125 results on '"Been, Laura E."'
Search Results
2. Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase
- Author
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Tabassum, Zarin, primary, Tseng, Jui-Heng, additional, Isemann, Camryn, additional, Tian, Xu, additional, Chen, Youjun, additional, Herring, Laura E., additional, and Cohen, Todd J., additional
- Published
- 2022
- Full Text
- View/download PDF
3. Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme
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Lee M. Graves, Emily M. Wilkerson, Ronald A. Merrill, Chian Ju Jong, Laura E. Herring, and Stefan Strack
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Models, Molecular ,0301 basic medicine ,Protein subunit ,Phosphatase ,macromolecular substances ,environment and public health ,Biochemistry ,Dephosphorylation ,03 medical and health sciences ,Holoenzymes ,Catalytic Domain ,Heterotrimeric G protein ,Chlorocebus aethiops ,Animals ,Humans ,Protein phosphorylation ,Protein Interaction Maps ,Protein Phosphatase 2 ,Phosphorylation ,Protein kinase A ,Molecular Biology ,030102 biochemistry & molecular biology ,Chemistry ,Cell Biology ,Protein phosphatase 2 ,Cell biology ,Protein Subunits ,HEK293 Cells ,030104 developmental biology ,COS Cells ,Protein Multimerization ,Signal Transduction - Abstract
Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.
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- 2020
4. The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer
- Author
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Diehl, J. Nathaniel, primary, Klomp, Jennifer E., additional, Snare, Kayla R., additional, Hibshman, Priya S., additional, Blake, Devon R., additional, Kaiser, Zane D., additional, Gilbert, Thomas S.K., additional, Baldelli, Elisa, additional, Pierobon, Mariaelena, additional, Papke, Björn, additional, Yang, Runying, additional, Hodge, Richard G., additional, Rashid, Naim U., additional, Petricoin, Emanuel F., additional, Herring, Laura E., additional, Graves, Lee M., additional, Cox, Adrienne D., additional, and Der, Channing J., additional
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- 2021
- Full Text
- View/download PDF
5. Chronic Alcohol Exposure Increases Circulating Bioactive Oxidized Phospholipids
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Yang, Lili, Latchoumycandane, Calivarathan, McMullen, Megan R., Pratt, Brian T., Zhang, Renliang, Papouchado, Bettina G., Nagy, Laura E., Feldstein, Ariel E., and McIntyre, Thomas M.
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- 2010
- Full Text
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6. High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry
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Claudia L.K. Hung, Laura E Bowie, Carly R. Desmond, Tamara Maiuri, Ray Truant, Jianrun Xia, and Susie Son
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Huntingtin ,DNA damage ,Nuclear Localization Signals ,Mutant ,Active Transport, Cell Nucleus ,Importin ,Biochemistry ,03 medical and health sciences ,mental disorders ,Huntingtin Protein ,Humans ,HMGB1 Protein ,Phosphorylation ,Molecular Biology ,Cells, Cultured ,Binding Sites ,030102 biochemistry & molecular biology ,Chemistry ,Endoplasmic reticulum ,Nuclear Proteins ,Molecular Bases of Disease ,Cell Biology ,Fibroblasts ,nervous system diseases ,Cell biology ,030104 developmental biology ,nervous system ,Nuclear transport ,Reactive Oxygen Species ,Nuclear localization sequence ,Protein Binding - Abstract
Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin β1– and 2–dependent proline–tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease.
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- 2019
7. Myc and ChREBP transcription factors cooperatively regulate normal and neoplastic hepatocyte proliferation in mice
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Jie Lu, James M. Dolezal, Huabo Wang, Jordan A. Mandel, Andrew W. Duncan, Frances Alencastro, Laura E. Jackson, Edward V. Prochownik, and Sucheta Kulkarni
- Subjects
Hepatoblastoma ,Ribosomal Proteins ,0301 basic medicine ,Transcription, Genetic ,Pyruvate Dehydrogenase Complex ,Mitochondrion ,Biology ,Biochemistry ,Oxidative Phosphorylation ,Proto-Oncogene Proteins c-myc ,Mice ,03 medical and health sciences ,Liver Neoplasms, Experimental ,Gene expression ,Animals ,Gene Regulation ,RNA, Messenger ,MLX ,Carbohydrate-responsive element-binding protein ,Molecular Biology ,Transcription factor ,Cell Proliferation ,Mice, Knockout ,Glutaminolysis ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Gene Expression Profiling ,Fatty Acids ,Nuclear Proteins ,Cell Biology ,Peroxisome ,Lipid Metabolism ,Cell biology ,Citric acid cycle ,030104 developmental biology ,Hepatocytes ,Transcription Factors - Abstract
Analogous to the c-Myc (Myc)/Max family of bHLH-ZIP transcription factors, there exists a parallel regulatory network of structurally and functionally related proteins with Myc-like functions. Two related Myc-like paralogs, termed MondoA and MondoB/carbohydrate response element–binding protein (ChREBP), up-regulate gene expression in heterodimeric association with the bHLH-ZIP Max-like factor Mlx. Myc is necessary to support liver cancer growth, but not for normal hepatocyte proliferation. Here, we investigated ChREBP's role in these processes and its relationship to Myc. Unlike Myc loss, ChREBP loss conferred a proliferative disadvantage to normal murine hepatocytes, as did the combined loss of ChREBP and Myc. Moreover, hepatoblastomas (HBs) originating in myc(−/−), chrebp(−/−), or myc(−/−)/chrebp(−/−) backgrounds grew significantly more slowly. Metabolic studies on livers and HBs in all three genetic backgrounds revealed marked differences in oxidative phosphorylation, fatty acid β-oxidation (FAO), and pyruvate dehydrogenase activity. RNA-Seq of livers and HBs suggested seven distinct mechanisms of Myc–ChREBP target gene regulation. Gene ontology analysis indicated that many transcripts deregulated in the chrebp(−/−) background encode enzymes functioning in glycolysis, the TCA cycle, and β- and ω-FAO, whereas those dysregulated in the myc(−/−) background encode enzymes functioning in glycolysis, glutaminolysis, and sterol biosynthesis. In the myc(−/−)/chrebp(−/−) background, additional deregulated transcripts included those involved in peroxisomal β- and α-FAO. Finally, we observed that Myc and ChREBP cooperatively up-regulated virtually all ribosomal protein genes. Our findings define the individual and cooperative proliferative, metabolic, and transcriptional roles for the “Extended Myc Network” under both normal and neoplastic conditions.
