Quantitative Analysis of RING E3 Ubiquitin Ligases

RING E3 泛素连接酶的定量分析

• 批准号: 7495363
• 负责人: Charles M Brenner
• 金额: $ 5.33万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495363
• 关键词: Active Sites; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological; Cell Cycle; Cells; Cellular Stress; Cellular biology; Class; Collection; Complex; Dependence; Enzymatic Biochemistry; Epithelial; Exclusion; Funding; Genetic; Health; Homologous Gene; Human; Human body; Kinetics; Link; Localized; Maize; Maps; Mass Spectrum Analysis; Mediating; Methods; Modification; Monoubiquitination; Polyubiquitin; Polyubiquitination; Protein Chemistry; Proteins; Proteolysis; Public Health; Reaction; Research Personnel; Resolution; Side; Signal Transduction; Site; Solvents; Specificity; Stress; System; Time; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Work; Yeasts; Zea mays; body system; gel electrophoresis; human PLK1 protein; in vivo; innovation; novel; programs; reconstitution; toe corn; tumor; ubiquitin-protein ligase

RING E3 ubiquitin ligases provide specificity to a huge fraction of biological ubiquitination reactions. Working in a manner that does not involve a covalent intermediate, these molecules bind substrates and specific E2 ubiquitin conjugating enzymes to effect transfer of ubiquitin to Lys residues on substrates, on the E3 (autoubiquitination), and on ubiquitin (polyubiquitination). The distribution, kinetics and specificity of these potentially competing reactions determine whether products will be targeted for proteolysis, differentially localized, and/or signal various forms of stress. We cloned Chfl and Chf2, the yeast homologs of the Chfr tumor suppressor, and developed genetic systems that indicate that Chf-imposed cell cycle delays depend on function of both Ubc4 and Mms2. We reconstituted purified ubiqutination reactions using Ubc4 and Ubc13/Mms2 as the ubiquitin conjugating enzymes and Chf1 and Chf2 as E3 and identified the reaction products by high resolution mass spectrometry. We have also mapped a specificity-determining region to the N-terminus of Chf1, identified large sets of Chf1 and Chf2 interactors, and developed novel methods to quantitate and provide a kinetic description of complex site-specific ubiquitination reactions. Specific Aims: 1) We will use quantitative mass spectrometry and enzymologyto define the sites, linkages and kinetics of Chf1 and Chf2 ubiquitination reactions with genetically validated ubiquitin conjugating enzymes and the proteins we have identified as Chf interactors. 2) We will determine the sites, linkages, biological consequences, and E2-dependence of Chf1 and Chf2 ubiquitination in vivo. This proposal has two long-term public health objectives. First, determining the mechanisms of function of yeast Chf1 and Chf2 is critical to understand function of Chfr, which is frequently inactivated in human tumors of epithelial origin. Second, innovations in analysis of RING E3 ubiquitin ligases are necessary to understand the specificity of function of RING E3 ubiquitin ligases, which have key functions in the health of every organ system in the human body.

环E3泛素连接酶为大部分生物泛素化反应提供了特异性。在职的 这些分子以不涉及共价中间体的方式结合底物和特定的E2 泛素结合酶有效泛素转移到底物上的Lys残基上，E3 （自夸泛素化）和泛素（多泛素化）。这些的分布，动力学和特异性 潜在的竞争反应决定了是否将产品用于蛋白水解，差异化 局部和/或信号各种形式的压力。 我们克隆了CHFL和CHF2，这是CHFR肿瘤的酵母同源物，并发展了遗传 表明CHF施加的细胞周期延迟的系统取决于UBC4和MMS2的功能。我们 使用UBC4和UBC13/MMS2作为泛素结合的重构纯化的Ubiqutination反应 酶，chf1和chf2作为E3，并通过高分辨率质量鉴定了反应产物 光谱法。我们还将特异性确定区域映射到CHF1的N端，确定 大量CHF1和CHF2相互作用者，并开发了新颖的方法来定量和提供动力学 描述复杂位点特异性泛素化反应。 具体目的： 1）我们将使用定量质谱和酶学来定义位点，链接和动力学 CHF1和CHF2泛素化反应与经遗传验证的泛素共轭酶和 我们已经确定为CHF相互作用的蛋白质。 2）我们将确定CHF1和CHF2的站点，链接，生物后果和E2依赖性 体内泛素化。 该提案具有两个长期的公共卫生目标。首先，确定功能机理 酵母CHF1和CHF2对于了解CHFR的功​​能至关重要，CHFR经常在人类中灭活 上皮来源的肿瘤。其次，在环E3泛素连接酶分析中的创新对于 了解环E3泛素连接酶功能的特异性，这些连接酶在健康中具有关键功能 人体中的每个器官系统。