Quantitative Analysis of RING E3 Ubiquitin Ligases

RING E3泛素连接酶为大部分生物泛素化反应提供特异性。工作 这些分子以不涉及共价中间体的方式结合底物和特异性E2 泛素缀合酶，以实现泛素转移到底物上的Lys残基，在E3上 （autoubiquitination）和泛素（polyubiquitination）。它们的分布、动力学和特异性 潜在的竞争性反应决定了产物是否将作为蛋白水解的目标， 局部的和/或信号各种形式的压力。 我们克隆了Chfr肿瘤抑制因子的酵母同源物Chf 1和Chf 2，并开发了遗传工程。 表明Chf施加的细胞周期延迟依赖于Ubc 4和Mms 2两者的功能的系统。我们 使用Ubc 4和Ubc 13/Mms 2作为泛素缀合物的重建纯化的泛素化反应 酶和Chf 1和Chf 2作为E3，并通过高分辨质量鉴定反应产物 光谱法我们还定位了Chf 1 N-末端的特异性决定区， 大量的Chf 1和Chf 2相互作用，并开发了新的方法来定量和提供动力学 描述复杂的位点特异性泛素化反应。 具体目标： 1)我们将使用定量质谱和酶学来确定酶的位点、连接和动力学。 Chf 1和Chf 2泛素化反应与遗传验证的泛素缀合酶和 这些蛋白质被鉴定为Chf相互作用物。 2)我们将确定Chf 1和Chf 2的位点、联系、生物学后果和E2依赖性 体内泛素化。 这项建议有两个长远的公共卫生目标。第一，确定功能的机制， 酵母Chf 1和Chf 2是了解Chfr功能的关键，Chfr在人体中经常失活 上皮源性肿瘤。其次，RING E3遍在蛋白连接酶分析的创新是必要的， 了解RING E3泛素连接酶功能的特异性，这些酶在健康中具有关键作用， 人体的每一个器官系统。

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.