Zhang Qing‐Yu , Ding Xinxin , Winkle Laura 
Published: June 2021

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens. Estrogens have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. It has been proposed that macrophage CYP19A1 can increase estrogen production at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes such as hyperproliferation and oncogenic transformation following xenobiotic‐induced toxicity. Our goal is to test this hypothesis using a cell‐specific Cyp19a1‐null mouse model. Here we report the generation and initial characterization of a myeloid cell (including macrophage) specific Cyp19a1‐null mouse model. A newly generated Cyp19a1‐lox mouse was intercrossed with a Cre‐transgenic mouse for myeloid cell specific deletion of a critical CYP19A1 exon. Cyp19a1 deletion was confirmed by PCR analysis of genomic DNA isolated from macrophages in lung bronchoalveolar lavage fluid; according to results of a quantitative PCR analysis, the deletion efficiency was greater than 90% in these cells. Expression of CYP19A1 protein was detected in alveolar macrophages from wild‐type mice, but not in macrophages from Cyp19a1‐null mice, by immunohistochemical and immunofluorescence staining with an anti‐CYP19A1 antibody. Identity of macrophages was confirmed by staining with an anti‐CD14 (macrophage biomarker) antibody. The ability of macrophages in lung bronchoalveolar lavage fluid from wild‐type mice to synthesize estradiol from testosterone was demonstrated using in vitro assays and LC‐MS based detection of estradiol. The aromatase activity was not detected in macrophages from the null mice. Tissue specificity of Cyp19a1 deletion was demonstrated by the lack of detection of the null allele by a quantitative PCR analysis of genomic DNAs from multiple other sources, including lung, liver, ovary, testis, and tail. Homozygous null mice are fertile and show normal growth and activity. They are currently studied for sensitivity to the lung toxicity of naphthalene, a ubiquitous pollutant and possible human lung carcinogen.Support or Funding InformationSupported in part by NIH grant ES020867This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of the Ca2+‐Mediated Endothelial Cell Migration by Connexin 43‐Formed Hemichannels
Authors: AMADOR HILDA ESPINOZA , CORDOVA ALEXANDRA , FIGUEROA XAVIER 
Published: June 2021

Endothelial cell migration is a key process in endothelium repair, tube formation and angiogenesis. The initiation and progress of endothelial cell migration is orchestrated by the generation of coordinated Ca2+ signals through Ca2+ release from the intracellular stores and Ca2+ entry. However, the molecular pathways involved in the Ca2+ influx have not been determined. Connexins (Cx) play a central role in the coordination of endothelial cell function, directly by cell‐to‐cell communication via gap junction and, indirectly, by the release of autocrine/paracrine signals through Cx‐formed hemmichannels. In addition to the release of signaling molecules, connexin hemichannels are permeable to Ca2+ and, thus, the opening of these channels may contribute to the Ca2+ influx observed during endothelial cell migration. Endothelial cells mainly express Cxs 37, 40 and 43 and, of these three connexin proteins, Cx43 has been reported to form hemichannels in endothelial cells. Interestingly, caveolae seems to be involved in the coordination of the Ca2+‐mediated endothelial cell migration and Cx43 has been found to be associated with caveolin‐1, a structural protein of the caveolae. However, the participation of Cx43 in endothelial cell migration has not been determined. Primary cultures of mesenteric endothelial cells were used to evaluate cell migration by the wound healing assay. Cx43 and caveolin‐1 distribution was assessed by immunofluorescence, activation of Cx hemichannels by measuring ethidium uptake and changes in intracellular Ca2+ concentration [Ca2+]i by loading the cells with Fluo‐4. The scratch of the endothelial cell monolayer with a micropipette tip induced a strong increase in [Ca2+]i and ethidium uptake by cells of the migration front, but not those of the monolayer. Treatment with 18‐b‐Glycyrrhetenic acid (bGA, 50 μM), a general connexin‐formed channel blocker, 37,43GAP27 (200 μM), a Cx37 and Cx43 blocking peptide, and TAT‐Gap19 (300 μM), a Cx43 hemichannels specific blocking peptide, blunted the increase in [Ca2+]i and, consistent with the participation of Cx43 in this process, 37,43GAP27 also reduced the increment in ethidium uptake. In addition, endothelial cell migration was associated with the re‐localization of Cx43 and caveolin‐1 to the rear part of the cells in the migration front, which was prevented by the treatment with TAT‐Gap19. As expected, bGA and TAT‐Gap19 also inhibited the endothelial cell migration rate. These results indicate that Cx43 hemichannels play a central role in the coordination of the Ca2+ signaling that mediates the endothelial cell migration.Support or Funding InformationGrants: FONDECYT 1150530 and CONICYT 21170977This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AIFm2 mediated retrograde stress signaling during ischemia‐reperfusion injury in heart: Role of 4‐HNE adduction
Authors: Sharma Sudha , Bhattarai Susmita , Subedi Utsab , Bess Ezra , Bhuiyan Md.Shenuarin , Panchatcharam Manikandan , Miriyala Sumitra 
Published: June 2021

Myocardial ischemia‐reperfusion injury results from restoration of normal coronary blood circulation after a prolonged period of circulatory arrest. Oxidative stress is one of the underlying mechanism for the pathogenesis of ischemia‐reperfusion injury. Oxidative stress results from a disturbance in the homeostatic balance between free radicle generation and availability of antioxidant molecule to scavenge them. Manganese superoxide dismutase (MnSOD), mitochondrial based antioxidant is capable of converting superoxide radical (O2•−) into oxygen. Reactive oxygen species cause lipid peroxidation and subsequent production of 4‐hydroxy‐2‐nonenal (4‐HNE) from polyunsaturated fatty acid (PUFA). 4‐HNE can modify signaling by causing adduction of cellular components such as proteins and nucleic acids. Mice lacking MnSOD specifically in cardiomyocyte (Myh6‐SOD2Δ) showed early mortality (~ 4 months) due to mitochondrial dysfunction and subsequent cardiomyopathy. Myh6‐SOD2Δ mice also had decreased mitochondrial respiration and cardiac performance shown by Seahorse XF analyzer and M‐mode echocardiography respectively. Following ischemia‐reperfusion injury, MnSOD expression was reduced shown by Real‐Time PCR array, Immunoblot, and Immunohistochemistry. Immunoblot analysis of AIFm2 showed increased expression in the ischemic heart. Normally AIFm2 localized to mitochondria, however, following ischemia 4‐HNE adducted AIFm2 translocated from mitochondria to nucleus thereby binding to DNA and resultant myocardial apoptosis. With the help of mass spectrometric analysis, it has been shown that His 174 and Cys 187 are two sites of AIFm2 where 4‐HNE adduction occurs. These results identify the role of 4‐HNE adduction in the regulation of mitochondrial retrograde signaling mediator protein apoptosis‐inducing factor associated mitochondrion protein 2 (AIFm2) during myocardial ischemia‐reperfusion injury.Support or Funding InformationNIH‐ AA025744; UL1RR033173; HL141998;NISBRE‐ COBRE1P20GM121307‐01A1; AAA‐FGAP‐5148; AHA ‐ 0950118G; 10SDG4190036This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.