TARGETING SCF SUBSTRATES TO THE PROTEASOME

将 SCF 底物靶向蛋白酶体

• 批准号: 6460809
• 负责人: DOROTA SKOWYRA
• 金额: $ 25.68万
• 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/6460809
• 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

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（申请人提供）：泛素介导的蛋白质降解 直到最近才被认为是细胞增殖的关键。 细胞生长和增殖中的信号传导。从那时起， 泛素介导的蛋白质水解已经涉及多个方面的 癌症的发病机制。这使得蛋白酶体成为一个有吸引力的目标， 可能的治疗干预。 这项工作的长期目标是了解 蛋白酶体募集底物并启动其 杀伤性有人建议通过生物化学解剖来实现这一目标， 体外蛋白质降解，使用纯化的底物和组分， 酵母S.酿酒酵母，被发现， 主要研究者小组的特点。该途径是保守的 并控制主要Gi细胞周期调节蛋白的降解， 从酵母到人类的生物体中的信号分子。获得的知识 因此，与酵母直接相关的理解SCF介导的 人体细胞中的蛋白质分解在本申请中，提出 揭示蛋白酶体的特征，可以作为靶点， 药理学调节其活性的步骤底物 识别和处理降级，而不是降级本身。 这些知识对于小说的发展具有重要的价值 针对癌症中蛋白酶体的策略。在这项建议中， 两个具体的目标：（1）确定SCF泛素连接酶的机制， 与蛋白酶体相关，并确定其在靶向底物中的作用， 降解观察到SCF靶向选定的蛋白质进行降解 两种可能的方式：（1）通过促进底物泛素化和（2）通过 促进其与蛋白酶体的直接接触。定义SCF的角色 在蛋白质周转中与蛋白酶体结合需要分离SCF突变体 不能结合蛋白酶体而保持泛素连接酶 活动为了分离和表征这样的突变体，使用体外系统， 已经开发出纯化的蛋白质，其为研究者提供了 独特的机会来解决蛋白质-蛋白质相互作用所需的 SCF/蛋白酶体结合。用这些试剂，泛素和SCF介导的 在体外和体内降解天然SCF底物，包括 定义基板识别的精确要求将是 研究了(2)表征底物解折叠步骤及其在制备中的作用。 底物复合物的非泛素化亚基的释放。在SCF中 底物多肽仅是紧密结合的多肽的一个组分。 靶向蛋白酶体的多蛋白复合物。提出要 研究底物展开作为潜在鉴别步骤的作用 在衬底选择中。这包括：（1）建立可靠的 用纯化的蛋白酶体进行底物解折叠试验，（2）鉴定 蛋白酶体亚基在底物解折叠中起作用， 蛋白酶体突变体，以及（3）确定这些亚基是否在蛋白酶体中起作用。 底物复合物的非泛素化组分的释放。