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协作项目已与蛋白质微含量化核心设施（PMCF）进行，其中大约有6000个样本，分析了57位代表来自8个实验室分支机构的主要研究人员的科学家。 一项巨大的努力是支持蛋白质表达核心设施（PECF）和Bob Petrovich博士。 PMCF的作用是在将PECF将材料移交给使用者之前确认蛋白质水平的基因表达。 其他仍在进行的未发表的项目包括： 在脂质和肌醇激酶上鉴定结合伴侣和翻译后修饰（PTMS）的位点-Steve Shears G蛋白C Lutz Birnbaumer上脂化的识别和定量 结合伴侣的鉴定和可能参与DNA复制和修复的蛋白质的翻译后修饰-Sam Wilson 在多种组织类型和/或条件下鉴定BAF复合物中蛋白质 - Trevor Archer p-选择素相互作用伙伴和治疗后磷酸蛋白酶的变化c Steve Akiyama Glis家族成员修改和约束伙伴C Anton Jetten 加工前后花生过敏原的PTM的表征（烤）C Geoffrey Mueller和Bob London 核心还在对亲和力技术（基于GFP的富集）进行增值研究，以帮助蛋白质，蛋白质结合伴侣和PTM鉴定。 媒体上最近发布的其他项目或项目包括： LDHC：生殖细胞特异性乳酸脱氢酶C基因（LDHC）导致由于精子功能的缺陷而导致男性不育症，包括精子ATP水平的迅速下降，进行性运动的降低以及未能发展出多激活的运动性。我们假设缺乏LDHC通过反馈抑制破坏了糖酵解，这是通过导致NAD（+）辅助因子在3-磷酸甘油甲醛脱氢酶活性至关重要的情况下引起缺陷，即精子（GAPDHS），或者是丙酮酸的积累。为了检验这些假设，使用核磁共振分析实时遵循标记的底物的利用。我们发现，在缺乏LDHC的精子中，葡萄糖消耗受到破坏，但是NAD：NADH比和丙酮酸水平不变，丙酮酸迅速代谢为乳酸。此外，用乳酸脱氢酶（LDH）抑制剂抑制剂诱导的代谢疾病与缺乏LDHC引起的代谢障碍不同。这支持了我们较早的结论，即鞭毛主要部分中存在的LDH同工酶是负责LDHC缺乏LDHC的残留LDH活性，但建议LDHC在维持精子中能量代谢的维持中具有额外的作用。通过共免疫沉淀与质谱法结合，我们确定了与LDHC相关的27种蛋白质。这些蛋白质中的大多数与ATP合成，利用，运输和/或隔离有关。这导致我们假设除了其在糖酵解中的作用外，LDHC是参与ATP稳态的复合体的一部分，该复合体因缺乏LDHC的精子而破坏。 -Mitch Eddy GR：糖皮质激素是通过核受体介导的基因调节维持稳态的应激激素。我们已经发现，其他细胞应激源与人糖皮质激素受体（GR）上的新型激素磷酸化位点Ser134与糖皮质激素信号传导整合在一起。 Ser134磷酸化是由p38促丝分裂原激活蛋白激酶（MAPK）依赖性方式的多种应激激活刺激诱导的。表达突变体糖皮质激素受体在Ser134（S134a-gr）处无能力的磷酸化的细胞，其激素依赖性基因组全基因组的转录反应和相关激素介导的细胞功能有显着改变。 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（14-3-3-3zeta）的ZetA同工型的缔合，从而导致所选基因的激素依赖性转录反应钝化。这些数据表明，Ser134的磷酸化充当GR上的分子传感器，从而通过改变的14-3-3zeta辅因子结合和启动子募集来监测细胞应激水平以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力信号决定对糖皮质激素的转录反应。 C JOHN CIDLOWSKI KV11.1：抑制KV11.1离子通道活性的突变导致心脏复极异常，从而导致QT2（LQT2）长时间心律失常和猝死。但是，对于大多数这些突变，关于将KV11.1故障与心脏死亡联系起来的分子机制一无所知。我们以前已经证明，与疾病相关的突变在离子通道上创建激酶的共识位点可以极大地改变离子通道活性。在这里，我们表明与LQT2相关的突变可以通过扰动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揭示了KV11.1离子通道的Ser890磷酸化。然后，我们通过磷酸检测免疫测定法结合了PKC磷酸化Ser890的遗传操纵。此外，我们表明SER890磷酸化与KV11.1膜密度的增加有关，随着失活的恢复改变。此外，在人乙醚内发现了新发现且尚未与之相关的LQT2相关的非同义单核苷酸多态性2660 GA-与Go-Go相关的基因1编码序列，该序列将精氨酸887替换为组氨酸固定（R8887H），对PKC依赖性均匀抑制了pkcccccccccccccceptionly 887最终抑制表面表达和电流密度。综上所述，我们的数据提供了此通道突变与LQT2之间的功能联系。 C Saverio Genitile（PMCF先前与Dave Armstrong合作进行的工作） 除了可忽略不足的资源所需的其他项目包括与阿姆斯特朗，黑皮，霍尔，胡，菲斯勒，J。Mason和R.S.所做的努力。威廉姆斯实验室。