PROPERTIES OF AN ATP-UBIQUITIN-DEPENDENT PROTEASE

ATP 泛素依赖性蛋白酶的特性

• 批准号: 2178633
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 21.7万
• 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 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178633
• 关键词: HeLa cells; active sites; adenosine triphosphate; antibody; crosslink; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; fluoresceins; fluorescence spectrometry; gel electrophoresis; laboratory rabbit; lysozyme; nucleic acid sequence; peptidases; phosphorylation; photochemistry; protein kinase; protein purification; tissue /cell culture; ubiquitin

Ubiquitin-dependent proteolysis plays a fundamental role in cell cycle regulation. Destruction of cyclin is required for cells to complete mitosis and evidence has recently been presented implicating the ubiquitin (Ub) pathway in this event. Four years ago this lab identified and purified a 26S protease capable of degrading ubiquitin-lysozyme conjugates. We proposed that the 26S protease was assembled from a well-known 20S protease (the multicatalytic protease or MCP) and polypeptides that confer ATP-dependence and ubiquitin recognition to the final complex. This application deals with the further characterization of this large ATP- stimulated multisubunit degradative enzyme. The isolation and sequencing of cDNAs that encode polypeptides in the 26S proteolytic particle will be continued. To study the assembly and disassembly of the 26S complex we have prepared fully active, fluoresceinated (F) 20S proteases and have shown that they can readily assemble into active 26S particles. We will continue to examine assembly/disassembly with the specific intention of testing "ribosome" versus "garbage disposal" models for protease mechanism. F-labeled 20S protease will also be employed to determine whether its subunits are in a state of dynamic equilibrium both in vitro and in living culture cells. Using photo-crosslinking approaches we identified two 26S subunits that bind ATP. Incubation of the 26S protease with gamma-32PO4- ATP produced three phosphorylated subunits. One of these is a 32K subunit in the 20S protease; we have evidence that another is a novel protein kinase inherent to the 26S particle. The role of phosphorylation in protease activity will be studied further. We will also use photo- crosslinking to identify 26S components that bind ubiquitin. Diubiquitin molecules linked by an isopeptide bond at lysine 48 will be derivatized and photocrosslinked to the 26S complex. Two years ago we developed a fast and very cheap method for synthesizing peptides as ubiquitin carboxyl extensions. This technology will be used to prepare chain-specific antibodies to components in the 26S complex and to prepare tethered substrates for probing active sites in the 20S protease. Finally, two separate enzymes that accurately process pentaUb to Ub have been identified. We will purify each enzyme, and we will continue our studies on poly-ubiquitin metabolism.

泛素依赖的蛋白分解在细胞周期中起着基础性的作用 监管。细胞需要细胞周期蛋白的破坏才能完成 有丝分裂和证据最近被提出与泛素有关 (UB)在此事件中的路径。四年前，这个实验室发现了 纯化了一种能够降解泛素-溶菌酶结合物的26S蛋白酶。 我们提出了26S蛋白酶是由一个众所周知的20S组装而成的 蛋白酶(多催化蛋白酶或MCP)和与之有关的多肽 ATP依赖和泛素识别到最终的复合体。这 申请涉及到这种大的ATP-的进一步表征- 刺激的多亚单位降解酶。细菌的分离和测序 在26S蛋白分解颗粒中编码多肽的cDNA将是 继续。为了研究26S复合体的组装和拆卸，我们 已经制备了完全活性的、荧光的(F)20S蛋白酶，并且已经 结果表明，它们可以很容易地组装成活性26S粒子。我们会 继续检查装配/拆卸，具体目的是 测试“核糖体”与“垃圾处理”模型的蛋白酶机制。 还将使用F标记的20S蛋白酶来确定其 亚基在体外和体内都处于动态平衡状态。 培养细胞。利用光交联法，我们鉴定了两个26S 结合三磷酸腺苷的亚基。26S水解酶与γ-32PO4-的孵育 三磷酸腺苷产生三个磷酸化亚基。其中一个是32K亚基。 在20s的蛋白酶中，我们有证据表明另一种是一种新的蛋白质 26S粒子所固有的激酶。磷酸化在细胞周期调控中的作用 水解酶活性有待进一步研究。我们还将使用照片- 交联化以识别与泛素结合的26S组分。泛素 通过赖氨酸48位的异肽键连接的分子将被衍生化并 与26S复合体发生光交联。两年前，我们开发了一种快速和 一种非常廉价的合成泛素羧基多肽的方法 分机。这项技术将用于制备特定于链的 抗26S复合体中各组分的抗体及制备 用于探测20S蛋白酶活性部位的底物。最后，两个 准确地将五联体转化为五联体的单独的酶是 确认身份。我们将提纯每一种酶，并继续我们的研究 关于多泛素代谢的研究。