Quantitative Analysis of RING E3 Ubiquitin Ligases

RING E3 泛素连接酶的定量分析

• 批准号: 7635714
• 负责人: Charles M Brenner
• 金额: $ 28.5万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7635714
• 关键词: Active Sites; Binding; Binding Proteins; Biochemical; Biological; Cell Cycle; Cells; Cellular Stress; Cellular biology; Collection; Dependence; Enzymatic Biochemistry; Epithelial; Exclusion; Funding; Genetic; Health; Homologous Gene; Human; Kinetics; Link; Mass Spectrum Analysis; Mediating; Methods; Modification; Monoubiquitination; Polyubiquitin; Polyubiquitination; Protein Chemistry; Proteins; Public Health; Reaction; Side; Signal Transduction; Site; Solvents; Specificity; System; Time; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Work; Yeasts; body system; gel electrophoresis; human PLK1 protein; in vivo; innovation; novel; reconstitution; tumor; ubiquitin-protein ligase

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.

环E3泛素连接酶是特定的结合蛋白，可介导泛素化的反应 泛素偶联酶（E2-UB）与靶蛋白，以从该活性位点的泛素转移 E2到目标的LYS侧链。因为靶蛋白经常通过几种等效物进行修改 泛素，环E3连接酶多次结合E2-UB。第二及以后的转移可能会发生在不同的 LYS残留物，产生一个多泛素化的靶标或以前转移的Lys残留物 泛素，产生一个多泛素的靶标（Passmore和Barford，2004年）。通过凝胶电泳，多个 单泛素化和多泛素化都作为梯子，其中靶蛋白通过 无处不在！ 〜8 kDa的单位，但反应的产物是不同的。新的定量质谱 最近已经开发了解决该问题的分析蛋白化学部分的方法 （Kirkpatrick等，2005a; Kirkpatrick等，2005b），尽管几乎没有综合努力来定义 恰好使用质谱，酶学，细胞的组合，E3 E3泛素连接酶进行 生物学和遗传学。 靶蛋白泛素化的复杂性是由于多种因素。首先，靶蛋白具有 多个暴露溶剂的Lys残基，可能是可访问的其他Lys残基 变性或泛素化的初始循环后。所有这些LYS残基都是潜在的主要目标 泛素化。其次，泛素有7个LYS残基，所有这些残留物在酵母提取物中均泛素化， Lys48和Lys63上的多泛素连接的原理位点（Peng等，2003； Kirkpatrick等，2005a）。第三， E2泛素结合酶具有不同的特异性，并有可能形成不同的靶标 蛋白质和多泛素连接与不同的E3泛素连接酶结合在一起，它们也具有独特的 特殊性。第四，已经提出了许多仅包含一类E3连接酶的环E3连接酶 除了修改或排除修改外，还要具有生物学上重要的自动泛素化反应 外部基材。例如，已经有证据表明CHFR是人类环E3泛素连接酶 在20-50％的人类肿瘤中，这被灭活（Scolnick和Halazonetis，2000; Mizuno等，2002; Shibata et; Al。，2002; Corn等，2003； Mariatos等人，2003年； Toyota等人，2003年），用UBC13/MMS2进行修改 本身具有lys63连接的聚偶像素，以信号细胞应激（Bothos等，2003； Matsusaka和Pines，2004）。 还提出了CHFR催化Ubc4和UBC5依赖性泛素化的泛素化的证据 激酶1（PLK1）导致PLK1的蛋白酶体降解以阻断细胞周期过渡（Kang等，2002）。在 CHFR的情况，功能是否取决于自动泛素化或底物泛素化或两者兼而有之？什么是 站点和链接？哪些E2真正涉及？修改的底物的集合是什么？ 为了确定与CHFR相关分子功能功能的生化和细胞机制，它变成了 开发剖析环E3连接酶功能的遗传系统所必需的（Bieganowski等，，， 2004年），重建纯化系统以表征反应的决定因素和产物，并 完善质谱方法来识别和量化位点和链接。申请人和共同申请人 已经开发了这些系统，结合了它们在遗传，生化和质量方面的专业知识 工作中的光谱分析通过有限的可支配资金而成为可能。酵母CHFR同源物，CHF1和 CHF2已被克隆，遗传表征，纯化并用于重建无细胞的泛素化 反应已通过质谱法分析。反应成分也已经 已经确定了经遗传验证的遗传和“相互作用”和CHF2的“相互作用”。 具体目的： 1）我们将使用定量质谱和酶学来定义站点，链接和 CHFL和CHF2泛素化反应的动力学与经遗传验证的泛素结合 我们已确定为CHF相互作用的酶和蛋白质。 2）我们将确定chf1和 CHF2在体内泛素化。 该提案具有两个长期的公共卫生目标。首先，确定作用机制 酵母CHF1和CHF2的理解至关重要 上皮来源的肿瘤。其次，在环E3泛素连接酶分析中的创新对于 了解环E3泛素连接酶功能的特异性，这些连接酶在健康中具有关键功能 每个器官系统。