PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

ATP/泛素依赖性蛋白酶的特性

• 批准号: 2608855
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 27.49万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608855
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; adenosine triphosphate; adenosinetriphosphatase; antibody; complementary DNA; electron microscopy; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; laboratory rabbit; lysozyme; molecular cloning; ornithine decarboxylase; peptidases; phosphorylation; protein degradation; protein kinase; protein purification; protein structure function; proteolysis; site directed mutagenesis; tissue /cell culture; ubiquitin

The 26S protease is a large multisubunit enzyme that degrades important regulatory proteins such as p53, cyclins, NFkappaB and ornithine decarboxylase. It is comprised of a regulatory complex containing at least 15 different subunits associated with the multicatalytic protease which contributes an additional 14 subunits. PCR-based strategies will be used to obtain cDNAs encoding four 26S protease subunits (S2, S9, S11 and S15) that remain unidentified. The cDNAs will be expressed in E. coli, and subunit specific antibodies will be generated from the recombinant proteins. Pulse-chase metabolic labeling and several immunoprecipitation approaches will be used to determine whether components of the 26S protease are in dynamic equilibrium. Antibodies specific to 26S protease subunits (S4, S5a, S5b, S6 and S12) are available. These antibodies and others generated in the near future will be used to determine the tissue distribution and intracellular location of the respective 26S subunits. In two separate projects, site-directed mutagenesis will be used to assess the importance of N-terminal coiled-coil regions of S4-like ATPases (subunits 4, 6, 7 and 8) in assembly of the 26S protease and in substrate selection. Direct filter binding assays will be used to identify proteins that interact with subunit 4. Deletional analysis of the ubiquitin- conjugate binding subunit (S5a) will be employed to determine which region(s) of the protein bind Ub tetramers and to determine how many binding sites are present. Finally, site-directed mutagenesis will be used to assess the importance of specific residues in the tetramer binding regions of S5a. The 26S protease is clearly involved in control of the cell cycle, and it is the enzyme most likely to generate antigenic peptides displayed on MHC Class l molecules. For these reasons, better understanding of the protease should have significant medical implications.

26S蛋白酶是一种大型多亚基酶，使重要的降解很重要 调节蛋白，例如p53，细胞周期蛋白，NFKAPPAB和鸟氨酸 脱羧酶。它由至少包含的调节复合物组成 与多催化蛋白酶相关的15个不同的亚基，这些亚基 另外14个亚基。将使用基于PCR的策略 获得编码四个26S蛋白酶亚基的cDNA（S2，S9，S11和S15） 那仍然不明。 cDNA将以大肠杆菌的形式表达 亚基特异性抗体将从重组产生 蛋白质。脉搏练习代谢标签和几个免疫沉淀 方法将用于确定26S的组件是否 蛋白酶处于动态平衡状态。针对26S蛋白酶的抗体 可提供亚基（S4，S5A，S5B，S6和S12）。这些抗体和 在不久的将来产生的其他人将用于确定组织 各个26S亚基的分布和细胞内位置。在 两个单独的项目，将使用站点定向的诱变来评估 S4样atpases的N末​​端盘绕圈区域的重要性 （亚基4、6、7和8）在26S蛋白酶和底物的组装中 选择。直接过滤器结合测定将用于识别蛋白质 与亚基相互作用4。泛素的缺失分析 共轭约束亚基（S5A）将使用哪个 蛋白质的区域结合UB四聚体，并确定多少 存在绑定位点。最后，将使用定向的诱变 评估特定残基在四聚体结合中的重要性 S5A地区。 26S蛋白酶显然参与了对 细胞周期，它是最有可能产生抗原的酶 在MHC类L分子上显示的肽。由于这些原因，更好 对蛋白酶的了解应该具有重要的医学 含义。