Energy production and utilization in the proteasome

蛋白酶体中的能量产生和利用

基本信息

批准号：
7474608
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
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2011-07-31
项目状态：
已结题

项目摘要

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）：分隔蛋白酶包含从其他细胞环境中隔离的蛋白水解位点。将折叠的蛋白质插入此类蛋白酶的蛋白水解腔是一个依赖能量的过程，其步骤包括识别，展开和渐进式进步进入蛋白水解腔。与这些蛋白酶相关的调节复合物的ATPase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的过程，这是真核细胞的主要细胞质和核蛋白酶。具体而言，我们打算研究19S调节复合物的ATPase亚基利用从ATP衍生的能量来驱动这些事件的机制。我们开发了蛋白酶体底物，其性质有助于研究力生产与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各种挑战，开发相关测定，以衡量蛋白酶体如何满足这些挑战，并检查蛋白酶体的子组件和突变体的能力来执行这些任务。特定目的1。测试能量生产和展开位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，并使用这些测定来测试蛋白酶体组件的功能。通过开发和应用生化测定法进行蛋白酶体介导的展开，将进一步扩展这些观察结果。特定目的3。检查影响插入停滞的遗传相互作用。将对保守ATPase基序的位置定向突变，以消除ATP水解，ATP结合，ATPases之间的通信和底物参与度。将制造和测试其他19S蛋白的随机突变。