Protein Degradation by the 26S Proteasome

26S 蛋白酶体的蛋白质降解

基本信息

批准号：
7263634
负责人：
YUENYU AMY LAM
金额：
$ 31.23万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7263634
关键词：
26S proteasome ATP Hydrolysis ATP phosphohydrolase ATP-Dependent Proteases Accounting Active Sites Address Affect Affinity Binding Biochemical Genetics Biochemistry Complex Consumption DHFR gene Dependence Development Dihydrofolate Reductase Effectiveness Endopeptidases Ensure Essential Genes Event Family Health Human In Vitro Individual Link Malignant Neoplasms Measures Mediating Methods Modeling Multiple Myeloma Mutagenesis Mutate Mutation Pathway interactions Peptide Hydrolases Personal Satisfaction Plasma Cells Polyubiquitin Proteasome Inhibitor Proteins Proteolysis Purpose Reaction Research Signal Transduction Tertiary Protein Structure Therapeutic Agents Time Ubiquitin United States Food and Drug Administration Variant Yeasts base connectin drug development genetic analysis multicatalytic endopeptidase complex mutant protein degradation protein folding yeast genetics

项目摘要

DESCRIPTION (provided by applicant): In the ubiquitin (Ub)/proteasome proteolytic pathway, substrate-linked polyUb is the specific targeting signal that leads proteins to be degraded by the multisubunit ATP-dependent protease complex called the 26S proteasome. The mechanisms by which the proteasome recognizes, unfolds, and translocates the substrate into its internal proteolytic chamber are poorly understood. In the proposal, in vitro biochemistry augmented by yeast genetics will be used to address how key events such as substrate unfolding and translocation are orchestrated by the six different ATPase subunits of the 19S regulatory complex of 26S proteasomes. For this purpose, well-defined polyUb-conjugates of wild-type and destabilized variants of two model proteins, dihydrofolate reductase and titin I27 domain, have been developed. By using these substrates to measure degradation and ATPase activities with purified wild-type and ATPase-mutated 26S proteasomes, we will be able to resolve the contributions that ATP hydrolysis and, potentially, individual ATPase subunits make toward the substrate binding, unfolding, and translocation steps of the degradation reaction. Our results will provide a basic understanding of how the 26S proteasome complex uses ATP to promote proteolysis. Overall, the results of the proposed research will significantly enhance our understanding of how the 26S proteasome degrades proteins and how the complex is regulated. The significance of proteasome research to human health has been highlighted by the recent FDA approval of the use of proteasome inhibitor as a treatment for multiple myeloma.

描述（由申请人提供）：在泛素 (Ub)/蛋白酶体蛋白水解途径中，底物连接的聚泛素 (polyUb) 是特异性靶向信号，导致蛋白质被称为 26S 蛋白酶体的多亚基 ATP 依赖性蛋白酶复合物降解。蛋白酶体识别、展开底物并将其转移到其内部蛋白水解室的机制尚不清楚。在该提案中，酵母遗传学增强的体外生物化学将用于解决26S蛋白酶体19S调节复合物的六个不同ATP酶亚基如何协调底物解折叠和易位等关键事件。为此，开发了两种模型蛋白（二氢叶酸还原酶和肌联蛋白 I27 结构域）的野生型和不稳定变体的明确多聚泛素结合物。通过使用这些底物来测量纯化的野生型和 ATP 酶突变的 26S 蛋白酶体的降解和 ATP 酶活性，我们将能够解决 ATP 水解以及潜在的单个 ATP 酶亚基对底物结合、解折叠和易位的贡献降解反应的步骤。我们的结果将提供对 26S 蛋白酶体复合物如何利用 ATP 促进蛋白水解的基本了解。总体而言，拟议研究的结果将显着增强我们对 26S 蛋白酶体如何降解蛋白质以及复合物如何调节的理解。 FDA 最近批准使用蛋白酶体抑制剂治疗多发性骨髓瘤，凸显了蛋白酶体研究对人类健康的重要性。