Quantitative Analysis of RING E3 Ubiquitin Ligases

E3环泛素连接酶是一种特异的结合蛋白，它介导泛素化的 泛素偶联酶(E2-Ub)与靶蛋白结合，将泛素从半胱氨酸的活性部位转移出来 E2连接到靶标的Lys侧链。因为靶蛋白经常被几个等价物修饰 泛素，E3环连接酶多次与E2-Ub结合。第二次和后续传输可以在不同的 Lys残基，产生倍增的单价靶标，或到先前转移的Lys残基 泛素，产生一种多泛素化的靶标(Passmore和Barford，2004)。通过凝胶电泳法，获得多个 单泛素化和多泛素化都以梯子的形式出现，在梯子中靶蛋白被 无所不在！约8 kDa，但反应产物不同。一种新型定量质谱学 最近已经发展出一些方法来解决这个问题的分析蛋白质化学部分。 (Kirkpatrick等人，2005a；Kirkpatrick等人，2005b)，尽管几乎没有综合的努力来定义 结合质谱学、酶学、细胞学研究E3环泛素连接酶的功能 生物学和遗传学。 靶蛋白泛素化的复杂性是多种因素共同作用的结果。首先，靶蛋白具有 多个暴露在溶剂中的赖氨酸残基，以及可能更多的可接触到的赖氨酸残基 在变性或泛素化的初始循环之后。所有这些赖氨酸残基都是潜在的主要靶点 泛素化。其次，泛素有7个赖氨酸残基，所有这些残基都在酵母提取物中泛素化， Lys48和Lys63上多泛素连接的主要位点(Peng等人，2003年；Kirkpatrick等人，2005a)。第三, E2泛素结合酶具有不同的特异性，并可能形成不同的靶点 蛋白质和多泛素与不同的E3泛素连接酶结合，这些连接酶也有不同的 具体细节。第四，已经提出了许多环状E3连接酶，它们只包含一类E3连接酶 进行生物上重要的自体素化反应，附加或排除其修饰 外部衬底。例如，有证据表明CHFR，一种人类环E3泛素连接酶 在20%-50%的人类肿瘤中，该基因被灭活(Scolnick和Halazonetis，2000；Mizuno等人，2002；柴田等人 等人，2002；Corn等人，2003；Mariatos等人，2003；Toyota等人，2003)，使用UBC13/MMS2功能来修改 它本身与Lys63连接的多泛素一起发出细胞压力信号(Bothos等人，2003年；Matsusaka和Pines，2004年)。 也有证据表明，Chfr催化Ubc4和Ubc5依赖的Polo样泛素化 激酶1(Plk1)导致Plk1的蛋白酶体降解以阻止细胞周期转变(Kang等人，2002年)。在……里面 在Chfr的情况下，功能依赖于自身泛素化还是底物泛素化，还是两者兼而有之？什么是 站点和链接？哪些E2真正参与其中？被修改的底物的集合是什么？ 为了确定CHFR相关分子的生化和细胞功能机制，它变成了 开发用于剖析环E3连接酶功能的遗传系统所必需的(Bieganowski等人， 2004)，重建纯化系统，以表征反应的决定因素和产物，并 改进质谱学方法，以确定和量化位点和连接。申请人和共同申请人 已经开发了这些系统，结合了他们在遗传、生化和质量方面的专业知识 有限的可自由支配资金使光谱分析工作成为可能。酵母Chfr同源物、CHF1和 CHF2基因已被克隆、鉴定、纯化，并用于重建无细胞泛素化 反应，用质谱仪进行了分析。反应组分也已经被 CHF1和CHF2的“相互作用”已经确定。 具体目标： 1)我们将使用定量质谱学和酶学来确定位点、连接和 CHF1和CHF2泛素化反应的动力学研究 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CHF1的位置、联系、生物学后果和对E2的依赖 体内CHF2泛素化。 这项提案有两个长期的公共卫生目标。一、确定作用机制 酵母CHF1和CHF2对于理解CHFR的功能至关重要，CHFR在人类中经常被灭活 上皮性肿瘤。其次，分析E3环泛素连接酶的创新是必要的 了解E3环泛素连接酶功能的特异性，它在人的健康中具有关键作用 每个器官系统。

RING E3 ubiquitin ligases are specific binding proteins that mediate the reaction of a ubiquitinated ubiquitin conjugating enzyme (E2-Ub) with a target protein to transfer ubiquitin from the active-site Cys of the E2 to a Lys side chain of the target. Because target proteins are frequently modified by several equivalents of ubiquitin, RING E3 ligases bind E2-Ub multiple times. Second and subsequent transfers can occur on different Lys residues, producing a multiply monoubiquitinated target, or to a Lys residue of a previously transferred ubiquitin, producing a polyubiquitinated target (Passmore and Barford, 2004). By gel electrophoresis, multiple monoubiquitination and polyubiquitination both appear as ladders in which the target protein is modified by ubiquity! units of ~8 kDa, yet the products of the reaction are distinct. Novel quantitative mass spectrometric methods have recently been developed to solve the analytical protein chemistry part of this problem (Kirkpatrick et al., 2005a; Kirkpatrick et al., 2005b), though there have been few integrated efforts to define precisely what RING E3 ubiquitin ligases do using a combination of mass spectrometry, enzymology, cell biology and genetics. The complexity of target protein ubiquitination is due to multiple factors. First, target proteins