Energy production and utilization in the proteasome

蛋白酶体中的能量产生和利用

• 批准号: 7264570
• 负责人: Philip Coffino
• 金额: $ 23.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7264570
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; Affect; Alanine; Be++ element; Beryllium; Binding; Biochemical; Biological Assay; Cells; Communication; Complex; Consumption; Development; Elements; Endopeptidases; Environment; Eukaryotic Cell; Event; Failure; Foundations; Genetic; Glycine; Impairment; Investigation; Kinetics; Laboratories; Length; Measurement; Measures; Mediating; Mutation; Nuclear; Peptide Hydrolases; Process; Production; Property; Proteins; Rate; Rest; Series; Site; Structure; System; Tertiary Protein Structure; Testing; Ubiquitination; Viral; Work; connectin; interest; multicatalytic endopeptidase complex; mutant; novel strategies; particle; protein folding

DESCRIPTION (provided by applicant): Compartmentalized proteases contain proteolytic sites sequestered from the rest of the cellular environment. Inserting a folded protein into the proteolytic chamber of such proteases is an energy-dependent process with steps that include recognition, unfolding and progressive advancement into the proteolytic chamber. ATPase subunits of regulatory complexes associated with these proteases produce the required force. We will examine the processes of substrate unfolding and translocation of the 26S proteasome, the major cytoplasmic and nuclear protease of the eukaryotic cell. Specifically, we intend to investigate the mechanism by which the ATPase subunits of the 19S regulatory complex utilize energy derived from ATP to drive these events. We have developed proteasome substrates whose properties facilitate examining the interactions between force production and consumption. Our recent findings lay the foundation for a novel approach to studying how proteasomes produce and consume energy. In these projected studies we will create a series of substrates that offer diverse and calibrated challenges to proteasome function, develop relevant assays to measure how well the proteasome meets these challenges, and examine the capacity of proteasome subassemblies and mutants to perform these tasks. Specific Aim 1. Test the properties of the interaction between sites of energy production and unfolding. Specific Aim 2. Develop alternate assays of substrate unfolding and fate and use these to test the function of proteasome components. These observations will be further extended by developing and applying biochemical assays for proteasome-mediated unfolding. Specific Aim 3. Examine genetic interactions affecting insertional arrest. Site-directed mutations of conserved ATPase motifs will be made to abrogate ATP hydrolysis, ATP binding, communication between ATPases and substrate engagement. Random mutations of additional 19S proteins will be made and tested.

描述（由申请人提供）：区隔化蛋白酶包含与细胞环境其余部分隔离的蛋白水解位点。将折叠的蛋白质插入到蛋白酶的蛋白水解腔中是一个依赖能量的过程，其步骤包括识别、展开和逐步进入蛋白水解腔。与这些蛋白酶相关的调节复合物的atp酶亚基产生所需的力。我们将研究26S蛋白酶体的底物展开和易位过程，26S蛋白酶体是真核细胞的主要细胞质和核蛋白酶。具体来说，我们打算研究19S调节复合体的ATP酶亚基利用ATP产生的能量来驱动这些事件的机制。我们已经开发了蛋白酶体底物，其性质有助于研究力产生和消耗之间的相互作用。我们最近的发现为研究蛋白酶体如何产生和消耗能量的新方法奠定了基础。在这些预计的研究中，我们将创建一系列底物，为蛋白酶体功能提供多样化和校准的挑战，开发相关的检测方法来测量蛋白酶体如何应对这些挑战，并检查蛋白酶体亚组件和突变体执行这些任务的能力。具体目标测试能源生产和展开地点之间相互作用的特性。具体目标2。开发底物展开和命运的替代测定方法，并使用这些方法来测试蛋白酶体成分的功能。这些观察结果将通过开发和应用蛋白酶体介导的展开的生化分析进一步扩展。具体目标3。检查影响插入停止的基因相互作用。保守的ATP酶基序的位点定向突变将取消ATP水解，ATP结合，ATP酶之间的通信和底物接合。另外的19S蛋白的随机突变将被制造和测试。