- Published
- 2018
8. The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer
- Author
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Devon R. Blake, Emanuel F. Petricoin, Thomas S. K. Gilbert, Zane D. Kaiser, Priya S. Hibshman, Elisa Baldelli, Adrienne D. Cox, Channing J. Der, Runying Yang, Lee M. Graves, Jennifer E. Klomp, J. Nathaniel Diehl, Kayla R. Snare, Laura E. Herring, Mariaelena Pierobon, Richard G. Hodge, Naim U. Rashid, and Björn Papke
- Subjects
MAPK/ERK pathway ,extracellular signal–regulated kinase ,mitogen-activated protein kinase ,endocrine system diseases ,STAT3, signal transducer and activator of transcription 3 ,PDAC, pancreatic ductal adenocarcinoma ,pancreatic cancer ,Cell Cycle Proteins ,SHP2, SH2 domain–containing phosphatase-2 ,ERK, extracellular signal–regulated kinase ,medicine.disease_cause ,Biochemistry ,Receptor tyrosine kinase ,DDR1 ,Kinome ,AM, acetoxymethyl ,cancer biology ,biology ,Kinase ,MEKi, MEK inhibitor ,MEK inhibitor ,kinome ,Protein-Tyrosine Kinases ,PLK1, polo-like kinase 1 ,Wee1 ,Mitogen-activated protein kinase ,KRAS ,RTK, receptor tyrosine kinase ,JAK, Janus kinase ,Carcinoma, Pancreatic Ductal ,Research Article ,MAP Kinase Signaling System ,mTOR, mammalian target of rapamycin ,MEK, mitogen-activated protein kinase/ERK kinase ,MIB, multiplexed inhibitor bead ,Proto-Oncogene Proteins p21(ras) ,PI, propidium iodide ,cDNA, complementary DNA ,proteomics ,ERKi, ERK inhibitor ,NCI, National Cancer Institute ,SOS1, SOS Ras/Rac guanine nucleotide exchange factor 1 ,Cell Line, Tumor ,medicine ,Humans ,WEE1 ,Molecular Biology ,RPPA, reverse phase protein array ,TBST, TBS with 0.05% Tween-20 ,LFQ, label-free quantification ,Cell Biology ,digestive system diseases ,EGFR, epidermal growth factor receptor ,Pancreatic Neoplasms ,Mutation ,biology.protein ,Cancer research ,RAS protein ,MAPK, mitogen-activated protein kinase ,DDR1, discoidin domain receptor 1 - Abstract
Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.
- Published
- 2021
9. Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme
- Author
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Jong, Chian Ju, primary, Merrill, Ronald A., additional, Wilkerson, Emily M., additional, Herring, Laura E., additional, Graves, Lee M., additional, and Strack, Stefan, additional
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- 2020
- Full Text
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10. Mass spectrometry–based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells
- Author
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McDonald, Ian M., primary, Grant, Gavin D., additional, East, Michael P., additional, Gilbert, Thomas S.K., additional, Wilkerson, Emily M., additional, Goldfarb, Dennis, additional, Beri, Joshua, additional, Herring, Laura E., additional, Vaziri, Cyrus, additional, Cook, Jeanette Gowen, additional, Emanuele, Michael J., additional, and Graves, Lee M., additional
- Published
- 2020
- Full Text
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11. Sequential adaptive changes in a c-Myc-driven model of hepatocellular carcinoma
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Laura E. Jackson, Edward V. Prochownik, Sucheta Kulkarni, Craig A. Byersdorfer, Sivakama S. Bharathi, Kevin Beezhold, Sarangarajan Ranganathan, Huabo Wang, Eric S. Goetzman, James M. Dolezal, and Jie Lu
- Subjects
Male ,0301 basic medicine ,Carcinoma, Hepatocellular ,DNA Repair ,Carcinogenesis ,medicine.medical_treatment ,Mice, Transgenic ,Biochemistry ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,medicine ,Animals ,Humans ,Glycolysis ,Gene Silencing ,Molecular Biology ,Electron Transport Complex I ,Innate immune system ,biology ,Electron Transport Complex II ,Gene Expression Profiling ,Succinate dehydrogenase ,Growth factor ,Liver Neoplasms ,Cancer ,Molecular Bases of Disease ,Mitochondrial Turnover ,Neoplasms, Experimental ,Cell Biology ,medicine.disease ,Tumor Burden ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Glutamine ,030104 developmental biology ,Liver ,Hepatocellular carcinoma ,biology.protein ,Cancer research ,Female ,Neoplasm Recurrence, Local ,Reactive Oxygen Species ,Function (biology) - Abstract
Hepatocellular carcinoma (HCC) is a common cancer that frequently overexpresses the c-Myc (Myc) oncoprotein. Using a mouse model of Myc-induced HCC, we studied the metabolic, biochemical, and molecular changes accompanying HCC progression, regression, and recurrence. These involved altered rates of pyruvate and fatty acid β-oxidation and the likely re-directing of glutamine into biosynthetic rather than energy-generating pathways. Initial tumors also showed reduced mitochondrial mass and differential contributions of electron transport chain complexes I and II to respiration. The uncoupling of complex II's electron transport function from its succinate dehydrogenase activity also suggested a mechanism by which Myc generates reactive oxygen species. RNA sequence studies revealed an orderly progression of transcriptional changes involving pathways pertinent to DNA damage repair, cell cycle progression, insulin-like growth factor signaling, innate immunity, and further metabolic re-programming. Only a subset of functions deregulated in initial tumors was similarly deregulated in recurrent tumors thereby indicating that the latter can “normalize” some behaviors to suit their needs. An interactive and freely available software tool was developed to allow continued analyses of these and other transcriptional profiles. Collectively, these studies define the metabolic, biochemical, and molecular events accompanyingHCCevolution, regression, and recurrence in the absence of any potentially confounding therapies.
- Published
- 2017
12. The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA
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Laura E. Newman, Anita H. Corbett, Callie P. Wigington, and Kevin J. Morris
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0301 basic medicine ,Cytoplasm ,Protein subunit ,Active Transport, Cell Nucleus ,RNA-binding protein ,Biology ,Poly(A)-Binding Protein I ,Poly(A)-Binding Proteins ,Biochemistry ,03 medical and health sciences ,PABPC1 ,RNA Precursors ,Humans ,ATP5G1 ,RNA Processing, Post-Transcriptional ,Nuclear protein ,Molecular Biology ,Cell Nucleus ,Zinc finger ,Gene knockdown ,ATP synthase ,Nuclear Proteins ,RNA-Binding Proteins ,Cell Biology ,Mitochondrial Proton-Translocating ATPases ,Molecular biology ,030104 developmental biology ,MCF-7 Cells ,Trans-Activators ,biology.protein ,RNA ,RNA Helicases - Abstract
Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function.
