TARGETING SCF SUBSTRATES TO THE PROTEASOME

将 SCF 底物靶向蛋白酶体

• 批准号: 6623058
• 负责人: DOROTA SKOWYRA
• 金额: $ 25.63万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623058
• 关键词: Saccharomyces cerevisiae; acid aminoacid ligase; adenosinetriphosphatase; cysteine endopeptidases; enzyme complex; enzyme mechanism; enzyme substrate complex; green fluorescent proteins; immobilized enzymes; proteasome; protein binding; protein degradation; protein folding; ubiquitin; Saccharomyces cerevisiae; acid aminoacid ligase; adenosinetriphosphatase; cysteine endopeptidases; enzyme complex; enzyme mechanism; enzyme substrate complex; green fluorescent proteins; immobilized enzymes; proteasome; protein binding; protein degradation; protein folding; ubiquitin

DESCRIPTION (provided by applicant): The ubiquitin-mediated protein degradation by the proteasome has been only recently recognized as critical for cellular signaling in cell growth and proliferation. Since then, perturbations of the ubiquitin-mediated proteolysis have been implicated in multiple aspects of the pathogenesis of cancer. This makes the proteasome an attractive target for possible therapeutical intervention. The long-term goal of the proposed work is to understand the molecular mechanisms by which the proteasome recruits substrates and initiates their destruction. It is proposed to address this goal by biochemical dissection of protein degradation in vitro, using purified substrates and components of the SCF ubiquitin ligase pathway of yeast S. cerevisiae, which were discovered and characterized by the principal investigator's group. This pathway is conserved and controls degradation of major Gi cell cycle regulatory proteins and signaling molecules in organisms from yeast to humans. The knowledge obtained with yeast is therefore directly relevant to the understanding of SCF-mediated proteolysis in human cells. In the current application, it is proposed to uncover features of the proteasome that could serve as targets for pharmacological regulation of its activity at the steps of substrate recognition and processing for degradation, but not the degradation itself. This knowledge will be of considerable value for development of novel strategies for targeting the proteasome in cancer. In this proposal there are two specific aims: (1) Identify the mechanism by which SCF ubiquitin ligase associates with the proteasome and define its role in targeting substrates for degradation. It was observed that SCF targets selected proteins for degradation in two possible ways: (1) by promoting substrate ubiquitination and (2) by facilitating its direct contact with the proteasome. Defining the role of SCF binding to the proteasome in protein turnover requires isolation of SCF mutants that cannot bind the proteasome while maintaining the ubiquitin ligase activity. To isolate and characterize such mutants, an in vitro system with purified proteins has been developed that provides the investigator with a unique opportunity to address the protein-protein interactions required for SCF/proteasome binding. With these reagents the ubiquitin and the SCF-mediated degradation of natural SCF substrates both in vitro and in vivo, including defining the precise requirements for substrate recognition will be investigated. (2) Characterize the substrate unfolding step and its role in the release of the non-ubiquitinated subunits of substrate complexes. In the SCF pathway, the substrate polypeptide is only one component of a tightly bound multi-protein complex that is targeted to the proteasome. It is proposed to investigate the role of substrate unfolding as a potential discriminatory step in substrate selection. This includes: (1) establishing a reliable substrate-unfolding assay with purified proteasomes, (2) identification of the proteasome subunits that play a role in substrate unfolding using purified proteasome mutants, and (3) defining whether these subunits play a role in the release of the non-ubiquitinated components of substrate complexes.

描述（由申请人提供）：泛素介导的蛋白质降解 蛋白酶体仅被最近被认为对细胞至关重要 细胞生长和增殖中的信号传导。从那以后， 泛素介导的蛋白水解已与多个方面有关 癌症的发病机理。这使得蛋白酶体成为有吸引力的目标 可能的治疗干预措施。 拟议工作的长期目标是了解分子 蛋白酶体募集底物并启动其蛋白酶的机制 破坏。建议通过生化解剖来解决这一目标 蛋白质在体外降解，使用纯化的底物和成分 酵母菌S.酿酒酵母的Scf泛素连接酶途径，被发现并 以首席研究员小组为特征。该途径是保守的 并控制主要的GI细胞周期调节蛋白和 从酵母到人类的生物体中的信号分子。获得的知识 因此，使用酵母与对SCF介导的理解直接相关 人类细胞中的蛋白水解。在当前的应用程序中，建议 发现可以作为目标的蛋白酶体的特征 在底物步骤中对其活性的药理调节 识别和处理降解，但没有降解本身。 这些知识对于开发新颖而具有很大的价值 针对癌症中蛋白酶体的策略。在这个建议中有 两个具体的目的：（1）确定SCF泛素连接酶的机制 与蛋白酶体相关联，并定义其在靶向基材中的作用 降解。据观察，SCF靶标选择了降解蛋白 通过两种可能的方式：（1）通过促进底物泛素化，（2） 促进其与蛋白酶体的直接接触。定义SCF的作用 与蛋白质更新中的蛋白酶体结合需要分离SCF突变体 在维持泛素连接酶的同时，无法结合蛋白酶体 活动。为了隔离和表征这种突变体，一种体外系统 已经开发了纯化的蛋白质，为研究者提供了 解决蛋白质 - 蛋白质相互作用所需的独特机会 SCF/蛋白酶体结合。使用这些试剂，泛素和SCF介导的 体外和体内的天然SCF底物的降解，包括 定义底物识别的确切要求将是 调查。 （2）表征底物展开步骤及其在 释放底物复合物的非泛素亚基。在SCF中 途径，底物多肽只是紧密结合的一个成分 针对蛋白酶体的多蛋白质复合物。提议 研究底物展开作为潜在歧视性步骤的作用 在底物选择中。这包括：（1）建立可靠的 用纯化蛋白酶体的底物无折叠测定，（2）鉴定 使用纯化的蛋白酶体亚基在底物展开中发挥作用 蛋白酶体突变体，以及（3）定义这些亚基是否在 底物复合物的非泛素化成分的释放。