PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

26S蛋白水解酶是一种大的多亚单位酶，它能降解重要的 调控蛋白如P53、细胞周期蛋白、NFkappaB和鸟氨酸 脱羧酶。它由一个监管复合体组成，其中至少包含 与多催化蛋白酶相关的15个不同亚基 额外贡献了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(S2、S9、S11和S15)的cDNA 这些人的身份仍未确定。这些DNA将在大肠杆菌中表达，并 亚基特异性抗体将从重组的 蛋白质。脉冲追逐代谢标记和几种免疫沉淀 将使用方法来确定26S的组件是否 蛋白酶处于动态平衡状态。26S蛋白酶特异性抗体 亚基(S4、S5a、S5b、S6和S12)可用。这些抗体和 在不久的将来产生的其他组织将被用于确定组织 26S亚基在细胞内的分布和定位。在……里面 两个独立的项目，定点突变将用于评估 S4样三磷酸腺苷酶N末端卷曲区域的重要性 (亚基4、6、7和8)在26S酶的组装和底物中 选择。直接过滤结合分析将被用来鉴定蛋白质 与亚基4相互作用。泛素的缺失分析- 将使用共轭结合亚基(S5a)来确定 结合Ub四聚体的蛋白质区域(S)和测定多少 存在结合位点。最后，将使用定点突变。 评估特定残基在四聚体结合中的重要性 S5a的区域。26S蛋白水解酶显然参与了对 细胞周期，它是最有可能产生抗原性的酶 显示在MHC类L分子上的多肽。出于这些原因，更好的 对蛋白水解酶的了解应该有重要的医学意义 这意味着什么。

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.