- Published
- 2016
13. Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations
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Kant, Sashi, primary, Davuluri, Gangarao, additional, Alchirazi, Khaled A., additional, Welch, Nicole, additional, Heit, Claire, additional, Kumar, Avinash, additional, Gangadhariah, Mahesha, additional, Kim, Adam, additional, McMullen, Megan R., additional, Willard, Belinda, additional, Luse, Donal S., additional, Nagy, Laura E., additional, Vasiliou, Vasilis, additional, Marini, Anna Maria, additional, Weiner, I. David, additional, and Dasarathy, Srinivasan, additional
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- 2019
- Full Text
- View/download PDF
14. High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry
- Author
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Son, Susie, primary, Bowie, Laura E., additional, Maiuri, Tamara, additional, Hung, Claudia L.K., additional, Desmond, Carly R., additional, Xia, Jianrun, additional, and Truant, Ray, additional
- Published
- 2019
- Full Text
- View/download PDF
15. Myc and ChREBP transcription factors cooperatively regulate normal and neoplastic hepatocyte proliferation in mice
- Author
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Wang, Huabo, primary, Dolezal, James M., additional, Kulkarni, Sucheta, additional, Lu, Jie, additional, Mandel, Jordan, additional, Jackson, Laura E., additional, Alencastro, Frances, additional, Duncan, Andrew W., additional, and Prochownik, Edward V., additional
- Published
- 2018
- Full Text
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16. Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis
- Author
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Sanjoy Rowchowdhury, Sangeeta R. Kashyap, John P. Kirwan, Laura E. Nagy, Megan R. McMullen, Becky M. Sebastian, Ariel E. Feldstein, Philip R. Schauer, and Antoinette D. Hillian
- Subjects
Male ,medicine.medical_specialty ,Adipose tissue ,Inflammation ,Biochemistry ,Mice ,Classical complement pathway ,Insulin resistance ,Internal medicine ,medicine ,Animals ,Homeostasis ,Glucose homeostasis ,Anaphylatoxin ,Molecular Biology ,Membrane Glycoproteins ,biology ,Molecular Bases of Disease ,Cell Biology ,medicine.disease ,Dietary Fats ,Receptors, Complement ,Complement system ,Mice, Inbred C57BL ,Glucose ,Endocrinology ,Adipose Tissue ,Liver ,biology.protein ,C3a receptor ,Insulin Resistance ,medicine.symptom - Abstract
Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance. Background: Complement is implicated in obesity and insulin resistance; however, the specific complement activation pathway involved is not known. Results: C1q in the classical pathway is required for activation of complement in response to high fat diets. Conclusion: C1q is an important contributor to high fat diet-induced insulin resistance. Significance: C1q may be an important therapeutic target for treating the derangements in metabolism associated with obesity.
- Published
- 2013
17. Salvador Protein Is a Tumor Suppressor Effector of RASSF1A with Hippo Pathway-independent Functions
- Author
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Farida Latif, Adrianna Henson, Nadia P.C. Allen, Thomas L. Dunwell, Howard Donninger, Laura E. Gordon, Geoffrey J. Clark, Andrew Williams, Jennifer Pogue, and Susannah Kassler
- Subjects
endocrine system ,Apoptosis ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Mice ,Cell Line, Tumor ,Animals ,Humans ,Tumor Protein p73 ,Phosphorylation ,Molecular Biology ,Transcription factor ,Hippo signaling pathway ,Kinase ,Effector ,Tumor Suppressor Proteins ,Cell Cycle ,fungi ,Nuclear Proteins ,Signal transducing adaptor protein ,Cell Biology ,Cell biology ,DNA-Binding Proteins ,body regions ,Drosophila melanogaster ,HEK293 Cells ,Signal transduction ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
The RASSF1A tumor suppressor binds and activates proapoptotic MST kinases. The Salvador adaptor protein couples MST kinases to the LATS kinases to form the hippo pathway. Upon activation by RASSF1A, LATS1 phosphorylates the transcriptional regulator YAP, which binds to p73 and activates its proapoptotic effects. However, although serving as an adaptor for MST and LATS, Salvador can also bind RASSF1A. The functional role of the RASSF1A/Salvador interaction is unclear. Although Salvador is a novel tumor suppressor in Drosophila and mice, its role in human systems remains largely unknown. Here we show that Salvador promotes apoptosis in human cells and that Salvador inactivation deregulates the cell cycle and enhances the transformed phenotype. Moreover, we show that although the salvador gene is seldom mutated or epigenetically inactivated in human cancers, it is frequently down-regulated posttranscriptionally. Surprisingly, we also find that although RASSF1A requires the presence of Salvador for full apoptotic activity and to activate p73, this effect does not require a direct interaction of RASSF1A with MST kinases or the activation of the hippo pathway. Thus, we confirm a role for Salvador as a human tumor suppressor and RASSF1A effector and show that Salvador allows RASSF1A to modulate p73 independently of the hippo pathway.
- Published
- 2011
18. Molecular Mechanism for Adiponectin-dependent M2 Macrophage Polarization
- Author
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Megan R. McMullen, Palash Mandal, Laura E. Nagy, Mark A. Barnes, and Brian T. Pratt
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medicine.medical_specialty ,Innate immune system ,Adiponectin ,medicine.medical_treatment ,Macrophage polarization ,Cell Biology ,Biology ,M2 Macrophage ,Biochemistry ,Cell biology ,Cytokine ,Endocrinology ,Internal medicine ,TLR4 ,medicine ,Tumor necrosis factor alpha ,Signal transduction ,Molecular Biology - Abstract
The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.
- Published
- 2011
19. High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors
- Author
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Steve L. Stice, Raymond S. Hurst, John E. Hambor, Jacob Bradley Schwarz, Robert J. Mather, John D. McNeish, Laura E. Zawadzke, Justin L Gazard, David W. Machacek, Robert A. Volkmann, Christopher J. O’Donnell, John T. Lazzaro, Marsha L. Roach, and Laura Weibley
- Subjects
Chemistry, Pharmaceutical ,Drug Evaluation, Preclinical ,AMPA receptor ,Pharmacology ,Biology ,Models, Biological ,Biochemistry ,Mice ,medicine ,Animals ,Humans ,Technology, Pharmaceutical ,Fluorometry ,Receptors, AMPA ,Receptor ,Induced pluripotent stem cell ,alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ,Molecular Biology ,Embryonic Stem Cells ,Neurons ,Drug discovery ,Glutamate receptor ,Cell Biology ,Immunohistochemistry ,Embryonic stem cell ,Cell biology ,medicine.anatomical_structure ,Receptors, Glutamate ,Drug Design ,Mutation ,Neuron ,Stem cell - Abstract
Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 x 10(6) compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10(6) compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.
