Quantitative Analysis of RING E3 Ubiquitin Ligases

RING E3 泛素连接酶的定量分析

• 批准号: 7477693
• 负责人: Charles M Brenner
• 金额: $ 36.32万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7477693
• 关键词: Active Sites; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological; Cell Cycle; Cells; Cellular Stress; Cellular biology; Class; Collection; Dependence; Enzymatic Biochemistry; Epithelial; Exclusion; Funding; Genetic; Health; Homologous Gene; Human; Human body; Kinetics; Link; Localized; Maize; Mass Spectrum Analysis; Mediating; Methods; Modification; Monoubiquitination; Polyubiquitin; Polyubiquitination; Protein Chemistry; Proteins; Proteolysis; Public Health; Reaction; Research Personnel; 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

DESCRIPTION (provided by applicant): 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 propecific Aims: 1) We will use quantitative mass spectrometry and enzymology to 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.

描述（由申请人提供）：环E3泛素连接酶为大部分生物泛素化反应提供了特异性。这些分子以不涉及共价中间体的方式进行工作，并结合底物和特定的E2泛素偶联酶，使泛素在E3（自夸夸夸有关）和泛素（泛素化）上的泛素转移至底物上的乳糖残基。这些潜在竞争反应的分布，动力学和特异性决定了产品是否将针对蛋白水解，差异定位和/或信号各种形式的压力。我们将CHFR抑制剂的酵母同源物克隆了CHFL和CHF2，并开发了遗传系统，表明CHF施加的细胞周期延迟取决于UBC4和MMS2的功能。我们使用UBC4和UBC13/MMS2作为泛素结合酶，将CHF1和CHF2作为E3重新定义了纯化的Ubiqutination反应，并确定了反应性的目标：1）我们将使用定量的质谱和酶学，与位点，Chft 2和Chftiminition和Chftimistion和Chftiminition和Chftimention和chff 2验证的泛素结合酶和我们已确定为CHF相互作用的蛋白质。 2）我们将确定chf1和chf2泛素化的位点，链接，生物后果和E2依赖性。该提案具有两个长期的公共卫生目标。首先，确定酵母CHF1和CHF2功能的机理对于了解CHFR的功​​能至关重要，CHFR经常在上皮来源的人类肿瘤中灭活。其次，对环E3泛素连接酶进行分析的创新对于了解环E3泛素连接酶功能的特异性是必要的，后者在人体每个器官系统的健康中具有关键功能。