ATP UBIQUITIN-DEPENDENT PROTEOLYSIS

ATP 泛素依赖性蛋白水解

• 批准号: 6018622
• 负责人: ARTHUR L HAAS
• 金额: $ 27.12万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018622
• 关键词: HeLa cells; active sites; adenosine monophosphate; adenosine triphosphate; affinity chromatography; chemical binding; chemical conjugate; chemical kinetics; conformation; covalent bond; enzyme complex; enzyme mechanism; enzyme substrate; isozymes; laboratory rabbit; molecular cloning; mutant; polymerase chain reaction; proteasome; protein degradation; protein sequence; protein structure function; site directed mutagenesis; stoichiometry; thioester; ubiquitin

The steady state intracellular concentration of proteins is regulated by the dynamic balance between rates of synthesis and degradation. The major pathway for degradation within eukaryotes is mediated by the multienzyme ATP, ubiquitin-dependent proteolytic pathway in which proteins are targeted for degradation by the 26S proteasome through their covalent conjugation to the 8.6 kDa polypeptide ubiquitin. Recent evidence indicates the ubiquitin/proteasome degradative pathway is required for a number of fundamental regulatory processes including proteolysis of abnormal proteins, mitotic progression, gene transcription and protein processing, developmentally-programmed cell death, the stress response, organelle biogenesis, and the turnover of various oncoproteins, tumor suppressors, and transcription factors. The long range goal of this proposal is to elucidate the enzymology and function of this pathway for potential therapeutic intervention. The immediate goals of this proposal constitute five specific aims: (1) Site-directed mutagenesis of ubiquitin will be utilized to identify key residues on the polypeptide required for its function as a means of mapping the active sites for key enzymes of the pathway; (2) Mutagenesis of specific residues within E1, the first enzyme of ubiquitin conjugation, will be exploited to identify the active site of this enzyme; (3) Deletion/mutation analysis of the major ubiquitin carrier protein E2/14K will be used to identify regions of the enzyme that interact with E1 and E3, the substrate recognition/conjugation enzyme; (4) Mutagenesis of a recently discovered ubiquitin carrier protein E2EPF, suggested to be required developmentally for the terminal differentiation of keratinocytes, will be utilized to identify the site of autoubiquitination proposed to regulate the self- targeting and intracellular concentration of this enzyme, followed by direct test of the hypothesis in cultured human cell lines by transient transfection of appropriate E2EPF mutants; (5) E2-affinity and two-hybrid screening methods will be used to isolate and clone cognate E3 isozymes requiring E2/14K and E2EPF, followed by their expression and kinetic analysis of the mechanisms for the recombinant conjugating enzymes.

蛋白质的稳态细胞内浓度受 合成速率和降解速率之间的动态平衡。 这 真核生物内降解的主要途径是由 多元素ATP，泛素依赖性蛋白水解途径，其中 蛋白质的靶向26S蛋白酶体通过其降解 与8.6 kDa多肽泛素的共价共轭。 最近的 证据表明泛素/蛋白酶体降解途径是 许多基本的监管程序所需的 异常蛋白质，有丝分裂进展，基因转录的蛋白水解 和蛋白质加工，发育编程的细胞死亡，应力 反应，细胞器生物发生和各种癌蛋白的周转， 肿瘤抑制剂和转录因子。 远程目标的 该建议是阐明该途径的酶学和功能 进行潜在的治疗干预。 直接目标 提案构成五个具体目的：（1）以地点为导向的诱变 泛素将用于识别多肽上的关键残基 其功能是映射键的活动站点所必需的 途径的酶； （2）E1中特定残基的诱变 泛素结合的第一种酶将被利用以识别 该酶的活跃部位； （3）缺失/突变分析 主要的泛素载体蛋白E2/14K将用于识别区域 与E1和E3相互作用的酶的底物 识别/共轭酶； （4）最近发现的诱变 泛素载体蛋白E2EPF，建议在发展中需要 对于角质形成细胞的末端分化，将被用作 确定提议调节自我的自动泛素化部位 该酶的靶向和细胞内浓度，然后是 直接检验瞬时培养的人类细胞系中的假设 转染适当的E2EPF突变体； （5）E2亲和力和两个杂交 筛选方法将用于隔离和克隆同源E3同工酶 需要E2/14K和E2EPF，然后是它们的表达和动力学 分析重组共轭酶的机制。