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.

泛素依赖性蛋白水解在细胞周期中发挥重要作用 规定。 细胞需要破坏细胞周期蛋白才能完成 有丝分裂和最近提出的证据表明泛素 (Ub) 此事件中的途径。 四年前，该实验室发现并 纯化了能够降解泛素-溶菌酶缀合物的 26S 蛋白酶。 我们提出26S蛋白酶是由著名的20S蛋白酶组装而成 蛋白酶（多催化蛋白酶或MCP）和多肽 最终复合物的 ATP 依赖性和泛素识别。 这 应用程序处理这个大ATP-的进一步表征 刺激多亚基降解酶。 分离和测序 编码 26S 蛋白水解颗粒中多肽的 cDNA 的数量将是 持续。 为了研究 26S 复合体的组装和拆卸，我们 已制备出完全活性的荧光素 (F) 20S 蛋白酶，并具有 结果表明它们可以很容易地组装成活性 26S 颗粒。 我们将 继续检查组装/拆卸，其具体目的是 测试蛋白酶机制的“核糖体”与“垃圾处理”模型。 F标记的20S蛋白酶也将用于确定其是否 亚基在体外和体内均处于动态平衡状态 培养细胞。 使用光交联方法，我们确定了两个 26S 结合 ATP 的亚基。 26S 蛋白酶与 gamma-32PO4- 一起孵育 ATP 产生三个磷酸化亚基。 其中之一是 32K 亚基 在20S蛋白酶中；我们有证据表明另一种是一种新蛋白质 26S 颗粒固有的激酶。 磷酸化的作用 蛋白酶活性将进一步研究。 我们还将使用照片- 交联以识别结合泛素的 26S 成分。 双泛素 由赖氨酸 48 处的异肽键连接的分子将被衍生化并 光交联至 26S 复合物。 两年前，我们开发了一种快速且 合成泛素羧基肽的非常便宜的方法 扩展。 该技术将用于制备链条专用的 针对 26S 复合物中成分的抗体并制备系留 用于探测 20S 蛋白酶活性位点的底物。 最后，两个 准确地将 pentaUb 加工成 Ub 的分离酶已被 确定。 我们将纯化每一种酶，我们将继续我们的研究 多聚泛素代谢。