BIOCHEMISTRY OF ENERGY-DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

能量依赖性（细胞内）蛋白质降解的生物化学

• 批准号: 3774309
• 负责人: M R MAURIZI
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3774309
• 关键词: Escherichia coli; active sites; adenosine triphosphate; adenosinetriphosphatase; allosteric site; bacterial proteins; bioenergetics; endopeptidases; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; isozymes; protein degradation; site directed mutagenesis

ATP-dependent proteases play critical roles in the post-translational regulation of the amounts and activities of important cellular proteins and in the removal of damaged and denatured proteins from the cell. Our research has focussed on the biochemical characterization of the ATP- dependent Clp and Lon proteases from E. coli and on the discovery of new ATP-dependent proteases. Site-directed mutagenesis of the two ATPase sites in ClpA demonstrated that the active form of ClpA is a hexamer and that interactions between the subunits affect the ATPase and protease- activating activity of ClpA. The domain I site was shown to be involved in assembly of the ClpA hexamer, and interaction with ClpP and the domain II site was needed for activation of the proteolytic activity ClpP against large proteins. Model peptide substrates have been synthesized and shown to be cleaved more rapidly than any previous substrates. These peptides interacted tightly with ClpA and activated its ATPase activity. The site on ClpA occupied by the peptides defines the allosteric site for proteins on the enzyme, and occupancy of this site appears to be necessary to open the active site of ClpP for large polypeptides and to accelerate the catalytic cleavage of peptide bonds. Using partially inactivated ClpP, it was demonstrated that processive cleavage of polypeptides and proteins required the array of active sites present in the dodecameric form of ClpP. Processive cleavage of model peptides was observed when non- hydrolyzable analogs of ATP were used to activate the enzyme, indicating that ATP hydrolysis does not play a direct role in processivity. Studies done in collaboration with Dr. Susan Gottesman have demonstrated that another ATPase from E. coli, ClpX, is evolutionarily related to ClpA and that this protein functions in vivo with ClpP as an essential component of a proteolytic system that degrades the highly unstable lambda O protein. Sequence and biochemical data now indicate the presence in E. coli of four ATPases, Clps A, B, X and Y and suggest that the energy-dependent proteolytic systems of even this simple organism are highly complex. In studies done in collaboration with Drs. Michael Gottesman and Nan Wang, it has been found that a close homolog of the ATP-dependent Lon protease occurs in human mitochondria. Further biochemical and immunochemical studies are underway to identify the properties and function of this protease in human cells.

依赖于ATP的蛋白水解酶在翻译后中起关键作用 对重要细胞蛋白的数量和活性的调节 以及从细胞中去除受损和变性的蛋白质。我们的 研究的重点是三磷酸腺苷的生化特性- 大肠杆菌依赖的CLP和Lon蛋白水解酶及其新的发现 依赖于ATP的蛋白水解酶。两个ATPase位点的定点突变 在ClpA中证明了ClpA的活性形式是六角体，并且 亚基之间的相互作用影响ATPase和蛋白酶- 激活ClpA的活性。域I站点被显示参与了 组装ClpA六聚体，并与ClpP和结构域II相互作用 ClpP蛋白水解酶活性的激活需要结合位点 大的蛋白质。合成了模拟多肽底物，并展示了 比任何以前的底物都要快地被切割。这些多肽 与ClpA紧密结合，激活其ATPase活性。该网站 多肽占据的ClpA决定了蛋白质的变构位置 在酶上，而占用这个站点似乎是必要的 ClpP对大分子多肽的活性部位和促进 多肽键的催化断裂。使用部分失活的ClpP，它 证明了多肽和蛋白质的连续裂解 所需的以十二进制形式存在的活动位置数组 ClpP。观察到模型多肽在非 ATP的可水解物被用来激活酶，表明 三磷酸腺苷的水解并不直接影响蛋白质的加工性。研究 与苏珊·戈特斯曼博士合作完成的研究证明了 来自大肠杆菌的另一种ATPase ClpX在进化上与ClpA和 这种蛋白质在体内发挥作用，而ClpP是 一种能降解高度不稳定的lambda O蛋白的蛋白分解系统。 序列和生化数据现在表明，在大肠杆菌中存在四种 ATPase、CLP A、B、X和Y，并提示能量依赖 即使是这种简单的有机体的蛋白质分解系统也是非常复杂的。在……里面 与Michael Gottesman博士和Nan Wang博士合作进行的研究，它 已发现依赖于ATP的Lon蛋白水解酶的一种近似物 发生在人类线粒体中。进一步的生化和免疫化学 目前正在进行研究，以确定该酶的性质和功能 人体细胞中的蛋白酶。