- Published
- 2010
20. Suppression of Lipopolysaccharide-stimulated Tumor Necrosis Factor-α Production by Adiponectin Is Mediated by Transcriptional and Post-transcriptional Mechanisms
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Laura E. Nagy, Pil Hoon Park, Palash Mandal, Megan R. McMullen, Honglian Huang, and Lei Sun
- Subjects
Lipopolysaccharides ,medicine.medical_specialty ,Time Factors ,Transcription, Genetic ,Lipopolysaccharide ,RNA Stability ,medicine.medical_treatment ,Biology ,Biochemistry ,Cell Line ,Mice ,chemistry.chemical_compound ,Tristetraprolin ,Internal medicine ,medicine ,Animals ,Humans ,Luciferase ,Promoter Regions, Genetic ,Molecular Biology ,Mitogen-Activated Protein Kinase 1 ,Messenger RNA ,Mitogen-Activated Protein Kinase 3 ,Adiponectin ,Tumor Necrosis Factor-alpha ,Macrophages ,Mechanisms of Signal Transduction ,Cell Biology ,Molecular biology ,Cytokine ,Endocrinology ,chemistry ,Cell culture ,Phosphorylation ,I-kappa B Proteins ,lipids (amino acids, peptides, and proteins) ,Tumor necrosis factor alpha - Abstract
Adiponectin is an adipokine with potent anti-inflammatory properties. Treatment of macrophages with adiponectin results in a suppression of lipopolysaccharide (LPS)-stimulated cytokine production. Here we investigated the transcriptional and post-transcriptional mechanisms by which adiponectin suppresses LPS-stimulated tumor necrosis factor (TNF)-alpha production. Treatment of RAW 264.7 macrophages with LPS increased TNF-alpha promoter-driven luciferase activity (TNF-alpha promoter/Luc activity) by 20-fold over basal. After culture with 1 mug/ml globular adiponectin (gAcrp) for 18 h, TNF-alpha promoter/Luc activity was increased even in the absence of LPS; further challenge with LPS only increased TNF-alpha promoter/Luc activity by 1.4-fold. Treatment with gAcrp decreased LPS-stimulated ERK1/2 phosphorylation and IkappaB degradation and suppressed the ability of LPS to increase the DNA binding activity of Egr-1 and p65. gAcrp also suppressed LPS-mediated stabilization of TNF-alpha mRNA. In controls cells, the half-life of TNF-alpha mRNA was increased from approximately 30 min at base line to approximately 80 min in response to LPS. After treatment with gAcrp for 18 h, LPS failed to increase TNF-alpha mRNA stability. This gAcrp-mediated loss of stimulus-induced stabilization of TNF-alpha mRNA required the presence of the TNF-alpha 3'-untranslated region and was associated with an increase in expression and RNA binding activity of tristetraprolin, an mRNA-binding protein that destabilizes TNF-alpha mRNA. In summary, these data characterize the complex transcriptional and post-transcriptional effects of gAcrp on LPS-stimulated TNF-alpha expression in macrophages. gAcrp treatment profoundly suppressed the ability of LPS to increase TNF-alpha transcription and reduced the stimulus-induced stabilization of TNF-alpha mRNA in response to LPS.
- Published
- 2008
21. A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network
- Author
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Francis, Joshua W., primary, Newman, Laura E., additional, Cunningham, Leslie A., additional, and Kahn, Richard A., additional
- Published
- 2017
- Full Text
- View/download PDF
22. Chronic Ethanol and Triglyceride Turnover in White Adipose Tissue in Rats
- Author
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Brian T. Pratt, James C. Alexander, Xiaocong Chen, Laura E. Nagy, Ilya Bederman, Li Kang, Stephen F. Previs, and Becky M. Sebastian
- Subjects
medicine.medical_specialty ,Triglyceride ,Chemistry ,Insulin ,medicine.medical_treatment ,Adipose tissue ,Cell Biology ,Triglyceride homeostasis ,White adipose tissue ,Glucose clamp technique ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Insulin resistance ,Endocrinology ,Internal medicine ,medicine ,Lipolysis ,Molecular Biology - Abstract
Chronic ethanol consumption disrupts whole-body lipid metabolism. Here we tested the hypothesis that regulation of triglyceride homeostasis in adipose tissue is vulnerable to long-term ethanol exposure. After chronic ethanol feeding, total body fat content as well as the quantity of epididymal adipose tissue of male Wistar rats was decreased compared with pair-fed controls. Integrated rates of in vivo triglyceride turnover in epididymal adipose tissue were measured using (2)H(2)O as a tracer. Triglyceride turnover in adipose tissue was increased due to a 2.3-fold increase in triglyceride degradation in ethanol-fed rats compared with pair-fed controls with no effect of ethanol on triglyceride synthesis. Because increased lipolysis accompanied by the release of free fatty acids into the circulation is associated with insulin resistance and liver injury, we focused on determining the mechanisms for increased lipolysis in adipose tissue after chronic ethanol feeding. Chronic ethanol feeding suppressed beta-adrenergic receptor-stimulated lipolysis in both in vivo and ex vivo assays; thus, enhanced triglyceride degradation during ethanol feeding was not due to increased beta-adrenergic-mediated lipolysis. Instead, chronic ethanol feeding markedly impaired insulin-mediated suppression of lipolysis in conscious rats during a hyperinsulinemic-euglycemic clamp as well as in adipocytes isolated from epididymal and subcutaneous adipose tissue. These data demonstrate for the first time that chronic ethanol feeding increased the rate of triglyceride degradation in adipose tissue. Furthermore, this enhanced rate of lipolysis was due to a suppression of the anti-lipolytic effects of insulin in adipocytes after chronic ethanol feeding.
- Published
- 2007
23. Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression
- Author
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Megan R. McMullen, Honglian Huang, Laura E. Nagy, Varsha Thakur, and Pil Hoon Park
- Subjects
Lipopolysaccharide ,Adiponectin ,medicine.medical_treatment ,Adipokine ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Interleukin 10 ,chemistry.chemical_compound ,Enzyme activator ,Cytokine ,chemistry ,medicine ,Macrophage ,Tumor necrosis factor alpha ,Molecular Biology ,hormones, hormone substitutes, and hormone antagonists - Abstract
Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-α expression in RAW264.7 macrophages; this TNF-α then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-α mRNA accumulation were associated with increased TNF-α promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFκB; mutation of either the Egr-1 or NFκB binding sites in the TNF-α promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IκB super-repressor prevented gAcrp-stimulated TNF-α promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-α promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-α production by macrophages via ERK1/2→Egr-1 and NFκB-dependent mechanisms; these increases in TNF-α in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.
