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.

RING E3 泛素连接酶是介导泛素化反应的特异性结合蛋白 泛素结合酶 (E2-Ub) 与靶蛋白结合，从活性位点 Cys 转移泛素 E2 至目标的 Lys 侧链。因为靶蛋白经常被几个等价物修饰 泛素，RING E3 连接酶多次结合 E2-Ub。第二次及后续传输可以发生在不同的 Lys 残基，产生多重单泛素化靶标，或先前转移的 Lys 残基 泛素，产生多泛素化靶标（Passmore 和 Barford，2004）。通过凝胶电泳，多重 单泛素化和多泛素化都以梯子的形式出现，其中靶蛋白被修饰 无处不在！单位约为 8 kDa，但反应产物是不同的。新型定量质谱 最近开发了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），尽管很少有综合努力来定义 RING E3 泛素连接酶结合了质谱、酶学、细胞学等技术，正是实现了这一点 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，目标蛋白具有 暴露于溶剂的多个赖氨酸残基以及可接近的潜在附加赖氨酸残基 变性或初始泛素化循环后。所有这些赖氨酸残基都是潜在的主要目标 泛素化。其次，泛素有7个Lys残基，所有这些残基在酵母提取物中都被泛素化，具有 Lys48 和 Lys63 处的多泛素连接的主要位点（Peng 等人，2003；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标集 蛋白质和多聚泛素连接与不同的 E3 泛素连接酶结合，也具有不同的特性 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 除了修饰或排除修饰之外，还具有生物学上重要的自泛素化反应 外部基板。例如，有证据表明 Chfr，一种人类 RING E3 泛素连接酶 在 20-50% 的人类肿瘤中失活（Scolnick 和 Halazonetis，2000 年；Mizuno 等人，2002 年；Shibata 等人） 等人，2002 年；玉米等人，2003；马里亚托斯等人，2003； Toyota et al., 2003)，使用 Ubc13/Mms2 修改功能 自身与 Lys63 连接的多聚泛素一起发出细胞应激信号（Bothos 等，2003；Matsusaka 和 Pines，2004）。 也有证据表明 Chfr 催化 Polo 样蛋白的 Ubc4 和 Ubc5 依赖性泛素化 激酶 1 (Plk1) 导致 Plk1 被蛋白酶体降解，从而阻止细胞周期转变 (Kang et al., 2002)。在 就 Chfr 而言，功能是否依赖于自身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 真正参与其中？被修饰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， 开发用于剖析 RING E3 连接酶功能的遗传系统是必要的（Bieganowski 等人， 2004），重建纯化系统来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联系。申请人及共同申请人 结合了他们在遗传、生化和质量方面的专业知识，开发了这些系统 有限的可自由支配资金使工作中的光谱分析成为可能。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泛素化 反应，已通过质谱分析。反应组分也已 经过遗传学验证，Chf1 和 Chf2 的“相互作用组”已确定。 具体目标： 1) 我们将使用定量质谱和酶学来定义位点、连接和 通过基因验证的泛素缀合进行 Chfl 和 Chf2 泛素化反应的动力学 我们已确定为 Chf 相互作用因子的酶和蛋白质。 2) 我们将确定 Chf1 和 E2 的位点、联系、生物学后果和 E2 依赖性 Chf2 体内泛素化。 该提案有两个长期公共卫生目标。一、确定作用机制 酵母 Chf1 和 Chf2 的研究对于了解 Chfr 的功能至关重要，Chfr 在人类中经常失活 上皮来源的肿瘤。其次，RING E3 泛素连接酶分析的创新对于 了解 RING E3 泛素连接酶的功能特异性，它对人体健康具有关键作用 每个器官系统。