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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.

项目概要/摘要调节蛋白质稳态（蛋白质稳态）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行性疾病。 Valosin 含蛋白 (VCP, p97)，AAA+ (ATPases) 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央调节因子之一。功能多样，包括从细胞器中展开泛素化蛋白、分离泛素化蛋白蛋白质复合物的产生和细胞器膜的重塑都归因于 VCP。这些功能由一组与 VCP 结合的“适配器”蛋白和泛素加工酶协调。尽管虽然这些蛋白质-蛋白质相互作用（PPI）对调节蛋白质稳态的重要性，但我们的研究还存在重大差距了解衔接蛋白如何将 VCP 的 ATP 酶活性与不同的细胞功能联系起来。此外， VCP 的点突变会导致一种致命的退行性疾病，称为多系统蛋白病 1 (MSP1)。 MSP1 与蛋白质稳态的多种改变有关，包括某些蛋白质降解的增加以及其他人的退化损失。 VCP 在 ATP 水解过程中经历较大的构象变化，并且 MSP1 突变改变 VCP 构象倾向。我们和其他人已经证明 PPI 也与 VCP 构象，导致 VCP 的 PPI 网络受 ATP 酶依赖性调节的假设构象动力学，并且 MSP1 突变通过改变这些而导致 PPI 网络失调动力学。因此，MSP1 疾病应被视为一种网络失调疾病。为了解决这个问题假设，我们需要新的工具来解决适配器如何与 VCP 构象结合并改变 ATPase 活性，以及构象依赖性结合如何影响 VCP 网络的细胞活动。我们将解决通过三个具体目标来弥补这些差距。 1) 我们已经证明接头蛋白能够感知 VCP 构象。我们将把这些观察扩展到至少八个接头/VCP复合物，并将评估 ATP酶活性和构象的适配器。该分析将提供哪些适配器的预测 MSP1 细胞中的依赖性功能增加或抑制。 2）我们利用了定点小分子一种称为二硫键捕获的发现方法，用于识别将 VCP 锁定为特定构象的化合物。我们假设抑制 VCP 动态会稳定一些 PPI，但会抑制生化和细胞依赖于 VCP 构象动力学的功能。 3）我们开发了N-的噬菌体展示文库 VCP 结构域来选择以高亲和力和选择性结合单个接头蛋白的突变体。我们假设阻断细胞中的单个接头/VCP复合物将导致VCP介导的变化途径和泛素蛋白质组（ubiquitinome）。结合 PPI 测量、小分子构象锁和基于蛋白质的 PPI 抑制剂将使我们能够预测哪些 VCP 途径导致（或减轻）MSP1 表型，并且应该利用它来开发癌症特异性 VCP 抑制剂。