Rewiring of the human protein homeostasis network in normal and disease contexts

 描述(由申请人提供)：功能蛋白质组对所有细胞和生物体都是至关重要的。维持蛋白质组完整性的细胞通路统称为蛋白质平衡网络。这个网络能够动态适应细胞的需求，并在包括癌症和神经退行性疾病在内的一系列疾病状态下重新连接，使其成为一个有希望的治疗靶点。然而，蛋白质平衡网络的动态、依赖于上下文的性质和规模是一个巨大的挑战，无法使用传统方法来解决。为了了解蛋白质平衡网络在正常和疾病状态下如何发挥作用，并准确定位作为治疗干预有效靶点的节点，需要一种系统的方法。在这里，我们建议通过整合我们最近开发的两项突破性技术来建立这样一种方法：哺乳动物细胞中的遗传相互作用图，揭示细胞路径；以及基于全基因组CRISPR的功能获得和丧失屏幕，它产生对基因功能的丰富、互补的见解。我们将把我们的策略扩展到基于FACS的荧光记者监测的细胞表型筛选。综上所述，这些创新将能够生成与背景相关的、多表型、功能获得和功能丧失的遗传交互作用图谱。我们的长期目标是使用这项技术和其他创新方法来了解正常和疾病背景下的蛋白质平衡网络，并利用其治疗潜力。在这项应用中，我们将使用我们的技术来解决三个对蛋白质平衡领域具有基本重要性和医学意义的问题：(A)Hsp70伴侣和辅助伴侣如何在不同的细胞环境中进行功能相互作用，以维持蛋白质稳定和生存？我们将确定不同癌细胞对不同Hsp70抑制剂易感性的遗传决定因素。这些结果意义重大，因为它们可以指导Hsp70抑制剂作为抗癌药物的未来治疗用途。(B)VCP/p97，蛋白平衡网络的中心多效性节点，如何协调不同的细胞过程，以及它的功能在疾病中是如何重新连接的？用CB-5083，一种目前作为抗癌药物进行临床试验的VCP抑制剂进行筛选，将揭示癌细胞对VCP抑制的易感性的决定因素。表达VCP突变等位基因的细胞中的遗传相互作用图有望揭示VCP在IBMPFD中被破坏的细胞角色，这些突变等位基因与与Paget病相关的包涵体肌病相关(IBMPFD)。(C)蛋白质平衡网络如何控制与神经退行性疾病相关的蛋白质聚集，以及有毒聚集体如何反过来重新连接蛋白质平衡网络？这一结果将指出疾病机制和潜在的治疗目标。我们在这里建议开发的技术可能会产生广泛的影响，因为它有可能改变生物医学研究的许多领域，在这些领域，由于缺乏阐明大的、上下文相关的细胞通路的方法，进展受到阻碍。

 DESCRIPTION (provided by applicant): A functional proteome is of paramount importance for all cells and organisms. The cellular pathways involved in maintaining the integrity of the proteome are collectively referred to as the proteostasis network. This network adapts dynamically to meet the requirements of the cell and is also rewired in a range of disease states, including cancer and neurodegenerative disease, making it a promising therapeutic target. However, the dynamic, context-dependent nature and size of the proteostasis network present a formidable challenge that cannot be addressed using traditional approaches. To understand how the proteostasis network functions in normal and disease states, and to pinpoint nodes that are effective targets for therapeutic intervention, a systems approach is called for. Here, we propose to establish such an approach by integrating two breakthrough technologies that we recently developed: Genetic interactions maps in mammalian cells, which reveal cellular pathways, and genome-wide CRISPR-based gain- and loss-of-function screens which yield rich, complementary insights into gene function. We will extend our strategy to FACS-based screening of cellular phenotypes monitored by fluorescent reporters. Taken together, these innovations will enable the generation of context-dependent, multi-phenotype, gain- and loss-of-function genetic interaction maps. Our long-term goal is to use this technology and other innovative approaches to understand the proteostasis network in normal and disease contexts and to harness its therapeutic potential. In this application, we will use our technology to address three questions with fundamental importance to the proteostasis field as well as medical significance: (A) How do Hsp70 chaperones and co-chaperones functionally interact in different cellular contexts to maintain proteostasis and survival? We will determine the genetic determinants of vulnerability to different Hsp70 inhibitors in different cancer cells. These result are significant since they can guide future therapeutic uses of Hsp70 inhibitors as anti-cancer drugs. (B) How does VCP/p97, a central pleiotropic node of the proteostasis network, coordinate different cellular processes, and how is its function rewired in disease? Screens with CB-5083, a VCP inhibitor currently in clinical trials as an anti-cancer drug, will reveal determinants of cancer cell vulnerability to VCP inhibition. Genetic interaction maps in cells expressing VCP mutant alleles associated with Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) are expected to uncover cellular roles of VCP that are disrupted in IBMPFD. (C) How does the proteostasis network control protein aggregation associated with neurodegenerative diseases, and how do toxic aggregates in turn rewire the proteostasis network? The results will point to disease mechanisms and potential therapeutic targets. The technology we propose to develop here is likely to have a broad impact due to its potential to transform many areas of biomedical research where progress has been hindered by the lack of approaches for the elucidation of large, context-dependent cellular pathways.