CNS MULTICATALYTIC PROTEINASE COMPLEX

CNS 多催化蛋白酶复合物

• 批准号: 2268025
• 负责人: SHERWIN WILK
• 金额: $ 21.09万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268025
• 关键词: Baculoviridae; acetylation; animal extract; animal tissue; caseins; catalyst; chromatography; endopeptidases; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; gene expression; genetic library; genetic regulatory element; imidazole; immunoaffinity chromatography; molecular cloning; nucleic acid sequence; phosphorylation; protein kinase A; protein sequence; radiotracer; recombinant DNA; stoichiometry

Proteolytic enzymes may play a role in the pathophysiology of degenerative diseases of the brain. The multicatalytic proteinase complex (MPC) first isolated and characterized in our laboratory from bovine pituitaries a decade ago, is a unique high molecular mass proteinase (700 kDa), composed of multiple non-identical subunits, and containing at least three distinct proteolytic activities. The primary structures of its cloned subunits which are highly conserved, bear no homology to any known protein but are homologous to each other. MPC is present in relatively high concentrations in brain and pituitary cytoplasm, where it likely participates in the extralysosomal degradation of ubiquitinated and non-ubiquitinated proteins. Many questions about the mechanism of its regulation and the relationship of its structure to its properties remain unanswered. We have found that chemical modification is a powerful approach to the study of structure- function relationships. Acetylation with N-acetylimidazole revealed that the initial degradation of casein may be catalyzed by a fourth component, distinct from the three components previously described and demonstrated that it is possible to prepare different catalytically active forms of the enzyme. The studies proposed in this application will employ chemical modification to characterize the individual components. N-acetylimidazole, citraconic anhydride and succinic anhydride will be used to determine the relationship of quaternary structure to function, and to prepare forms of the enzyme for characterization of the substrate specificities of each of the components. Active-site directed inhibitors will be synthesized. MPC has been isolated in both latent and activated forms. The mechanisms underlying its activation remain unknown. We have demonstrated that several MPC subunits are phosphorylated by a cAMP-dependent protein kinase that co-purifies with the proteinase. Phenyl Sepharose chromatography removes the kinase as well as the heaviest MPC subunit, while activating the degradation of casein. The possible role of phosphorylation and of the heaviest MPC subunit in the regulation of MPC activity will be explored by the application of classical and molecular biological techniques. It is likely that MPC abnormalities underlie some brain pathologies. The proposed studies should aid in our understanding of this biologically significant macromolecule.

蛋白水解酶可能在退化的病理生理中起作用 大脑疾病。 多催化蛋白酶复合物（MPC）首先 在我们的实验室中孤立并从牛垂体中进行特征 十年前，是一种独特的高分子质量蛋白酶（700 kDa），组成 多个非相同亚基，至少包含三个不同 蛋白水解活性。 克隆亚基的主要结构 高度保守的，对任何已知蛋白质都没有同源性，而是 彼此同源。 MPC以相对较高的浓度存在 在大脑和垂体细胞质中，它可能参与 泛素化和非泛素化蛋白的外染色体降解。 有关其监管机制及关系的许多问题 其属性的结构仍未得到解答。 我们发现 化学修饰是研究结构的强大方法 - 功能关系。 N-乙酰咪唑用乙酰化表明， 酪蛋白的初始降解可以由第四个成分催化， 与先前描述和证明的三个组成部分不同 可以准备不同的催化活性形式的 酶。 本应用中提出的研究将采用化学 修改以表征各个组件。 N-乙酰咪唑，， 柑橘酸酐和琥珀酸酐将用于确定 第四纪结构与功能的关系，并准备形式 表征每个的底物特异性的酶 组件。 将合成活跃位点的定向抑制剂。 MPC 已经以潜在和激活形式隔离。 机制 其基本激活仍然未知。 我们已经证明了 几个MPC亚基被cAMP依赖性蛋白激酶磷酸化 这与蛋白酶共磨。 苯基色糖色谱法 在激活时去除激酶以及最重的MPC亚基 酪蛋白的退化。 磷酸化和 MPC活动调节中最重的MPC亚基将由 经典和分子生物学技术的应用。 这是 MPC异常可能是一些大脑病理的基础。 这 拟议的研究应有助于我们对这一生物学的理解 显着的大分子。