- Published
- 2007
24. The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA
- Author
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Wigington, Callie P., primary, Morris, Kevin J., additional, Newman, Laura E., additional, and Corbett, Anita H., additional
- Published
- 2016
- Full Text
- View/download PDF
25. High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry.
- Author
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Susie Son, Bowie, Laura E., Maiuri, Tamara, Hung, Claudia L. K., Desmond, Carly R., Jianrun Xia, and Truant, Ray
- Subjects
- *
REACTIVE oxygen species , *HUNTINGTIN protein , *NUCLEAR energy , *HUNTINGTON disease , *ENDOPLASMIC reticulum - Abstract
Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin β1– and 2–dependent proline–tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
26. Functional Analysis of the Plant Disease Resistance Gene Pto Using DNA Shuffling
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Laura E. Rose, Neil H. Willits, Richard W Michelmore, Alexander Kozik, Yao Luo, Muriel Guittet, Qilin Pan, Elena Kochetkova, Adriana Bernal, and Jeffrey S. Pollack
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Models, Molecular ,Protein Conformation ,Blotting, Western ,Molecular Sequence Data ,Mutant ,Pseudomonas syringae ,Mutagenesis (molecular biology technique) ,Virulence ,Protein Serine-Threonine Kinases ,Biology ,Genes, Plant ,Biochemistry ,law.invention ,Solanum lycopersicum ,Gene Expression Regulation, Plant ,law ,Two-Hybrid System Techniques ,Point Mutation ,Cloning, Molecular ,Molecular Biology ,Gene ,Gene Library ,Plant Diseases ,Plant Proteins ,Recombination, Genetic ,Genetics ,DNA Shuffling ,DNA ,Cell Biology ,Phenotype ,Immunity, Innate ,Protein Structure, Tertiary ,DNA shuffling ,Plant Leaves ,Genetic Techniques ,Mutagenesis, Site-Directed ,Recombinant DNA ,Plasmids ,Protein Binding ,Rhizobium ,Signal Transduction - Abstract
Pto is a serine/threonine kinase that mediates resistance in tomato to strains of Pseudomonas syringae pv. tomato expressing the (a)virulence proteins AvrPto or AvrPtoB. DNA shuffling was used as a combinatorial in vitro genetic approach to dissect the functional regions of Pto. The Pto gene was shuffled with four of its paralogs from a resistant haplotype to create a library of recombinant products that was screened for interaction with AvrPto in yeast. All interacting clones and a representative sample of noninteracting clones were sequenced, and their ability to signal downstream was tested by the elicitation of a hypersensitive response in an AvrPto-dependent or -independent manner in planta. Eight candidate regions important for binding to AvrPto or for downstream signaling were identified by statistical correlations between individual amino acid positions and phenotype. A subset of the regions had previously been identified as important for recognition, confirming the validity of the shuffling approach. Three novel regions important for Pto function were validated by site-directed mutagenesis. Several chimeras and point mutants exhibited a differential interaction with (a)virulence proteins in the AvrPto and VirPphA family, demonstrating distinct binding requirements for different ligands. Additionally, the identification of chimeras that are both constitutively active as well as capable of binding AvrPto indicates that elicitation of downstream signaling does not involve a conformational change that precludes binding of AvrPto, as previously hypothesized. The correlations between phenotypes and variation generated by DNA shuffling paralleled natural variation observed between orthologs of Pto from Lycopersicon spp.
- Published
- 2005
27. Ric-3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7 Subunit in Mammalian Cells
- Author
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Laura E. Chavez-Noriega, Charles J. Cohen, Mark E. Williams, Bill Burton, Jayashree Aiyar, Arturo Urrutia, and Anatoly Shcherbatko
- Subjects
alpha7 Nicotinic Acetylcholine Receptor ,Xenopus ,Receptor expression ,Blotting, Western ,Receptors, Nicotinic ,Biology ,Biochemistry ,Cell Line ,Ganglion type nicotinic receptor ,hemic and lymphatic diseases ,Muscarinic acetylcholine receptor ,Muscarinic acetylcholine receptor M5 ,Animals ,Humans ,Immunoprecipitation ,RNA, Messenger ,Receptor ,Molecular Biology ,Neurons ,HEK 293 cells ,Electric Conductivity ,Intracellular Signaling Peptides and Proteins ,Proteins ,Cell Biology ,Bungarotoxins ,Molecular biology ,Rats ,Protein Subunits ,Nicotinic acetylcholine receptor ,Gene Expression Regulation ,Oocytes ,Alpha-4 beta-2 nicotinic receptor ,Protein Binding - Abstract
Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.
- Published
- 2005
28. Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition
- Author
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Nikolai Lissin, Emma Gostick, Bruno Laugel, Annelise Vuidepot, Daniel C. Douek, Joris Hemelaar, David Price, Andrew K. Sewell, Bent K. Jakobsen, Yi Li, Jonathan M. Boulter, and Laura E. Crotty
- Subjects
Antigen Targeting ,T-Lymphocytes ,T cell ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,Cross Reactions ,Major histocompatibility complex ,Biochemistry ,Epitope ,Antigen ,medicine ,Humans ,Cytotoxic T cell ,Antigens ,Antigen-presenting cell ,Molecular Biology ,biology ,T-cell receptor ,Cell Biology ,Surface Plasmon Resonance ,Flow Cytometry ,Molecular biology ,Recombinant Proteins ,Kinetics ,medicine.anatomical_structure ,Solubility ,biology.protein - Abstract
The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.
- Published
- 2005
29. Interleukin (IL)-15 and IL-2 Reciprocally Regulate Expression of the Chemokine Receptor CX3CR1 through Selective NFAT1- and NFAT2-dependent Mechanisms
- Author
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Laura E. Via, Jana Barlic, David H. McDermott, Maya N. Merrell, Philip M. Murphy, and Jacqueline Gonzales
- Subjects
Time Factors ,Transcription, Genetic ,Biochemistry ,Mice ,Chemokine receptor ,Genes, Reporter ,Luciferases ,Promoter Regions, Genetic ,Interleukin-15 ,Regulation of gene expression ,Mice, Inbred BALB C ,Nuclear Proteins ,Interleukin ,NFAT ,Exons ,Chromatin ,Recombinant Proteins ,Up-Regulation ,DNA-Binding Proteins ,Interleukin 15 ,Receptors, Chemokine ,Plasmids ,medicine.drug ,Interleukin 2 ,Chromatin Immunoprecipitation ,Molecular Sequence Data ,CX3C Chemokine Receptor 1 ,Mice, Transgenic ,Biology ,Transfection ,Cell Line ,Open Reading Frames ,medicine ,Animals ,Humans ,Immunoprecipitation ,Molecular Biology ,Transcription factor ,Crosses, Genetic ,Base Sequence ,Dose-Response Relationship, Drug ,Models, Genetic ,NFATC Transcription Factors ,Membrane Proteins ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Gene Expression Regulation ,Mutagenesis ,Mutation ,Leukocytes, Mononuclear ,Interleukin-2 ,RNA ,Transcription Factors - Abstract
We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2Rbetagamma, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a kappaB-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the kappaB-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1-/- mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a kappaB-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.
- Published
- 2004
30. Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens
- Author
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John B. Rafferty, Patrice Courvalin, Iain A. Murray, Laura E. Kehoe, and Jaruwaree Snidwongse
- Subjects
Models, Molecular ,Streptogramins ,Protein Conformation ,Stereochemistry ,medicine.medical_treatment ,Enterococcus faecium ,Drug Resistance ,Streptogramin ,Dalfopristin ,Crystallography, X-Ray ,Ligands ,Virginiamycin ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Acetyltransferases ,polycyclic compounds ,medicine ,Histidine ,Molecular Biology ,Gram-Positive Bacterial Infections ,Alanine ,Binding Sites ,biology ,Quinupristin ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,Anti-Bacterial Agents ,Enzyme binding ,Quinupristin/dalfopristin ,Models, Chemical ,chemistry ,Mutagenesis, Site-Directed ,Protein Binding ,medicine.drug - Abstract
Synercid®, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 A resolution in the absence of ligands, at 2.8 A resolution bound to dalfopristin, and at 3.0 A resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >105-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid® resistance.
