ROLE OF ATP-DEPENDENT PROTEOLYSIS IN MAMMALIAN CELLS

哺乳动物细胞中 ATP 依赖性蛋白水解的作用

• 批准号: 3456859
• 负责人: JULIE M FAGAN
• 金额: $ 10.02万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3456859
• 关键词: adenosine triphosphate; affinity chromatography; chemical conjugate; chickens; enzyme complex; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; genetic translation; histochemistry /cytochemistry; immunochemistry; laboratory rabbit; laboratory rat; messenger RNA; muscle cells; muscle disorders; muscle metabolism; muscle proteins; nonhistone nucleoprotein; peptidases; protease inhibitor; protein sequence; tissue /cell culture; ubiquitin

Since the rate at which a cell grows depends on a net balance between protein synthesis and protein breakdown, the mechanisms and control of both processes are of great biomedical importance. Over the years, much has been learned about the protein synthetic process but the notion still remains that the degradation of proteins occurs primarily in lysosomes by acid hydrolases. However, recent evidence indicates that a nonlysosomal degradative process, one which requires ATP, may catalyze the rate-limiting step in the breakdown of both abnormal and short- lived normal proteins in mammalian tissue. A cytoplasmic ATP- dependent protease, which we first isolated from reticulocytes and more recently from skeletal muscle and liver, is involved in the degradation of proteins conjugated to ubiquitin and may function to prevent the accumulation of such proteins in these cells. The structure and mechanism of this novel class of enzymes has not been investigated. To help clarify the role of the ATP-dependent degradative pathway in protein breakdown and propose to 1) purify the ATP- dependent proteases from muscle and identify their physiological substrates and inhibitors. 2) The purified enzymes will be used to produce polyclonal antibodies for an ELISA assay to monitor levels of the enzyme during periods of rapid muscle loss (e.g. denervation, starvation, disuse, infection, and cancer). 3) Antibodies to ubiquitin will identify which subcellular compartments and components of the cell contain ubiquitin- protein conjugates. 4) Characterization of the purified ATP- dependent proteases will help identify specific inhibitors to this proteolytic pathway and will also provide a tool or investigating the mechanism of action of these proteases. 5) By in vitro physiological techniques, we hope to clarify the relative importance of the ATP-dependent proteases and the other proteolytic system in muscle with inhibitors to these pathways. We will test whether rates of protein breakdown correlate with levels of a ATP-dependent proteases and the extent of ubiquitination. 6) mRNAs which code for the ATP-dependent proteases will be isolated and used to study the biosynthesis and the regulation of these proteases at the genetic level. cDNA clones will be isolated using synthetic oligonucleotides as well as antibodies to obtain hybridization probes. It will then be possible to measure whether changes in enzyme levels under different physiological conditions are due to regulation of the steady-state mRNA levels or to changes in gene copy.

由于细胞生长的速率取决于净余额 在蛋白质合成和蛋白质分解之间 对这两个过程的控制非常重要。 多年来，关于蛋白质合成的知识已经很多 过程，但仍然存在降解的观念 蛋白质主要发生在酸水解酶的溶酶体中。 但是，最近的证据表明非血液体系 降解过程（需要ATP）可能会催化 异常和短期的分解中的限速步骤 在哺乳动物组织中生命正常蛋白质。 细胞质ATP- 依赖蛋白酶，我们首先从网状细胞中分离出来 最近来自骨骼肌和肝脏，参与 结合泛素的蛋白质降解，可能 功能以防止这些蛋白在这些中积累 细胞。 这个新颖类的结构和机制 酶尚未研究。 帮助阐明依赖ATP的降解的作用 蛋白质分解中的途径，并提出1）净化ATP- 肌肉的依赖蛋白酶并识别其生理 底物和抑制剂。 2）纯化酶将用于 产生ELISA测定的多克隆抗体以监测 在快速肌肉丧失期间的酶水平（例如 去神经，饥饿，废弃，感染和癌症）。 3） 泛素的抗体将识别哪个亚细胞 细胞的隔室和成分包含泛素 - 蛋白结合物。 4）表征纯化的ATP- 依赖蛋白酶将有助于确定特定的抑制剂 蛋白水解途径，还将提供工具或研究 这些蛋白酶的作用机理。 5）在体外 生理技术，我们希望澄清亲戚 依赖ATP的蛋白酶和其他的重要性 肌肉中具有抑制剂的蛋白水解系统。 我们将测试蛋白质分解的发生率是否与 ATP依赖性蛋白酶的水平和 泛素化。 6）mRNA代码为ATP依赖性 蛋白酶将被分离并用于研究生物合成和 这些蛋白酶在遗传水平上调节。 cDNA 克隆将使用合成寡核苷酸以及 获得杂交探针的抗体。 那将有可能 衡量不同的酶水平的变化 生理条件是由于稳态的调节 mRNA水平或基因拷贝变化。