PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

26 S蛋白酶是一种大的多亚基酶，其降解重要的 调节蛋白如p53、细胞周期蛋白、NF κ B和鸟氨酸 脱羧酶它由一个调控复合物组成，至少含有 15种不同的亚基与多催化蛋白酶相关， 又增加了14个亚单位。将使用基于PCR的策略 获得编码四个26 S蛋白酶亚基（S2、S9、S11和S15）的cDNA， 仍无法确认cDNA将在E.杆菌和 亚单位特异性抗体将由重组体产生 proteins.脉冲追踪代谢标记和几种免疫沉淀 将使用各种方法来确定26 S的组件是否 蛋白酶处于动态平衡。26 S蛋白酶特异性抗体 子单元（S4、S5 a、S5 b、S6和S12）可用。这些抗体和 在不久的将来产生的其他物质将用于确定组织 分布和细胞内的位置的各个26 S亚基。在 两个独立的项目，定点诱变将用于评估 S4-like ATP酶N端卷曲螺旋区的重要性 （亚基4、6、7和8）在26 S蛋白酶的组装中和在底物中 选择.直接过滤器结合试验将用于识别蛋白质 与第四亚单位相互作用。泛素的缺失分析- 缀合物结合亚基（S5 a）将用于确定 蛋白质的一个或多个区域结合Ub四聚体，并确定有多少 存在结合位点。最后，将使用定点诱变 为了评估特定残基在四聚体结合中的重要性， S5 A的区域。26 S蛋白酶显然参与了对蛋白质的控制。 细胞周期，是最有可能产生抗原的酶。 在MHC I类分子上展示的肽。由于这些原因， 对蛋白酶的了解应该有重大的医学意义 影响

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.