Energy production and utilization in the proteasome

描述（由申请人提供）：区室化蛋白酶含有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此类蛋白酶的蛋白水解室中是能量依赖性过程，其步骤包括识别、解折叠和逐步推进到蛋白水解室中。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胞质和核蛋白酶。具体来说，我们打算调查的机制，其中ATP酶亚基的19S调节复合物利用能源来自ATP驱动这些事件。我们已经开发了蛋白酶体底物，其性质有利于检查力的生产和消费之间的相互作用。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。在这些计划的研究中，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满足这些挑战，并检查蛋白酶体突变体执行这些任务的能力。具体目标1。测试能量产生和展开位点之间相互作用的性质。具体目标2。开发底物展开和命运的替代测定法，并使用这些方法来测试蛋白酶体组分的功能。这些观察结果将进一步扩展，通过开发和应用蛋白酶体介导的展开的生化测定。具体目标3。检查影响插入停滞的遗传相互作用。将进行保守ATP酶基序的定点突变以消除ATP水解、ATP结合、ATP酶之间的通讯和底物接合。将对额外的19S蛋白进行随机突变并进行检测。

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.