“Regulation of Protein Quality Control by the VCP AAA-ATPase”

项目摘要 蛋白质质量控制对于维持细胞蛋白质稳态至关重要，尤其是在 压力、疾病状态和衰老。这些网络包括：1)帮助蛋白质折叠的伴侣 和中间体的稳定以防止聚集，以及2)降解途径，如泛素 蛋白酶体系统和自噬，以消除功能次佳的蛋白质和细胞器。……的下降 衰老细胞中的蛋白质质量控制有助于许多在遗传和临床上截然不同的 神经退行性疾病，如肌萎缩侧索硬化症、阿尔茨海默氏症、帕金森氏症、亨廷顿氏症 以及普恩病毒病。含有Valosin的蛋白(VCP，也称为p97)是一种AAA-ATPase，它发挥着 泛素蛋白酶体系统通过指定的接头捕获泛素化底物的重要作用 调节底物降解。VCP突变会导致几种神经退行性疾病，包括ALS和 罕见的多系统疾病、包涵体肌病、骨派杰氏病和额颞部痴呆 (IBMPFD)。VCP最常见的突变区域位于接头结合部位，并存在于某些 实例这些突变改变了绑定到VCP的适配器星座，使其目标偏向于 底物。VCP突变的一个共同细胞特征是蛋白质聚集体的形成和持续存在 未能被清理的公司。我们已经确定了一个针对侵略者的特定VCP-适配器复合体，一个 隔离错误折叠的在细胞应激期间形成的聚集蛋白质的结构。而侵略者则是 通常是细胞保护的，在它们持续存在的条件下，例如在老化的神经元中 蛋白质质量控制途径，它们最终会变成细胞毒性。VCP适配器耗尽会导致 侵袭体形成的缺陷，泛素化底物的稳定和触发内质网应激。我们假设 VCP通过转接子特异性靶向介导侵袭体清除，以实现特定错误折叠的清除 或聚集的底物。我们将结合靶向分子研究、定量蛋白质组学和 为实现高分辨率成像，提出了这一建议的主要目标。我们将(1)描述VCP 侵袭体靶向复合体，以确定靶向机制及其在侵袭体内的功能(2) 利用泛素残基识别VCP复合体靶向侵袭体的泛素化细胞靶点 捕获蛋白质组学和(3)确定与疾病相关的VCP突变是否影响其与 侵袭体复合体，并测试VCP接头是否能够识别和清除疾病相关蛋白 集合体。这项提议的成功实施将确定可为 开发能够在许多神经退行性疾病中进行聚集清除的药物。

Project Abstract Protein quality control is of critical importance in maintaining cellular protein homeostasis, especially during stress, disease states and aging. These networks are comprised of: 1) chaperones that assist in protein folding and stabilization of intermediates to prevent aggregation, and 2) degradation pathways, such as the ubiquitin proteasome system and autophagy, to eliminate proteins and organelles with sub-optimal function. A decline in protein quality control in aging cells contributes to the development of numerous genetically and clinically distinct neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's, Parkinson's, Huntington's and prion diseases. Valosin containing protein (VCP, also known as p97) is a AAA-ATPase that plays an important role in the ubiquitin proteasome system to capture ubiquitylated substrates via designated adaptors to mediate substrate degradation. Mutation of VCP causes several neurodegenerative disorders including ALS and a rare multi-system disorder, inclusion body myopathy, Paget’s disease of the bone and frontotemporal dementia (IBMPFD). The most commonly mutated region of VCP resides at the site of adaptor binding and in certain instances these mutations alter the constellation of adaptors that are bound to VCP skewing its targeting to substrates. A common cellular feature of VCP mutations is the formation and persistence of protein aggregates that fail to be cleared. We have identified a specific VCP-adaptor complex that is targeted to the aggresome, a structure that sequesters misfolded aggregated proteins formed during cell stress. While aggresomes are generally cyto-protective, under conditions where they persist, for example in aging neurons with declining protein quality control pathways, they eventually become cytotoxic. Depletion of the VCP adaptor leads to a deficit in aggresome formation, stabilization of ubiquitylated substrates and triggers ER stress. We hypothesize that VCP mediates aggresome clearance by adaptor specific targeting to enable clearance of specific misfolded or aggregated substrates. We will use a combination of targeted molecular studies, quantitative proteomics and high-resolution imaging to achieve the main objectives of this proposal. We will (1) characterize the VCP aggresome targeted complex to determine mechanisms of targeting and its functions within the aggresome (2) identify ubiquitylated cellular targets of the VCP complex targeted to the aggresome using ubiquitin remnant capture proteomics and (3) determine whether disease-relevant VCP mutations impact its association with the aggresome complex and test if the VCP adaptor can recognize and enable clearance of disease relevant protein aggregates. Successful implementation of this proposal will identify new targets that can be pursued for the development of agents that enable aggregate clearance in a number of neurodegenerative disorders.