Protein Microcharacterization

蛋白质微观表征

• 批准号: 8554148
• 负责人: Jason Williams
• 金额: $ 74.79万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554148
• 关键词: ATP Synthesis Pathway; Affinity; Allergens; Arginine; Arrhythmia; Binding; Cardiac Death; Cell physiology; Cells; Cellular Stress; Chromatin; Code; Collaborations; Complex; Congenital Heart Defects; Consensus; Consumption; Core Facility; Coupled; DNA Repair; DNA biosynthesis; Data; Defect; Detection; Disease; Energy Metabolism; Ethers; Failure; Family member; Feedback; Flagella; GTP-Binding Proteins; Gene Expression; Gene Expression Regulation; Genes; Germ Cells; Glucocorticoid Receptor; Glucocorticoids; Glucose; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Hand; Histidine; Histocompatibility Testing; Homeostasis; Hormones; Human; Immunoassay; Inositol; Intramural Research; Ion Channel; Isoenzymes; Label; Laboratories; Lactate Dehydrogenase; Lead; Link; Lipids; London; Maintenance; Male Infertility; Mass Spectrum Analysis; Mediating; Membrane; Metabolic Diseases; Modification; Molecular; Molecular Weight; Monitor; Mutation; NADH; National Institute of Environmental Health Sciences; Nuclear Magnetic Resonance; Nuclear Receptors; P-Selectin; Peanuts - dietary; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Modification Site; Post-Translational Protein Processing; Process; Promoter Regions; Protein Binding; Protein C; Protein Isoforms; Protein Kinase C; Proteins; Publishing; Pyruvate; Recovery; Research; Research Personnel; Residual state; Resources; Role; Sampling; Scientist; Services; Signal Transduction; Signaling Protein; Single Nucleotide Polymorphism; Site; Sodium; Stimulus; Stress; Sudden Death; Surface; Techniques; Testing; Time; Work; base; cell motility; cofactor; density; genetic manipulation; genome-wide; human MAPK14 protein; inhibitor/antagonist; mutant; promoter; protein expression; response; sensor; sperm cell; sperm function; stressor

A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF) with approximately 6000 samples analyzed from 57 scientists representing 29 principle investigators from 8 laboratory branches. One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases - Steve Shears Identification and quantitation of lipidation on G-proteins C Lutz Birnbaumer Identification of binding partners and post-translational modification of proteins that may be involved in DNA replication and repair - Sam Wilson Identification of proteins in the BAF complexes under a variety of tissue types and/or conditions - Trevor Archer P-selectin interaction partners and changes in phosphoproteome upon treatment C Steve Akiyama Glis family members modifications and binding partners C Anton Jetten Characterization of the PTMs of peanut allergens before and after processing (roasting) C Geoffrey Mueller and Bob London The core also is performing value added research in affinity techniques (GFP-based enrichment) to aid in protein, protein binding partner, and PTM identifications. Other recently published projects or projects in press include: LDHC: Germ cell-specific lactate dehydrogenase C gene (Ldhc) leads to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. -Mitch Eddy GR: Glucocorticoids are stress hormones that maintain homeostasis through gene regulation mediated by nuclear receptors. We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Cells expressing a mutant glucocorticoid receptor incapable of phosphorylation at Ser134 (S134A-GR) had significantly altered hormone-dependent genome-wide transcriptional responses and associated hormone-mediated cellular functions. The phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform of the 14-3-3 class of signaling proteins (14-3-3zeta) on chromatin promoter regions, resulting in a blunted hormone-dependent transcriptional response of select genes. These data argue that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to redirect glucocorticoid-regulated signaling through altered 14-3-3zeta cofactor binding and promoter recruitment. This posttranslational modification allows prior cellular stress signals to dictate the transcriptional response to glucocorticoids. C John Cidlowski Kv11.1: Mutations that inhibit Kv11.1 ion channel activity contribute to abnormalities of cardiac repolarization that can lead to long QT2 (LQT2) cardiac arrhythmias and sudden death. However, for most of these mutations, nothing is known about the molecular mechanism linking Kv11.1 malfunction to cardiac death. We have previously demonstrated that disease-related mutations that create consensus sites for kinases on ion channels can dramatically change ion channel activity. Here, we show that a LQT2-associated mutation can inhibit Kv11.1 ion channel activity by perturbing a consensus site for the Ser/Thr protein kinase C (PKC). We first reveal by mass spectrometry analysis that Ser890 of the Kv11.1 ion channel is phosphorylated. Then, we demonstrate by a phospho-detection immunoassay combined with genetic manipulation that PKC phosphorylates Ser890. Furthermore, we show that Ser890 phosphorylation is associated with an increase in Kv11.1 membrane density with alteration of recovery from inactivation. In addition, a newly discovered and as yet uncharacterized LQT2-associated nonsynonymous single nucleotide polymorphism 2660 GA within the human ether--go-go-related gene 1 coding sequence, which replaces arginine 887 with a histidine residue (R887H), strongly inhibits PKC-dependent phosphorylation of residue Ser890 on Kv11.1, and ultimately inhibits surface expression and current density. Taken together, our data provide a functional link between this channel mutation and LQT2. C Saverio Genitile (work previously performed by the PMCF in collaboration with Dave Armstrong) Additional projects that have required more than negligible resources include efforts performed with the Armstrong, Blackshear, Hall, Hu, Fessler, J. Mason, and R.S. Williams laboratories.

蛋白质微表征核心设施（PMCF）已经或正在开展蛋白质表征方面的各种服务和合作项目，分析了来自8个实验室分支的57名科学家代表29名主要研究人员的约6000份样品。