- Published
- 2003
31. Calmodulin Oxidation and Methionine to Glutamine Substitutions Reveal Methionine Residues Critical for Functional Interaction with Ryanodine Receptor-1
- Author
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David D. Thomas, Bradley R. Fruen, Razvan L. Cornea, Rachel A. Bloomquist, Edward M. Balog, Charles F. Louis, D.J. Black, and Laura E. Norton
- Subjects
animal structures ,Calmodulin ,Swine ,Glutamine ,Protein subunit ,Biochemistry ,Mass Spectrometry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Methionine ,medicine ,Animals ,Molecular Biology ,RYR1 ,biology ,Ryanodine receptor ,Circular Dichroism ,Endoplasmic reticulum ,Skeletal muscle ,Ryanodine Receptor Calcium Release Channel ,Cell Biology ,musculoskeletal system ,Sarcoplasmic Reticulum ,medicine.anatomical_structure ,chemistry ,biology.protein ,Calcium ,Electrophoresis, Polyacrylamide Gel ,Oxidation-Reduction ,tissues - Abstract
Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.
- Published
- 2003
32. Nutritional Control of mRNA Stability Is Mediated by a Conserved AU-rich Element That Binds the Cytoplasmic Shuttling Protein HuR
- Author
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Ibrahim Yaman, Bedabrata Sarkar, Martin D. Snider, James Fernandez, Laura E. Nagy, Maria Hatzoglou, and Robert J. Schneider
- Subjects
Cytoplasm ,RNA Stability ,Molecular Sequence Data ,ELAV-Like Protein 1 ,Electrophoretic Mobility Shift Assay ,RNA-binding protein ,Biology ,Biochemistry ,Article ,Tumor Cells, Cultured ,Protein biosynthesis ,Animals ,RNA, Messenger ,Amino acid transporter ,Amino Acids ,3' Untranslated Regions ,Molecular Biology ,Cationic Amino Acid Transporter 1 ,chemistry.chemical_classification ,AU-rich element ,Base Sequence ,RNA-Binding Proteins ,Translation (biology) ,Cell Biology ,MRNA stabilization ,Rats ,Amino acid ,ELAV Proteins ,chemistry ,Antigens, Surface - Abstract
The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3′-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.
- Published
- 2002
33. Chronic Ethanol Increases Lipopolysaccharide-stimulated Egr-1 Expression in RAW 264.7 Macrophages
- Author
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Megan R. McMullen, Liang Shi, Raj Kishore, and Laura E. Nagy
- Subjects
Messenger RNA ,Ethanol ,Lipopolysaccharide ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,chemistry ,Transcription (biology) ,Phosphorylation ,Tumor necrosis factor alpha ,Binding site ,Molecular Biology ,Transcription factor - Abstract
Increased production of tumor necrosis factor α (TNFα) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor α (TNFα) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFα promoter activity contribute to increased TNFα expression. Binding of Egr-1 to theTNFα promoter was increased by 2.5-fold after ethanol exposure, whereas NFκB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFκB site to LPS-stimulated TNFα promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFα mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulatedEgr-1 promoter-driven CAT expression and transcription ofEgr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFα. Increased Egr-1 promoter activity and TNFα mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFα production after chronic ethanol exposure.
- Published
- 2002
34. Stabilization of Tumor Necrosis Factor α mRNA by Chronic Ethanol
- Author
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Megan R. McMullen, Laura E. Nagy, and Raj Kishore
- Subjects
Lipopolysaccharide ,Kinase ,p38 mitogen-activated protein kinases ,Cell Biology ,MRNA stabilization ,Biology ,Biochemistry ,Molecular biology ,Pathogenesis ,chemistry.chemical_compound ,chemistry ,Mitogen-activated protein kinase ,biology.protein ,Tumor necrosis factor alpha ,Signal transduction ,Molecular Biology - Abstract
Increased expression of tumor necrosis factor α (TNFα) in response to chronic ethanol has been implicated in the pathogenesis of alcoholic liver disease. However, the molecular mechanisms by which ethanol increases the levels of TNFα are not well characterized. Utilizing Kupffer cells isolated from rats fed an ethanol containing diet and a murine macrophage cell line, RAW264.7, exposed to ethanol in culture, we have demonstrated that exposure to chronic ethanol results in an enhanced expression of lipopolysaccharide (LPS)-induced TNFα. While chronic ethanol had no effect on the rate of LPS-induced TNFα transcription as measured by nuclear run-on experiments, TNFα mRNA half-life was increased by chronic ethanol. Chronic ethanol also potentiated the activation of LPS-induced p38 mitogen-activated protein (MAP) kinase in Kupffer cells, as well as in RAW264.7 cells. Specific inhibition of p38 MAP kinase activation by SB203580 in Kupffer cells or by overexpression of dominant negative p38 MAP kinase in RAW264.7 cells blocked ethanol-mediated TNFα mRNA stabilization. Furthermore, using chimeric reporter constructs, we have shown that A + U-rich elements in the 3′-untranslated region of TNFα mRNA are not sufficient to impart ethanol-mediated stabilization on TNFα mRNA.
- Published
- 2001
35. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains
- Author
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Brown, Laura E., primary, Nicholson, Martin W., additional, Arama, Jessica E., additional, Mercer, Audrey, additional, Thomson, Alex M., additional, and Jovanovic, Jasmina N., additional
- Published
- 2016
- Full Text
- View/download PDF
36. Brk, a Breast Tumor-derived Non-receptor Protein-tyrosine Kinase, Sensitizes Mammary Epithelial Cells to Epidermal Growth Factor
- Author
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Helen E. Jolin, Martin J. Page, Barry A. Gusterson, Laura E. Jackson, Christopher J. Dean, Philip Mitchell, Mark R. Crompton, Karen Barker, and Tahereh Kamalati
- Subjects
Biology ,Biochemistry ,Epithelium ,3T3 cells ,Mice ,Epidermal growth factor ,medicine ,Animals ,Humans ,Breast ,Kinase activity ,Receptor ,Molecular Biology ,Cells, Cultured ,Epidermal Growth Factor ,Kinase ,3T3 Cells ,Cell Biology ,Protein-Tyrosine Kinases ,Neoplasm Proteins ,ErbB Receptors ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Cancer research ,Female ,PTK6 ,Tyrosine kinase ,Cell Division ,Proto-oncogene tyrosine-protein kinase Src - Abstract
brk (breast tumor kinase) shows homology to the src family of non-receptor protein-tyrosine kinases and is expressed in breast carcinomas. In order to investigate the role of brk in breast tumor development, we have examined the growth and transformation properties of human mammary epithelial cells engineered to overexpress Brk. Interestingly, like c-Src, overexpression of Brk leads to sensitization to EGF, and also results in a partially transformed phenotype. Further investigation of the latter activity was attempted by mutational analysis, targeting key residues known to affect tyrosine kinase activity in Src-like kinases. Mutation of amino acid residue Lys-219 to Met, by analogy to Src, abolished both kinase activity and transformation capacity. Mutation of amino acid residue Tyr-447 to Phe, however, resulted in a decrease in transforming potential without affecting kinase activity. These results suggest that while Src and Brk share some functional properties, they act differently during transformation. These differences are discussed in the context of the mechanisms underlying breast cancer development.
