Protein Degradation by the 26S Proteasome

26S 蛋白酶体的蛋白质降解

• 批准号: 7769848
• 负责人: YUENYU AMY LAM
• 金额: $ 30.9万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 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/7769848
• 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; 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; Plasma Cells; Polyubiquitin; Proteasome Inhibitor; Proteins; Proteolysis; Reaction; Research; Signal Transduction; Tertiary Protein Structure; Therapeutic Agents; Time; Ubiquitin; Variant; Yeasts; base; connectin; drug development; genetic analysis; multicatalytic endopeptidase complex; mutant; protein complex; protein degradation; protein folding; yeast genetics

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，底物链接的Polyub是特定的靶向信号，它导致蛋白质被多生育ATP ATP依赖性蛋白酶复合物降解，称为26S蛋白酶体。蛋白酶体识别，展开和易位的底物进入其内部蛋白水解腔的机制知之甚少。在该提案中，将使用酵母遗传学增强的体外生物化学来解决26S蛋白酶体的19S调节络合物的六个不同ATPase亚基等关键事件（例如底物展开和易位）如何策划。为此，已经开发了两种模型蛋白的野生型和不稳定变体的明确定义的多核偶联物，即二氢叶酸还原酶和TITIN I27结构域。通过使用这些底物来测量具有纯化的野生型和ATPase-Mutated 26S蛋白酶体的降解和ATPase活性，我们将能够解决ATP水解的贡献，并且可能是单个ATPase亚基对底物结合，展开，展开的脱位反应的易位。我们的结果将提供对26S蛋白酶体复合物如何使用ATP促进蛋白水解的基本理解。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如何调节复合物的理解。最近，FDA批准使用蛋白酶体抑制剂作为多发性骨髓瘤的治疗方法，蛋白酶体对人类健康的重要性得到了强调。