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; 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起着基本作用 规定。 细胞完成需要破坏细胞周期蛋白 最近提出了有丝分裂和证据，暗示了泛素 （UB）此事件中的途径。 四年前，这个实验室确定了 纯化的26S蛋白酶能够降解泛素 - 淋巴结缀合物。 我们提出26S蛋白酶是从众所周知的20s组装的 蛋白酶（多催化蛋白酶或MCP）和赋予多肽 ATP依赖性和泛素识别最终复合物。 这 应用程序涉及这个大型ATP的进一步表征 刺激的多亚基降解酶。 隔离和测序 编码26S蛋白水解颗粒中多肽的cDNA将是 持续。 研究26S综合体的组装和拆卸我们 已经制备了完全活跃的荧光（F）20S蛋白酶，并具有 表明它们可以轻松地组装成活性26S颗粒。 我们将 继续检查组装/拆卸的特定意图 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是否 亚基在体外和生活中处于动态平衡状态 培养细胞。 使用照片跨链接方法，我们确定了两个26s 结合ATP的亚基。 将26S蛋白酶与γ-32PO4-孵育 ATP产生了三个磷酸化亚基。 其中之一是一个32K亚基 在20年代的蛋白酶中；我们有证据表明另一种是一种新型蛋白质 26S粒子固有的激酶。 磷酸化在 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。 二丁纤维素 通过赖氨酸48处的无肽键连接的分子将被衍生，并 光叠链接链接到26S复合体。 两年前，我们开发了一个快速的 将肽合成为泛素羧基的非常便宜的方法 扩展。 该技术将用于准备特定于链的技术 在26S复合物中的组件抗体并制备束缚 用于探测20S蛋白酶活性位点的底物。 最后，两个 精确处理pentaub到Ub的单独酶已经 确定。 我们将净化每个酶，我们将继续学习 关于多泛素代谢。