MOLECULAR CHAPERONES AND PROTEIN DEGRADATION

分子伴侣和蛋白质降解

• 批准号: 6011876
• 负责人: ALFRED L GOLDBERG
• 金额: $ 40.86万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6011876
• 关键词: Archaea; Escherichia coli; adenosinetriphosphatase; bacterial proteins; cell free system; electron microscopy; fungal proteins; mass spectrometry; molecular chaperones; proteasome; protein degradation; protein structure; stress proteins; ubiquitin; yeasts

DESCRIPTION: Bacterial and animal cells selectively degrade proteins with abnormal conformations. This process helps prevent the intracellular accumulation of unfolded proteins in genetic diseases, during heat shock, and with aging. Molecular chaperones, in addition to catalyzing protein folding and translocation, play essential roles in this degradative process. During the current funding period the PI has shown that in E. coli the chaperones DnaK, DnaJ, and GrpE are necessary for the rapid degradation of certain unfolded model polypeptides while GroEL, GroES, and Trigger Factor function together in the breakdown of other (abnormal) polypeptides. These chaperones associate with the substrates and appear to release or present them in a conformation that facilitates proteolytic digestion. In yeast the PI has shown that the DnaJ homolog Ydj1 and Hsp70 of the SSA family are essential for ubiquitin conjugation to abnormal proteins and certain regulatory proteins by binding and enhancing the susceptibility for ubiquitination. The current grant is comprised of four specific aims. Specific Aim A will continue studies on the roles of GroEL/ES/TF in the degradation of the recombinant model substrate CRAG by delineating discrete steps in the initial fragmentation. These studies will combine proteolytic susceptibility studies, mass spec analysis of peptide products, and direct structure determination by EM tomography in collaboration Wolfgang Baumeister. Other studies will test whether the model CRAG substrate is degraded through an analogous chaperone dependent pathway in yeast, allowing the use of yeast genetics to dissect steps. Specific Aim B will focus on clarifying the functions of DnaK and DnaJ in the degradation of the unfolded protein PhoA in E. coli. These studies will use His tagged PhoA and protease dificient strains to isolate intact substrate-chaperone complexes. Specific Aim C will determine the roles of the DnaJ and Hsp70 families in the ubiquitin-proteasome pathway in yeast using model substrates carrying defined degradation signals. This line of investigation will be extended to defined cell free studies as well as studies of the potential role of Sis1 in binding to ubiquitin conjugates prior to degradation by the proteasome. Specific Aim D will define the chaperone like function of the PAN and HslU ATPases which support protein breakdown by proteasome homologs in archeabacteria and E. coli.

描述：细菌和动物细胞选择性地降解蛋白质 异常构象。此过程有助于防止细胞内 在遗传疾病，热休克和 随着衰老。分子伴侣，除了催化蛋白质折叠和 易位，在此降解过程中扮演重要角色。在 当前资金期PI表明，在大肠杆菌中，DNAK， DNAJ和GRPE对于某些展开的快速降解是必需的 在凹槽，凹槽和触发因数一起函数的同时，模拟多肽 其他（异常）多肽的分解。这些伴侣与 底物并似乎以一种构象来释放或呈现它们 促进蛋白水解消化。在酵母中，PI表明DNAJ SSA家族的同源性YDJ1和HSP70对于泛素至关重要 通过结合和 增强对泛素化的敏感性。当前的赠款包括 在四个特定目标中。具体目标将继续研究 在重组模型底物crag降解中的凹槽/ES/TF通过 在初始碎片中描述离散步骤。这些研究会 结合蛋白水解敏感性研究，肽的质量分析 产品，以及通过EM层析成像进行的直接结构确定 Wolfgang Baumeister。其他研究将测试模型crag底物是否 通过酵母中的类似伴侣依赖途径降解，允许 使用酵母遗传学来剖析步骤。特定的目标B将重点放在 阐明DNAK和DNAJ在展开的降解中的功能 大肠杆菌中的蛋白质phoa。这些研究将使用他标记的phoa和蛋白酶 可分离完整的底物链酮复合物的多种菌株。具体目标 C将确定DNAJ和HSP70家族在 使用定义的模型基板在酵母中的泛素蛋白 - 蛋白酶体途径 退化信号。这一调查将扩展到定义 无细胞研究以及SIS1在结合中的潜在作用的研究 在蛋白酶体降解之前，将泛素结合物。具体目标d 将定义PAN和HSLU ATPases的伴侣功能 支持细菌和大肠杆菌中蛋白酶体同源物的蛋白质分解。