have multiple Lys residues that are solvent-exposed and potentially additional Lys residues that become accessible after denaturation or initial cycles of ubiquitination. All such Lys residues are potential primary targets of ubiquitination. Second, ubiquitin has 7 Lys residues, all of which are ubiquitinated in yeast extracts, with the principle sites of polyubiquitin linkage at Lys48 and Lys63 (Peng et al., 2003; Kirkpatrick et al., 2005a). Third, the E2 ubiquitin conjugating enzymes have different specificities and potentially form different sets of target protein and polyubiquitin linkages in combination with different E3 ubiquitin ligases, which also have distinct specificities. Fourth, many RING E3 ligases, which comprise just one class of E3 ligases, have been proposed to have biologically important autoubiquitination reactions in addition to, or to the exclusion of, modification of external substrates. For example, evidence has been presented that Chfr, a human RING E3 ubiquitin ligase that is inactivated in 20-50% of human tumors (Scolnick and Halazonetis, 2000; Mizuno et al., 2002; Shibata et al., 2002; Corn et al., 2003; Mariatos et al., 2003; Toyota et al., 2003), functions with Ubc13/Mms2 to modify itself with Lys63-linked polyubiquitin to signal cellular stress (Bothos et al., 2003; Matsusaka and Pines, 2004). Evidence has also been presented that Chfr catalyzes Ubc4 and Ubc5-dependent ubiquitination of Polo-like kinase 1 (Plk1) leading to proteasomal degradation of Plk1 to block cell cycle transition (Kang et al., 2002). In the case of Chfr, does function depend on autoubiquitination or substrate-ubiquitination or both? What are the sites and linkages? Which E2s are really involved? What is the collection of substrates that are modified? To determine the biochemical and cellular mechanisms of function of Chfr-related molecules, it became necessary to develop genetic systems with which to dissect RING E3 ligase functions (Bieganowski et al., 2004), to reconstitute purified systems to characterize the determinants and products of the reactions, and to refine mass spectrometric methods to identify and to quantify sites and linkages. The applicant and coapplicant have developed these systems, combining their expertise in genetic, biochemical and mass spectrometric analysis in work made possible by limited discretionary funding. Yeast Chfr homologs, Chf1 and Chf2, have been cloned, characterized genetically, purified, and used to reconstitute cell-free ubiquitination reactions, which have been analyzed by mass spectrometry. The reaction components have also been validated genetically and the "interactomes" of Chf1 and Chf2 have been determined. Specific Aims: 1) We will use quantitative mass spectrometry and enzymology to define the sites, linkages and kinetics of Chfl 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 action of yeast Chf1 and Chf2 is critical to understand the 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.