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Protein and small-molecule tools to probe the conformational dependence of the VCP/p97 protein-protein interaction network

用于探测 VCP/p97 蛋白质-蛋白质相互作用网络构象依赖性的蛋白质和小分子工具

基本信息

批准号：
10228038
负责人：
Michelle Arkin
金额：
$ 34.07万
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：
2018
资助国家：
美国
起止时间：
2018-09-05 至 2023-07-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT Modulating the functions of key players in protein homeostasis (proteostasis) could lead to therapies for diseases from cancer to neurodegeneration. Valosin Containing Protein (VCP, p97), a member of the AAA+ (ATPases associated with various cellular activities) family of enzymes, is one of the cell’s central regulators of proteostasis. Functions as diverse as unfolding of ubiquitinated proteins from organelles, segregation of ubiquitinated proteins from protein complexes, and remodeling of organelle membranes have been ascribed to VCP. These functions are coordinated by a set of “adaptor” proteins and ubiquitin-processing enzymes that bind to VCP. Despite the importance of these protein-protein interactions (PPI) to regulated proteostasis, there are major gaps in our understanding of how adaptor proteins link VCP’s ATPase activity to diverse cellular functions. Furthermore, point mutations in VCP cause a fatal, degenerative disease called Multisystem Proteinopathy 1 (MSP1). MSP1 is associated with multiple alterations in proteostasis that include both increased degradation of some proteins and loss of degradation of others. VCP undergoes large conformational changes during ATP hydrolysis, and MSP1 mutations alter VCPs conformational propensity. We and others have shown that PPI are also linked to VCP conformation, leading to the hypothesis that VCP’s PPI network is modulated by ATPase-dependent conformational dynamics, and that MSP1 mutations lead to dysregulation of the PPI network by altering these dynamics. MSP1 disease should therefore be viewed as a disease of network dysregulation. To address this hypothesis, we need new tools that address how adaptors bind to VCP conformations and alter ATPase activity, and how conformational-dependent binding affects the cellular activities of the VCP network. We will address these gaps through three Specific Aims. 1) We have shown that adaptor proteins sense VCP conformation. We will extend these observations to at least eight adaptor/VCP complexes and will also evaluate the effect of adaptors on ATPase activity and conformation. This analysis will provide predictions for which adaptor- dependent functions are increased or inhibited in MSP1 cells. 2) We have utilized a site-directed small-molecule discovery approach called disulfide-trapping to identify compounds that lock VCP into specific conformations. We hypothesize that inhibiting VCP dynamics will stabilize some PPI, but will inhibit biochemical and cellular functions that rely on VCP conformational dynamics. 3) We have developed phage-displayed libraries of the N- domain of VCP to select mutants that bind with high affinity and selectivity to single adaptor proteins. We hypothesize that blocking individual adaptor/VCP complexes in cells will lead to changes in VCP-mediated pathways and the ubiquitin proteome (ubiquitinome). Combining PPI measurements, small-molecule conformational locks, and protein-based PPI inhibitors will allow us to predict which VCP pathways lead to (or mitigate) MSP1 phenotypes, and which should be harnessed to develop cancer-specific VCP inhibitors.

项目总结/摘要调节蛋白质稳态（蛋白质稳态）中关键参与者的功能可能会导致疾病的治疗从癌症到神经退化Valosin Containing Protein（VCP，p97）是AAA+（ATP酶）的成员与各种细胞活动相关）酶家族，是细胞蛋白质稳态的中心调节因子之一。功能多样，如从细胞器中展开泛素化蛋白，分离泛素化蛋白从蛋白质复合物，和细胞器膜的重塑已被归因于VCP。这些功能由一组“衔接”蛋白和结合VCP的泛素加工酶协调。尽管这些蛋白质-蛋白质相互作用（PPI）的重要性，以调节蛋白质稳态，有很大的差距，在我们的了解衔接蛋白如何将VCP的ATP酶活性与多种细胞功能联系起来。此外，委员会认为， VCP中的点突变导致称为多系统蛋白质病1（MSP 1）的致命的退行性疾病。MSP1 与蛋白质稳态的多种改变有关，包括一些蛋白质降解增加以及其他人的堕落。VCP在ATP水解过程中经历大的构象变化， MSP 1突变改变VCP的构象倾向。我们和其他人已经表明，PPI也与 VCP构象，导致假设VCP的PPI网络是由ATP酶依赖的 MSP 1突变通过改变这些构象动力学，导致PPI网络的失调，动力学因此，MSP 1疾病应被视为网络失调的疾病。为了解决这个假设，我们需要新的工具来解决衔接子如何结合VCP构象并改变ATP酶活性，以及构象依赖性结合如何影响VCP网络的细胞活性。我们将解决这些差距通过三个具体目标。1)我们已经表明，衔接蛋白的意义VCP构象。我们将这些观察扩展到至少八个适配器/VCP复合物，并将评估衔接子对ATP酶活性和构象的影响。该分析将提供预测哪个适配器- 在MSP 1细胞中依赖性功能增加或抑制。2)我们利用定点小分子一种称为二硫化物捕获的发现方法，用于识别将VCP锁定为特定构象的化合物。我们假设，抑制VCP动力学将稳定一些PPI，但将抑制生化和细胞依赖于VCP构象动力学的功能。3)我们已经开发了N- 结构域的VCP选择突变体，结合具有高亲和力和选择性的单一衔接蛋白。我们假设阻断细胞中的单个接头/VCP复合物将导致VCP介导的泛素蛋白质组（ubiquitinome）。结合PPI测量，小分子构象锁，和蛋白质为基础的PPI抑制剂将使我们能够预测哪些VCP途径导致（或缓解）MSP 1表型，并应利用其开发癌症特异性VCP抑制剂。