Quantitative Analysis of RING E3 Ubiquitin Ligases

描述（由申请人提供）：RING E3泛素连接酶为大部分生物泛素化反应提供特异性。以不涉及共价中间体的方式工作，这些分子结合底物和特异性E2泛素缀合酶，以实现泛素转移到底物上、E3上（autoubiquitination）和泛素上（polyubiquitination）的Lys残基。这些潜在竞争反应的分布、动力学和特异性决定了产物是否将被靶向蛋白水解、差异定位和/或发出各种形式的应激信号。我们克隆了Chfl和Chf 2，Chfr肿瘤抑制因子的酵母同源物，并开发了遗传系统，表明Chf施加的细胞周期延迟取决于Ubc 4和Mms 2的功能。我们用Ubc 4和Ubc 13/Mms 2作为泛素结合酶，Chf 1和Chf 2作为E3，重建纯化的泛素化反应，并鉴定反应的特异性目的：1）我们将使用定量质谱和酶学来确定Chf 1和Chf 2与遗传上验证的泛素结合酶和我们已经鉴定为Chf相互作用物的蛋白质的泛素化反应的位点、连接和动力学。2)我们将确定的网站，链接，生物学后果，和E2依赖性的Chf 1和Chf 2在体内的泛素化。这项建议有两个长远的公共卫生目标。首先，确定酵母Chf 1和Chf 2的功能机制对于理解Chfr的功能至关重要，Chfr在上皮来源的人类肿瘤中经常失活。其次，RING E3遍在蛋白连接酶分析的创新对于了解RING E3遍在蛋白连接酶功能的特异性是必要的，RING E3遍在蛋白连接酶对人体每个器官系统的健康具有关键功能。

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.