SPECIFICITY OF INTRACELLULAR PROTEOLYSIS IN EUKARYTOES

真核生物细胞内蛋白水解的特异性

• 批准号: 3293164
• 负责人: ROBERT E. COHEN
• 金额: $ 17.15万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293164
• 关键词: Saccharomyces; chemical conjugate; conformation; cytochrome c; enzyme substrate; high performance liquid chromatography; laboratory rabbit; mutant; nonenzyme proteolytic agent; nonhistone nucleoprotein; nuclear magnetic resonance spectroscopy; peptidases; protein sequence; protein structure; trypsin inhibitors; ubiquitin

Nearly all aspects of cell growth and metabolism rely upon precise control of protein levels. This balance is not achieved solely through the regulation of protein synthesis, but depends as well upon the rates of protein degradation. Metabolic shifts, environmental stress, and cellular differentiation all are accompanied bby selective proteolysis as the cell adapts to its new condition. To accomplish this, the machinery for intracellular proteolysis must possess both specificity and great flexibility. In eukaryotes, one pathway of intracellular proteolysis involves a 76 amino acid polypeptide, ubiquitin (Ub), which is attached covalently to proteins prior to their degradation. Two questions concerning this system will be addressed: what features of the target protein make it a substrate for ubiquitination, and what features of the resultant conjugate are recognized for subsequent proteolysis. A series of native and modified forms of cytochrome c's and bovine pancreatic trypsin inhibitor will be used as substrates to examine: 1) selectivity among a well-defined set of target proteins that represent folded, misfolded and unfolded conformations; 2) characteristics of ubiquitination sites -- sequence, tertiary structure, and flexibility; 3) involvement of the substrate N-terminus in target recognition; 4) the apparent cooperativity or processivity of ubiquitination. The structures of free Ub and various Ub-amide and Ub-protein conjugates will be compared by one- and two-dimensional 1H nuclear magnetic resonance spectroscopy. Ubiquitinated degradation intermediates of apo-cytochrome c will be sought in yeast. Enzymes of the yeast Ub pathway will be purified and an in vitro ubiquitination system established. Ub conjugation to various mutant apo-cytochrome c's in vitro will be compared with their in vivo turnover rates.

细胞生长和新陈代谢的几乎所有方面都取决于精确的控制 蛋白质水平。 这种平衡不是仅通过 调节蛋白质合成，但也取决于 蛋白质降解。 代谢移位，环境应力和细胞 分化都伴随BBY选择性蛋白水解为细胞 适应其新状况。 为此， 细胞内蛋白水解必须具有特异性既具有特异性 灵活性。 在真核生物中，细胞内蛋白水解的一个途径涉及76氨基 酸多肽，泛素（UB），该蛋白与蛋白质共同附着 在降解之前。 有关此系统的两个问题将是 发表：目标蛋白的哪些特征使其成为基材 泛素化以及所得共轭的哪些特征被识别 随后的蛋白水解。 细胞色素C和牛的一系列天然和改良形式 胰腺胰蛋白酶抑制剂将用作检查的底物： 1）一组定义明确的靶蛋白的选择性 折叠，错误折叠和展开的构象； 2）泛素化位点的特征 - 序列，三级结构， 和灵活性； 3）底物N末端参与目标识别； 4）泛素化的明显合作或加工性。 自由UB以及各种UB-酰胺和UB蛋白结合的结构 将通过一维和二维的1H核磁共振比较 光谱法。 将寻求泛素化降解中间体 在酵母中。 酵母UB途径的酶将被纯化并在体外纯化 建立了泛素化系统。 UB连接到各种突变体 将Apo-Cytochrome C的体外C与其体内周转率进行比较 费率。