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。这些分子以一种不涉及共价中间体的方式工作，结合底物和特定的E2泛素结合酶，以影响泛素转移到底物、E3（自泛素化）和泛素（多泛素化）上的赖氨酸残基。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会成为蛋白水解的目标、差异定位和/或各种形式的应激信号。我们克隆了Chfr肿瘤抑制因子的酵母同源物Chfl和Chf2，并建立了遗传系统，表明chf施加的细胞周期延迟依赖于Ubc4和Mms2的功能。我们以Ubc4和Ubc13/Mms2为泛素偶联酶，Chf1和Chf2为E3，重构了纯化的泛素化反应，并确定了反应的特异性目的：1)我们将使用定量质谱和酶学来确定基因验证的泛素偶联酶和我们确定的Chf相互作用蛋白的Chf1和Chf2泛素化反应的位点、键和动力学。2)我们将确定体内Chf1和Chf2泛素化的位点、联系、生物学后果和e2依赖性。这项建议有两个长期的公共卫生目标。首先，确定酵母Chf1和Chf2的功能机制对于了解Chfr的功能至关重要，Chfr在人类上皮源性肿瘤中经常失活。其次，为了了解RING E3泛素连接酶的功能特异性，需要在分析RING E3泛素连接酶方面进行创新，RING E3泛素连接酶对人体各器官系统的健康都具有关键作用。

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.