ATP UBIQUITIN DEPENDENT PROTEOLYSIS

ATP 泛素依赖性蛋白水解

• 批准号: 2177256
• 负责人: ARTHUR L HAAS
• 金额: $ 22.23万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177256
• 关键词: Manduca; active sites; adenosine triphosphate; affinity chromatography; chemical binding; chemical conjugate; chemical kinetics; circular dichroism; computer graphics /printing; conformation; crystallization; enzyme complex; enzyme inhibitors; enzyme mechanism; genetic manipulation; high performance liquid chromatography; isozymes; laboratory rabbit; mutant; polymerase chain reaction; protein degradation; protein folding; protein purification; protein sequence; protein structure function; site directed mutagenesis; stoichiometry; thermodynamics; ubiquitin; western blottings

The immediate goal of this grant is to examine the mechanism of ubiquitin- protein conjugation as part of a continuing interest in the multi-enzyme pathway of ATP, ubiquitin-dependent protein degradation. This system is implicated in a spectrum of fundamental regulatory processes within the cell for which an understanding of the mechanism and specificity of this novel post-translational modification is critical. The Specific Aims of the proposal are: (1)Characterize the E1 inhibitor of Manduca sexta intersegmental muscle. The low molecular weight (8 kDa) heat-stable E1 inhibitor proposed to regulate conjugation within ISM during eclosion will be purified and its mechanism of inhibition characterized. The putative high molecular weight antagonist of the inhibitor will also be examined. (2)Map the function of specific ubiquitin amino acid residues. Kinetic, thermodynamic, and site-directed mutagenesis will be exploited to map the function(s) of residues on ubiquitin with respect to contact surfaces for binding enzymes of the pathway, the contribution of such binding energy to the catalytic efficiency of E1, and the structural correlates on ubiquitin that define linkage specificity in E3-independent multi-ubiquitination. (3)Examine the mechanism of E214K. Kinetic and thermodynamic studies will be used to examine the relative affinities of E2 and its ubiquitin thiolester to activating enzyme and isopeptide ligase to test hypotheses on the competition of different isozymes for binding to these proteins. Site- directed mutagenesis will be used to elucidate the mechanism of transthiolation and conjugate formation. (4)Develop E2 affinity methods for isolating E3 isozymes. Affinity methods will be developed for using E2-linked and E2-ubiquitin thiolester analog- linked affinity columns to isolate putative E2-specific E3 isozymes.

这笔赠款的直接目标是检查泛素 - 蛋白质结合是对多酶的持续兴趣的一部分 ATP的途径，泛素依赖性蛋白质降解。 这个系统是 与一系列基本监管过程有关 对此的理解的理解和特异性的细胞 新型的翻译后修饰至关重要。 具体目的 该提案是： （1）表征甘达·塞克斯塔（Manduca Sexta）段肌肉的E1抑制剂。 提出的低分子量（8 kDa）热稳定E1抑制剂提议 在ECOLOSION期间，ISM内的共轭将被纯化，并将其纯化 抑制机制的特征。 推定的高分子量 还将检查抑制剂的拮抗剂。 （2）绘制特异性泛素氨基酸残基的功能。 动力学， 热力学和定向诱变将被利用以绘制 泛素残基的功能相对于接触表面 途径的结合酶，这种结合能对 E1的催化效率以及泛素的结构相关 这定义了与E3无关的多泛素化中的链接特异性。 （3）检查E214K的机制。 动力学和热力学研究将 用于检查E2及其泛素的相对亲和力 硫酯激活酶和等肽连接酶以检验假设 不同同工酶与这些蛋白质结合的竞争。 地点- 定向诱变将用于阐明 转硫醇和结合形成。 （4）开发用于分离E3同工酶的E2亲和力方法。 亲和力方法 将开发用于使用E2连接和E2-泛素硫素类似物 连接的亲和力柱与隔离假定的E2特异性E3同工酶。