TARGETING SCF SUBSTRATES TO THE PROTEASOME

将 SCF 底物靶向蛋白酶体

• 批准号: 6727672
• 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/6727672
• 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 蛋白酶体直到最近才被认为对细胞 细胞生长和增殖中的信号传导。从那时起，微扰的 泛素介导的蛋白分解参与了多个方面的 癌症的发病机制。这使得蛋白酶体成为一个有吸引力的靶标 可能的治疗干预。 这项拟议工作的长期目标是理解分子 蛋白酶体招募底物和启动底物的机制 毁灭。建议通过生化解剖来解决这一目标 蛋白质的体外降解，使用纯化的底物和成分 酿酒酵母SCF泛素连接酶途径的研究 以首席调查组为特征的。这条途径是保守的 并控制主要GI细胞周期调节蛋白的降解和 生物体中的信号分子从酵母到人类。所获得的知识 因此与酵母直接相关的是对SCF介导的理解 人类细胞中的蛋白质分解作用。在当前的应用程序中，建议 发现可作为靶点的蛋白酶体的特征 其活性在底物台阶上的药理调节 对退化的识别和处理，而不是退化本身。 这些知识将对小说的发展具有相当大的价值 针对癌症中的蛋白酶体的策略。在这项建议中，有以下几点 两个特定的目标：(1)确定SCF泛素连接酶的机制 与蛋白酶体结合，并确定其在靶向底物中的作用 退化。观察到，SCF针对选定的蛋白质进行降解。 以两种可能的方式：(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。明确SCF的角色定位 结合蛋白质周转中的蛋白酶体需要分离SCF突变体 在维持泛素连接酶的同时不能结合蛋白酶体 活动。为了分离和鉴定这样的突变体，一个体外系统 纯化的蛋白质已经被开发出来，为研究人员提供了一种 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。通过这些试剂，泛素和干细胞因子介导的 天然干细胞因子底物的体外和体内降解，包括 定义衬底识别的精确要求将是 调查过了。(2)表征底物展开步骤及其在 底物复合体的非泛素化亚单位的释放。在SCF中 途径中，底物多肽只是紧密结合的一个成分 以蛋白酶体为靶点的多蛋白质复合体。现建议： 研究底物展开作为潜在歧视步骤的作用 在底物选择上。这包括：(1)建立可靠的 用纯化的蛋白酶体进行底物去折叠试验(2)鉴定 在底物展开过程中起作用的蛋白酶体亚基 蛋白酶体突变体，以及(3)定义这些亚基是否在 底物复合体的非泛素化成分的释放。