Energy production and utilization in the proteasome

描述(申请人提供)：分隔的蛋白水解酶包含与细胞环境的其余部分隔离的蛋白水解点。将折叠的蛋白质插入这类酶的蛋白水解室是一个能量依赖的过程，包括识别、展开和逐步进入蛋白水解室的步骤。与这些酶相关的调节复合体的ATPase亚单位产生所需的力量。我们将研究26S蛋白酶体的底物展开和转位过程，26S蛋白酶体是真核细胞的主要细胞质和核蛋白。具体地说，我们打算研究19S调控复合体的ATPase亚单位利用来自ATP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，其性质有助于检验力生产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奠定了基础。在这些计划中的研究中，我们将创建一系列底物来对蛋白酶体功能提出不同的和校准的挑战，开发相关的分析来衡量蛋白酶体如何很好地应对这些挑战，并检查蛋白酶体亚组件和突变体执行这些任务的能力。具体目的1.测试能量产生部位和展开部位之间相互作用的性质。具体目的2.建立底物展开和去向的替代检测方法，并用这些方法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质酶体介导解折叠的生化分析方法来进一步扩展。具体目标3.研究影响插入停滞的遗传交互作用。保守的ATPase基序将进行定点突变，以消除ATP水解、ATP结合、ATPase之间的通讯和底物结合。将对更多的19S蛋白质进行随机突变并进行测试。

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.