TARGETING SCF SUBSTRATES TO THE PROTEASOME

将 SCF 底物靶向蛋白酶体

基本信息

批准号：
6871240
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
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2007-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6871240
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

项目摘要

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直到最近才被认为对细胞至关重要细胞生长和增殖中的信号传导。从此以后，各种扰动泛素介导的蛋白水解作用涉及多种方面癌症的发病机制。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。拟议工作的长期目标是了解分子蛋白酶体招募底物并启动其作用的机制破坏。建议通过生化解剖来实现这一目标使用纯化的底物和组分进行体外蛋白质降解酿酒酵母的 SCF 泛素连接酶途径，已被发现并以主要研究者的小组为特征。这条路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，与酵母的结合与理解 SCF 介导的人体细胞中的蛋白水解作用。在当前的应用中，建议揭示蛋白酶体的特征，可作为目标其活性在底物步骤中的药理调节识别和处理退化，但不是退化本身。这些知识对于小说的开发具有相当大的价值。针对癌症中的蛋白酶体的策略。这份提案中有两个具体目标：（1）确定 SCF 泛素连接酶的机制与蛋白酶体结合并定义其在靶向底物中的作用降解。据观察，SCF 针对选定的蛋白质进行降解有两种可能的方式：（1）促进底物泛素化；（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，同时维持泛素连接酶活动。为了分离和表征此类突变体，体外系统纯化的蛋白质已被开发出来，为研究人员提供了解决所需的蛋白质-蛋白质相互作用的独特机会 SCF/蛋白酶体结合。使用这些试剂，泛素和 SCF 介导的天然 SCF 底物在体外和体内的降解，包括定义基材识别的精确要求将是调查了。 (2) 表征底物展开步骤及其在底物复合物的非泛素化亚基的释放。在SCF中途径中，底物多肽只是紧密结合的多肽的一个组成部分靶向蛋白酶体的多蛋白复合物。建议研究底物展开作为潜在歧视步骤的作用在基材选择上。这包括：（1）建立可靠的使用纯化的蛋白酶体进行底物解折叠测定，（2）鉴定使用纯化的蛋白酶体亚基在底物解折叠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。