Proteomics of Cell Signaling in Embryogenesis.

胚胎发生中细胞信号转导的蛋白质组学。

• 批准号: 9309758
• 负责人: STEVEN P GYGI
• 金额: $ 62.07万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9309758
• 关键词: Address; Adult; Affinity; Aging; Animal Cap; Autoimmunity; Bayesian Method; Behavior; Biological Models; Biology; Cell Differentiation process; Cell physiology; Cells; Chemistry; Childhood; Collaborations; Communities; Complex; Confidence Intervals; Consensus; Data; Data Set; Databases; Decision Making; Defect; Detection; Development; Disease; Ectoderm; Embryo; Embryonic Development; Erinaceidae; Experimental Designs; Funding; Genetic Transcription; Genome; Genotype; Goals; Grant; Heritability; Human; Hybrids; Immune System Diseases; Individual; Knowledge; Label; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Mathematics; Measurement; Measures; Methods; Mitogen-Activated Protein Kinases; Modernization; Modification; Mutate; Neurons; Organism; Outcome; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Phosphoproteins; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pluripotent Stem Cells; Population; Post-Translational Protein Processing; Procedures; Process; Protein Dynamics; Proteins; Proteomics; RNA; Reagent; Regenerative Medicine; Regulatory Pathway; Resources; Sampling; Signal Pathway; Signal Transduction; Site; Specificity; System; Systems Biology; Technology; Testing; Time; Tissues; Translating; Translations; Xenopus; Xenopus laevis; base; differential expression; embryo stage 2; experimental study; genetic regulatory protein; genome-wide; improved; inhibitor/antagonist; innovation; insight; kinase inhibitor; knock-down; mathematical analysis; mathematical methods; novel strategies; phosphoproteomics; protein expression; protein profiling; receptor; relating to nervous system; response; scaffold; stem; tool; ubiquitin ligase; vertebrate embryology

Project Summary Most cellular processes in both the embryo and the adult are controlled by either differential expression or modification of regulatory proteins. We will develop proteomic tools to study quantitatively the effect of protein expression levels and post-translational modification on key signaling pathways in cells, and probe the complex behavior of these pathways in vertebrate embryonic development. Our premise is that highly reliable and very accurate quantitative profiling of protein levels and phosphorylation along with proper mathematical analysis will lead to insights into how signaling pathways communicate information and enable cells to make decisions on a cellular and tissue level. Phosphorylation is thought to be the major regulatory modification of proteins. At least half of all mammalian proteins are phosphorylated, yet for most phospho-sites we do not know which kinase is responsible for the phosphorylation, and hence how the target protein is regulated. We propose to dramatically advance this situation via a systems biology framework for studying phosphorylation cascades in mammalian cells in culture and in early vertebrate embryonic development, using Xenopus laevis embryos as a model system. We have shown how manipulation of kinase activity, with poly-specific kinase inhibitors, can identify the relationship between a phenotype and a specific kinase. By measuring phosphorylation sites quantitatively by MS (using each individual phosphosite as its own phenotype) we plan to provide a kinase- substrate map at a genome scale for both human and Xenopus, and use this to connect kinase activity to cellular phenotype. Our team is comprised of a close and well established collaboration between an expert embryologist/cell biologist/biochemist (Kirschner), an expert in systematic analysis and statistical inference (Peshkin) and an expert in proteomic mass spectrometry (Gygi). Our recently developed methods will allow us to reach unprecedented depth of quantification of proteins and protein post-translational modifications. In this grant we will develop further innovations in experimental design, analytical improvements and improvements in MS. The large size of Xenopus embryos permits organism-level studies of such sensitivity that they can be used on a relatively small number of experimentally manipulated embryos. Our efforts will produce systems- level knowledge of the dynamic protein states in cells and in cell populations in early vertebrate development. The resulting data sets and methods should be a powerful resource for vertebrate embryology and could be informative for heritable defects in human embryos. Many embryonic pathways, such as MAPkinase, Wnt, and hedgehog, are also very important in childhood development, tissue and cell turnover in adults, regenerative medicine, and diseases of the immune system and cancer. As many of these pathways and kinase relationships seem conserved across stages of development and across vertebrate species, there is promise of rapid transfer from model system to human patients. In particular we expect that our approach will also serve to facilitate systems-level pharmacology approaches to therapy.

项目概要 胚胎和成体中的大多数细胞过程都是由差异表达或 调节蛋白的修饰。我们将开发蛋白质组学工具来定量研究蛋白质的作用 细胞中关键信号通路的表达水平和翻译后修饰，并探测复杂的 这些途径在脊椎动物胚胎发育中的行为。我们的前提是高度可靠且非常 蛋白质水平和磷酸化的准确定量分析以及适当的数学分析 将深入了解信号通路如何传递信息并使细胞能够做出决策 在细胞和组织水平上。磷酸化被认为是蛋白质的主要调节修饰。在 至少一半的哺乳动物蛋白质被磷酸化，但对于大多数磷酸化位点，我们不知道是哪个 激酶负责磷酸化，从而决定靶蛋白的调节方式。我们建议 通过研究磷酸化级联的系统生物学框架极大地推进了这种情况 使用非洲爪蟾胚胎作为培养物和早期脊椎动物胚胎发育中的哺乳动物细胞 模型系统。我们已经展示了如何使用多特异性激酶抑制剂来操纵激酶活性 确定表型和特定激酶之间的关系。通过测量磷酸化位点 通过 MS 定量（使用每个单独的磷酸位点作为其自己的表型），我们计划提供一种激酶- 人类和非洲爪蟾基因组规模的底物图谱，并用它来将激酶活性与 细胞表型。我们的团队由专家之间密切且良好的合作组成 胚胎学家/细胞生物学家/生物化学家（Kirschner），系统分析和统计推断专家 （Peshkin）和蛋白质组质谱专家（Gygi）。我们最近开发的方法将使我们能够 达到前所未有的蛋白质和蛋白质翻译后修饰定量深度。在这个 资助我们将在实验设计、分析改进和改进方面进一步创新 在女士。非洲爪蟾胚胎的大尺寸允许在生物体水平上进行敏感性研究，使得它们可以 用于相对少量的实验操作胚胎。我们的努力将产生系统—— 对早期脊椎动物发育过程中细胞和细胞群中动态蛋白质状态的了解。 由此产生的数据集和方法应该成为脊椎动物胚胎学的强大资源，并且可以 为人类胚胎的遗传缺陷提供信息。许多胚胎途径，例如 MAPkinase、Wnt 和 刺猬，对于儿童发育、成人组织和细胞更新、再生也非常重要 医学、免疫系统疾病和癌症。由于许多这些途径和激酶关系 似乎在不同的发育阶段和不同的脊椎动物物种中都是保守的，因此有望实现快速转移 从模型系统到人类患者。我们特别希望我们的方法也将有助于促进 系统级药理学治疗方法。