Proteasome substrate processing

描述（由申请人提供）：蛋白酶体是控制蛋白质降解的泛素-蛋白酶体系统的效应器，是结构和功能复杂的分子机器，包含30多种不同的蛋白质。不稳定调节蛋白的选择性降解对细胞调节至关重要；蛋白酶体还可以消除潜在的有毒错误折叠蛋白质。蛋白质水解的错误调控与多种疾病机制有关，蛋白酶体已被证实是癌症化疗的靶点。该项目的总体目标是更好地了解蛋白酶体底物降解的机制。了解蛋白酶体作用的详细机制需要更充分地定义这一多步骤过程中的中间状态，并了解蛋白酶体的不同蛋白质和亚组件之间的相互作用如何协调并参与底物加工。我们将利用一组对蛋白酶体降解有不同要求的底物。这就要求蛋白酶体的识别和/或降解功能逐渐受限，如下所示：泛素化底物：蛋白酶体必须执行所有内在功能。承载泛素非依赖性降解标签的底物：泛素控制功能冗余。底物连接到蛋白酶体：捕获多余的，下游事件所需。将执行下列具体目标以促进这些目标：目标1。确定蛋白酶体如何识别和捕获一个不依赖泛素的脱蛋白，生化和遗传策略将用于识别鸟氨酸脱羧酶的降解信号脱蛋白的受体。目标2。定义底物机械稳定性是否以及如何影响加工时间和ATP消耗上述三类中的每一类代表将用于研究加工动力学，底物机械稳定性和ATP水解之间的相互作用。目标3。确定底物通过蛋白酶体的途径和在加工过程中亚基接触的变化。我们将使用上述底物来研究蛋白酶体成分和底物在加工过程中如何改变它们的相互作用。目标4。我们将使用定义的蛋白酶体突变，包括atp酶和α环门的突变，来改变降解的轨迹，从而促进多步骤途径中中间状态的检测和排序。公共卫生相关性：细胞必须识别和破坏它们的某些蛋白质成分——那些异常的或已经完成编程任务的蛋白质成分。破坏的引擎被称为蛋白酶体。这个大复合体包含大约30种不同的蛋白质，必须协调其多种酶的活性，才能首先识别被标记为破坏的蛋白质，并最终将其分解成小片段。我们建议研究这一加工途径背后的一系列生化事件。在包括癌症在内的一系列疾病中，指定蛋白质破坏的系统被错误调节，蛋白酶体已被证实是癌症化疗的目标。

DESCRIPTION (provided by applicant): Proteasomes, the effectors of the ubiquitin-proteasome system that controls protein degradation, are structurally and functionally complex molecular machines containing more than 30 distinct proteins. Selective degradation of labile regulatory proteins is essential to cellular regulation; proteasomes also eliminate potentially toxic misfolded proteins. The misregulation of proteolysis is implicated in a broad array of disease mechanisms, and the proteasome has been validated as a target of cancer chemotherapy. The overall goal of this project is to understand better the mechanism of proteasome substrate degradation. Detailed mechanistic understanding of proteasome action requires more fully defining the intermediate states in this multi-step process and understanding how interactions among the different proteins and subassemblies of the proteasome are coordinated and participate in substrate processing. We will make use of a set of substrates with distinct requirements for proteasomal degradation. These require progressively restricted subsets of proteasome functions for their recognition and/or degradation, as follows: Ubiquitinated substrates: Proteasome must perform all intrinsic functions. Substrates bearing ubiquitin-independent degradation tag: Ubiquitin control functions redundant. Substrates tethered to proteasome: Capture redundant, downstream events required. The following Specific Aims will be carried out in furtherance of those goals: Aim 1. Determine how the proteasome recognizes and captures an ubiquitin-independent degron Biochemical and genetic strategies will be used to identify the receptor for the degron, the degradation signal, of ornithine decarboxylase. Aim 2. Define whether and how substrate mechanical stability affects processing time and ATP consumption Representatives of each of the three classes described above will be used to investigate the interactions among processing kinetics, substrate mechanical stability and ATP hydrolysis. Aim 3. Determine the pathway of a substrate through the proteasome and changes in subunit contacts during its processing. We will use the substrates described above to examine how proteasome components and substrates change their interactions during processing. Aim 4. Perturb the substrate processing pathway by means of site-directed proteasome mutations We will use defined proteasome mutations, including those of ATPases and the alpha ring gate, to alter the trajectory of degradation, thereby facilitating the detection and ordering of intermediate states within the multi-step pathway PUBLIC HEALTH RELEVANCE: Cells must recognize and destroy certain of their protein components- those that are aberrant or have completed a programmed task. The engine of destruction is termed the proteasome. This large complex encompasses about 30 different kinds of proteins and must coordinates its multiple enzyme activities so as to first recognize proteins marked for destruction and finally reduce them to small fragments. We are proposing to study the series of biochemical events that underlie this processing pathway. The system which specifies protein destruction is mis-regulated in a broad array of disease, including cancer, and the proteasome has been validated as a target of cancer chemotherapy.