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.

蛋白质表征方面的各种服务和合作项目已经或正在利用蛋白质微表征核心设施 (PMCF) 进行，分析了来自 8 个实验室分支机构的 29 位主要研究人员的 57 名科学家的约 6000 个样品。 其中一项重大工作是支持蛋白质表达核心设施 (PECF) 和 Bob Petrovich 博士。 PMCF 的作用是在 PECF 将材料交给用户之前在蛋白质水平上确认基因表达。 其他仍在进行中的未发布项目包括： 脂质和肌醇激酶上结合伴侣和翻译后修饰 (PTM) 位点的鉴定 - Steve Shears G 蛋白 C Lutz Birnbaumer 脂化的鉴定和定量 鉴定可能参与 DNA 复制和修复的结合伴侣和蛋白质翻译后修饰 - Sam Wilson 在各种组织类型和/或条件下鉴定 BAF 复合物中的蛋白质 - Trevor Archer P-选择素相互作用伙伴和治疗后磷酸化蛋白质组的变化 C Steve Akiyama Glis 家族成员修饰和结合伙伴 C Anton Jetten 花生过敏原加工（烘烤）前后 PTM 的表征 C Geoffrey Mueller 和 Bob London 该核心还在亲和技术（基于 GFP 的富集）方面进行增值研究，以帮助蛋白质、蛋白质结合伴侣和 PTM 鉴定。 其他最近发表的项目或正在出版的项目包括： LDHC：生殖细胞特异性乳酸脱氢酶 C 基因 (Ldhc) 由于精子功能缺陷而导致男性不育，包括精子 ATP 水平快速下降、前向活力下降以及无法发展过度活跃的活力。我们假设缺乏 LDHC 会通过反馈抑制来破坏糖酵解，要么导致对 3-磷酸甘油醛脱氢酶、精子 (GAPDHS) 活性至关重要的 NAD(+) 辅因子更新缺陷，要么导致丙酮酸积累。为了测试这些假设，使用核磁共振分析来实时跟踪标记底物的利用。我们发现，在缺乏 LDHC 的精子中，葡萄糖消耗受到干扰，但 NAD:NADH 比率和丙酮酸水平没有变化，并且丙酮酸迅速代谢为乳酸。此外，乳酸脱氢酶（LDH）抑制剂草酸钠治疗引起的代谢紊乱与缺乏LDHC引起的代谢紊乱不同。这支持了我们之前的结论，即 LDHA（一种存在于鞭毛主片中的 LDH 同工酶）是缺乏 LDHC 的精子中残留 LDH 活性的原因，但也表明 LDHC 在维持精子能量代谢方面具有额外的作用。通过免疫共沉淀结合质谱法，我们鉴定了 27 种与 LDHC 相关的蛋白质。这些蛋白质中的大多数与 ATP 合成、利用、运输和/或隔离有关。这使我们推测，LDHC 除了在糖酵解中发挥作用外，还是参与 ATP 稳态的复合物的一部分，而缺乏 LDHC 的精子中该复合物会被破坏。 ——米奇·艾迪 GR：糖皮质激素是一种应激激素，通过核受体介导的基因调节维持体内平衡。我们发现其他细胞应激源通过人类糖皮质激素受体 (GR) 上的一个新的独立于激素的磷酸化位点 Ser134 与糖皮质激素信号传导整合。 Ser134 磷酸化是由多种应激激活刺激以 p38 丝裂原激活蛋白激酶 (MAPK) 依赖性方式诱导的。表达不能在 Ser134 位点磷酸化的突变糖皮质激素受体 (S134A-GR) 的细胞显着改变了激素依赖性全基因组转录反应和相关激素介导的细胞功能。 Ser134 的磷酸化显着增加了 GR 与染色质启动子区域上 14-3-3 类信号蛋白 (14-3-3zeta) 的 zeta 亚型的关联，导致所选基因的激素依赖性转录反应减弱。这些数据表明，Ser134 的磷酸化充当 GR 上的分子传感器，监测细胞应激水平，通过改变 14-3-3zeta 辅因子结合和启动子招募来重定向糖皮质激素调节的信号传导。这种翻译后修饰允许先前的细胞应激信号决定对糖皮质激素的转录反应。 C约翰·西德洛夫斯基 Kv11.1：抑制 Kv11.1 离子通道活性的突变会导致心脏复极异常，从而导致长 QT2 (LQT2) 心律失常和猝死。然而，对于大多数突变，Kv11.1 功能障碍与心源性死亡之间的分子机制尚不清楚。我们之前已经证明，在离子通道上创建激酶共有位点的疾病相关突变可以显着改变离子通道活性。在这里，我们发现 LQT2 相关突变可以通过干扰 Ser/Thr 蛋白激酶 C (PKC) 的共有位点来抑制 Kv11.1 离子通道活性。我们首先通过质谱分析发现Kv11.1离子通道的Ser890被磷酸化。然后，我们通过磷酸检测免疫分析结合基因操作证明 PKC 磷酸化 Ser890。此外，我们发现 Ser890 磷酸化与 Kv11.1 膜密度的增加以及失活恢复的改变相关。此外，人ether-go-go相关基因1编码序列中新发现且尚未表征的LQT2相关非同义单核苷酸多态性2660 GA，用组氨酸残基（R887H）取代精氨酸887，强烈抑制Kv11.1上残基Ser890的PKC依赖性磷酸化， 并最终抑制表面表达和电流密度。总而言之，我们的数据提供了该通道突变与 LQT2 之间的功能联系。 C Saverio Genitile（之前由 PMCF 与 Dave Armstrong 合作完成的工作） 其他需要的资源可忽略不计的项目包括与 Armstrong、Blackshear、Hall、Hu、Fessler、J. Mason 和 R.S. 合作进行的项目。威廉姆斯实验室。