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

项目摘要/摘要 调节蛋白质稳态(蛋白质稳态)中的关键角色的功能可能会导致疾病的治疗 从癌症到神经退化。含有Valosin的蛋白(VCP，p97)，AAA(ATPase)的成员 与各种细胞活动相关的)酶家族，是细胞蛋白质稳定的中央调节因子之一。 功能多样，如从细胞器中解开泛素化蛋白质，分离泛素化蛋白质 从蛋白质复合体出发，细胞器膜的重塑被归因于VCP。这些函数 由一系列与VCP结合的“接头”蛋白和泛素处理酶来协调。尽管 这些蛋白质-蛋白质相互作用(PPI)对调节蛋白平衡的重要性，在我们的 了解接头蛋白如何将VCP的ATPase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 与蛋白平衡的多种改变有关，包括某些蛋白质的降解增加 和丧失他人的堕落。VCP在ATP水解过程中经历了较大的构象变化， MSP1突变改变了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网络受ATPase依赖的调制 构象动力学，以及MSP1突变通过改变这些而导致PPI网络的失调 动力学。因此，MSP1疾病应该被视为一种网络调节失调的疾病。要解决这个问题 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， 以及构象依赖的结合如何影响VCP网络的细胞活性。我们将解决 这些差距是通过三个具体目标实现的。1)我们已经证明接头蛋白可以感知VCP的构象。 我们将把这些观察扩展到至少8个接头/VCP复合体，并将评估 ATPase活性和构象的适配子。该分析将提供对哪个适配器的预测- MSP1细胞的依赖功能增加或被抑制。2)我们利用了一种定点小分子 发现方法称为二硫化物捕捉法，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)构建了N-噬菌体展示文库 VCP的结构域，以选择与单个接头蛋白高亲和力和选择性结合的突变体。我们 假设阻断细胞中单个适配器/VCP复合体将导致VCP介导的改变 途径和泛素蛋白质组(泛素组)。结合PPI测量，小分子 构象锁定和基于蛋白质的PPI抑制剂将使我们能够预测哪些VCP途径导致(或 缓解)MSP1表型，应该利用这些表型来开发癌症特异性的VCP抑制剂。

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.