Quantitative Analysis of RING E3 Ubiquitin Ligases

RING E3泛素连接酶是特异性结合蛋白，其介导泛素化的蛋白质与泛素化的蛋白质的反应。 泛素缀合酶（E2-Ub）与靶蛋白结合，以将泛素从靶蛋白的活性位点Cys转移至靶蛋白。 E2与靶标的Lys侧链连接。因为靶蛋白经常被几个当量的 泛素、RING E3连接酶多次结合E2-Ub。第二次和随后的传输可以发生在不同的 Lys残基，产生多重单泛素化的靶标，或与先前转移的靶的Lys残基结合。 泛素，产生多泛素化的靶标（Passmore和Barford，2004）。通过凝胶电泳， 单泛素化和多聚泛素化都表现为梯状结构，其中靶蛋白被 无处不在！单位的~8 kDa，但反应的产物是不同的。新型定量质谱 最近已经开发了一些方法来解决这个问题的分析蛋白质化学部分 （柯克帕特里克等人，2005 a;柯克帕特里克等人，2005年b），但很少有综合努力， RING E3泛素连接酶的作用，结合质谱、酶学、细胞 生物学和遗传学。 靶蛋白泛素化的复杂性是由多种因素造成的。首先，靶蛋白具有 暴露于溶剂的多个Lys残基和可能变得可接近的其他Lys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标， 泛素化第二，泛素具有7个赖氨酸残基，所有这些残基在酵母提取物中被泛素化， 在Lys 48和Lys 63处的多聚泛素连接的主要位点（Peng等，2003;柯克帕特里克等人，2005年a）。第三、 E2泛素结合酶具有不同特异性，并可能形成不同的靶点 蛋白质和多聚泛素连接与不同的E3泛素连接酶组合，其也具有不同的 特殊性第四，已经提出了许多仅包含一类E3连接酶的RING E3连接酶 具有生物学上重要的autoubiquitination反应，除了或排除，修饰 外部基板。例如，已有证据表明，人RING E3泛素连接酶Chfr 其在20-50%的人肿瘤中失活（Scolnick和Halazonetis，2000; Mizuno等，2002;柴田et 例如，2002; Corn等人，2003; Mariatos等人，2003; Toyota等人，2003），函数与Ubc 13/Mms 2修改 其本身与Lys 63连接的多聚泛素一起发出细胞应激信号（Bothos等，2003;松坂和派恩斯，2004）。 也有证据表明，Chfr催化Ubc 4和Ubc 5依赖的Polo样蛋白的泛素化， 激酶1（Plk 1）导致Plk 1的蛋白酶体降解以阻断细胞周期转换（Kang等人，2002年）。在 在Chfr的情况下，功能是否依赖于自泛素化或底物泛素化或两者兼而有之？有哪些 网站和链接？哪些E2真的参与了？被修改的底物的集合是什么？ 为了确定Chfr相关分子功能的生化和细胞机制， 这是开发用于剖析RING E3连接酶功能的遗传系统所必需的（Bieganowski等， 2004），以重建纯化的系统，以表征反应的决定因素和产物，并 改进质谱方法，以确定和量化位点和连接。申请人和共同申请人 已经开发出这些系统，结合他们在遗传、生物化学和质量方面的专业知识， 在有限的可自由支配的资金下，工作中的光谱分析成为可能。酵母Chfr同系物，Chf 1和 chf 2已被克隆、遗传学表征、纯化并用于重构无细胞泛素化 反应，已通过质谱分析。反应组分也已被 基因验证和“相互作用组”的Chf 1和Chf 2已被确定。 具体目标： 1)我们将使用定量质谱和酶学来确定位点，连接和 Chf 1和Chf 2泛素化反应的动力学与遗传验证的泛素缀合 酶和蛋白质，我们已经确定为Chf相互作用。 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。 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。第一，确定作用机制 酵母Chf 1和Chf 2的表达对于了解Chfr的功能至关重要，Chfr在人体中经常失活 上皮源性肿瘤。其次，RING E3泛素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， 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.