- Published
- 1996
37. NMR studies of peptides derived from the putative binding regions of cartilage proteins. No evidence for binding to hyaluronan
- Author
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Laura E. Lerner, P J Hajduk, David A. Horita, and Paul F. Goetinck
- Subjects
Circular dichroism ,Protein structure ,Biochemistry ,Tandem repeat ,Chemistry ,Sequence analysis ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Binding site ,Molecular Biology ,Protein secondary structure ,Peptide sequence - Abstract
Previous work has implicated sequences within the tandem repeats of cartilage link protein in the interaction of link protein with hyaluronan. This conclusion was based on competitive inhibition experiments using synthetic peptides (Goetinck, P. F., Stirpe, N. S., Tsonis, P. A., and Carlone, D. (1987) J. Cell Biol. 105, 2403-2407). Further investigation of this system using high resolution nuclear magnetic resonance, circular dichroism, and competitive inhibition with other peptides indicates that the previously observed inhibition of link protein-hyaluronan binding was not caused by peptide-hyaluronan interactions. Instead, nonspecific aggregation of the peptides with link protein is proposed to account for all of the experimental data. Consequently, there is no direct experimental evidence to support the conclusion that these sequences in the tandem repeats of link protein are responsible for the link protein-hyaluronan interaction. If these peptides do represent the hyaluronan binding regions of link protein, these results imply a highly structure-dependent interaction between link protein and hyaluronan. Conformational analysis of the peptides using two-dimensional nuclear Overhauser spectroscopy indicates that the linear peptides do not adopt any stable secondary structure. However, several residues in the disulfide-looped peptides exhibit connectivities, suggesting a relatively long-lived extended chain conformation, consistent with predictions of secondary structure based on sequence analysis.
- Published
- 1994
38. Mapping the IκB kinase β (IKKβ)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKβ-mediated activation of nuclear factor κB
- Author
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Stephen C. Graham, Geoffrey L. Smith, Camilla T.O. Benfield, Jonathan M. Grimes, Laura E. McCoy, M.W. Bahar, Asa P. Oldring, Daniel S. Mansur, Brian J. Ferguson, and David I. Stuart
- Subjects
Viral protein ,Protein subunit ,Mutant ,Active Transport, Cell Nucleus ,Mutation, Missense ,Vaccinia virus ,IκB kinase ,Biology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Viral Proteins ,Gene expression ,medicine ,Viral Protein ,Humans ,NF-kappaB ,Receptor ,Molecular Biology ,Transcription factor ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,030302 biochemistry & molecular biology ,NF-kappa B ,Cell Biology ,Innate Immunity ,Cell biology ,I-kappa B Kinase ,HEK293 Cells ,Amino Acid Substitution ,Protein Structure and Folding ,biology.protein ,Immunosuppressor ,Pox Viruses ,Protein Multimerization ,Protein A ,Hydrophobic and Hydrophilic Interactions - Abstract
The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to proinflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
- Published
- 2011
39. Regulation of interleukin 8 gene expression by oxidant stress
- Author
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A M Preston, Daniel G. Remick, J Kenney, Laura E. DeForge, Laurence A. Boxer, and E. Takeuchi
- Subjects
Lipopolysaccharide ,Activator (genetics) ,medicine.medical_treatment ,Inflammation ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,Cytokine ,Immune system ,chemistry ,Gene expression ,Immunology ,medicine ,Tumor necrosis factor alpha ,Interleukin 8 ,medicine.symptom ,Molecular Biology - Abstract
Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.
- Published
- 1993
40. Utilization of an alternative transcription initiation site of somatic cytochrome c in the mouse produces a testis-specific cytochrome c mRNA
- Author
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Norman B. Hecht and Laura E. Hake
- Subjects
Regulation of gene expression ,Untranslated region ,Messenger RNA ,Cytochrome ,Cytochrome c ,Promoter ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,biology.protein ,RNase H ,Molecular Biology ,Gene - Abstract
The differential regulation of somatic and testis-specific cytochromes c during spermatogenesis in the mouse is accompanied by changes in mRNA length (Hake, L. E., Alcivar, A. A., and Hecht, N. B. (1990) Development 110, 249-257). In spermatogenic stem cells through early meiotic cells, we detect four somatic cytochrome c (cyt cs) mRNAs of 1.3, 1.1, and 0.7-0.5 kilobases (kb), whereas in postmeiotic cells we detect a larger cyt cs mRNA of 1.7 kb. Oligonucleotide-directed RNase H cleavage of cyt cs mRNA revealed that the 1.7-kb mRNA contains over 1 kb of 5'-untranslated region which is not present in the four shorter cyt cs mRNAs. RNase protection assays indicate that this additional sequence arises from the utilization of an alternative transcription initiation site of the functional cyt cs gene which is 1085 base pairs upstream of the initiation site for the four shorter cyt cs mRNAs. To analyze the promoter for the 1.7-kb mRNA, a genomic clone containing the cyt cs gene and 5 kb of 5'-flanking DNA was isolated. Sequence comparison of the putative promoter region with promoters of other postmeiotically expressed genes reveals several conserved regions. Utilization of this alternative initiation site may be involved in the down-regulation of cytochrome cs during spermatogenesis.
- Published
- 1993
41. The Essential Autophagy Gene ATG7 Modulates Organ Fibrosis via Regulation of Endothelial-to-Mesenchymal Transition
- Author
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Singh, Krishna K., primary, Lovren, Fina, additional, Pan, Yi, additional, Quan, Adrian, additional, Ramadan, Azza, additional, Matkar, Pratiek N., additional, Ehsan, Mehroz, additional, Sandhu, Paul, additional, Mantella, Laura E., additional, Gupta, Nandini, additional, Teoh, Hwee, additional, Parotto, Matteo, additional, Tabuchi, Arata, additional, Kuebler, Wolfgang M., additional, Al-Omran, Mohammed, additional, Finkel, Toren, additional, and Verma, Subodh, additional
- Published
- 2015
- Full Text
- View/download PDF
42. Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly
- Author
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Kent A. Willadsen, Larry S. Tobacman, Laura E. Hill, and Carol A. Butters
- Subjects
Troponin T ,Cooperativity ,macromolecular substances ,Cell Biology ,Biology ,musculoskeletal system ,Biochemistry ,Troponin ,Tropomyosin ,Molecular biology ,Tropomyosin binding ,Troponin I ,Biophysics ,biology.protein ,Binding site ,tissues ,Molecular Biology ,Actin - Abstract
Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.
- Published
- 1992
43. Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules
- Author
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Laura E. Hill, Larry S. Tobacman, John P. Mehegan, and Carol A. Butters
- Subjects
chemistry.chemical_classification ,genetic structures ,biology ,Troponin T ,Cooperativity ,macromolecular substances ,Cell Biology ,musculoskeletal system ,Biochemistry ,Troponin ,Tropomyosin ,Amino acid ,Tropomyosin binding ,chemistry ,Troponin I ,biology.protein ,tissues ,Molecular Biology ,Actin - Abstract
The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.
- Published
- 1992
44. Increased levels of junB and c-jun mRNAs in male germ cells following testicular cell dissociation. Maximal stimulation in prepuberal animals
- Author
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Laura E. Hake, Acacia A. Alcivar, Norman B. Hecht, and M P Hardy
- Subjects
Messenger RNA ,JUNB ,Cellular differentiation ,c-jun ,Cell Biology ,Testicle ,Biology ,Biochemistry ,Molecular biology ,Interstitial cell ,medicine.anatomical_structure ,MRNA polyadenylation ,hemic and lymphatic diseases ,medicine ,Northern blot ,Molecular Biology - Abstract
We have examined the relative transcript levels of the junB and c-jun proto-oncogenes during development of the mouse testis. junB and c-jun mRNA levels are low in total RNA from intact immature or mature testes. Dissociation of testicular cells, however, increases the levels of junB and c-jun mRNAs, with higher increases in the dissociated cells from testes of 8-day-old mice than from 17-day-old or sexually mature mice. These differences in junB and c-jun mRNA levels localize to specific cell types. In testes from 8-day-old mice, the mRNA levels for both proto-oncogenes are higher in type B spermatogonia and in the interstitial cell fraction than in type A spermatogonia. In testes of 17-day-old mice, the highest mRNA levels for both proto-oncogenes are seen in preleptotene spermatocytes and interstitial cells, with decreasing levels in leptotene/zygotene spermatocytes and prepuberal pachytene spermatocytes. junB and c-jun mRNAs are nearly undetectable in pachytene spermatocytes, round spermatids, and residual bodies/cytoplasts. The increased junB mRNA levels originate not only from the expected 2.1-kilobase transcript but from a more slowly migrating transcript of about 2.3 kilobases. RNase H analysis demonstrates that this migration change was due to an increase in mRNA polyadenylation. The low levels of junB and c-jun mRNAs in intact testes and the much higher levels in isolated cells from identical testes suggest that the disruption of cell-to-cell contact increases the amount of junB and c-jun transcripts in specific cells of the testis. Coupled with this increase, structural changes are seen with the junB mRNA.
- Published
- 1990
45. Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis
- Author
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Frank M. Raushel, Laura E. Post, and David J. Post
- Subjects
ATP synthase ,Active site ,Cell Biology ,Biology ,Carbamyl Phosphate ,Carbamoyl phosphate synthetase ,Biochemistry ,chemistry.chemical_compound ,Adenosine diphosphate ,chemistry ,Carbamoyl phosphate ,biology.protein ,Site-directed mutagenesis ,Molecular Biology ,Adenosine triphosphate - Abstract
The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.
- Published
- 1990
46. Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter
- Author
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Laura E. Nagy, Adrienne S. Gordon, Christopher Franklin, David J. Casso, and Ivan Diamond
- Subjects
Adenosine transport ,biology ,Cell Biology ,Nucleoside transporter ,Purinergic signalling ,Biochemistry ,Isoleucine transport ,Adenosine ,Adenylyl cyclase ,Adenosine A1 receptor ,chemistry.chemical_compound ,chemistry ,biology.protein ,Extracellular ,medicine ,Molecular Biology ,medicine.drug - Abstract
Chronic exposure to ethanol results in heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory guanine nucleotide regulatory protein. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization (Nagy, L. E., Diamond, I., Collier, K., Lopez, L., Ullman, B., and Gordon, A. S., Mol. Pharmacol., in press). To understand the mechanism underlying ethanol-induced increases in extracellular adenosine, we examined the interaction of ethanol with the adenosine transport system in S49 lymphoma cells. We found that ethanol inhibited nucleoside uptake without affecting deoxyglucose or isoleucine transport. Inhibition of adenosine uptake was due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake. Consequently, ethanol no longer increased extracellular adenosine concentrations. Taken together with our previous studies, these results suggest that ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which causes an initial increase in intracellular cAMP levels and subsequent development of heterologous desensitization of cAMP signal transduction.
- Published
- 1990
47. Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly
- Author
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Mitsche, Matthew A., primary, Packer, Laura E., additional, Brown, Jeffrey W., additional, Jiang, Z.Gordon, additional, Small, Donald M., additional, and McKnight, C.James, additional
- Published
- 2014
- Full Text
- View/download PDF
48. Mice lacking C1q are protected from high fat diet-induced hepatic insulin resistance and impaired glucose homeostasis.
- Author
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Hillian, Antoinette D., primary, McMullen, Megan R., additional, Sebastian, Becky M., additional, Roychowdhury, Sanjoy, additional, Kashyap, Sangeeta R., additional, Schauer, Philip R., additional, Kirwan, John P., additional, Feldstein, Ariel E., additional, and Nagy, Laura E., additional
- Published
- 2013
- Full Text
- View/download PDF
49. Mice lacking C1q are protected from high fat diet-induced hepatic insulin resistance and impaired glucose homeostasis
- Author
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Megan R. McMullen, Antoinette D. Hillian, Philip R. Schauer, Laura E. Nagy, Ariel E. Feldstein, Sangeeta R. Kashyap, John P. Kirwan, Becky M. Sebastian, and Sanjoy Roychowdhury
- Subjects
medicine.medical_specialty ,Endocrinology ,Insulin resistance ,Chemistry ,Internal medicine ,medicine ,Glucose homeostasis ,Additions and Corrections ,High fat diet ,Cell Biology ,medicine.disease ,Molecular Biology ,Biochemistry - Published
- 2013
50. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains.
- Author
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Brown, Laura E., Nicholson, Martin W., Arama, Jessica E., Mercer, Audrey, Thomson, Alex M., and Jovanovic, Jasmina N.
- Subjects
- *
GABA receptors , *N-terminal residues , *GABAERGIC neurons , *SYNAPSES , *SYNAPTOGENESIS , *CELL culture - Abstract
The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type ofα/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, andγ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs andα1/β2/γ2-expressing HEK293 cells, theα1,β2, orγ2 ECDeach caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
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