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

正常和疾病背景下人类蛋白质稳态网络的重新布线

• 批准号: 8954850
• 负责人: Martin Kampmann
• 金额: $ 232.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8954850
• 关键词: Address; Alleles; Antineoplastic Agents; Area; Biomedical Research; Cell physiology; Cells; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Disease; Equilibrium; Frontotemporal Dementia; Future; Generations; Genes; Genetic; Genetic Determinism; Goals; Homeostasis; Human; Inclusion Bodies; Investigation; Malignant Neoplasms; Mammalian Cell; Maps; Medical; Molecular Chaperones; Monitor; Myopathy; Nature; Neurodegenerative Disorders; Organism; Osteitis Deformans; Pathway interactions; Phenotype; Proteins; Proteome; Public Health; Reporter; Research; Role; System; Technology; Therapeutic; Therapeutic Intervention; Therapeutic Uses; base; cancer cell; gene discovery; gene function; genome-wide; inhibitor/antagonist; innovation; innovative technologies; insight; loss of function; meetings; mutant; novel therapeutics; protein aggregation; public health relevance; screening; therapeutic target

 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.

 描述（由申请人提供）：功能性蛋白质组对所有细胞和生物体都至关重要。参与维持蛋白质组完整性的细胞途径统称为蛋白质稳态网络。该网络动态地适应以满足细胞的要求，并且还在一系列疾病状态中重新连接，包括癌症和神经退行性疾病，使其成为有希望的治疗靶点。然而，动态的，上下文依赖的性质和规模的蛋白质稳定网络提出了一个艰巨的挑战，不能使用传统的方法来解决。为了了解蛋白质稳态网络在正常和疾病状态下的功能，并确定治疗干预的有效靶点，需要系统方法。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，它揭示了细胞通路，以及全基因组基于CRISPR的功能获得和丧失筛选，它产生了对基因功能的丰富，互补的见解。我们将把我们的策略扩展到基于流式细胞仪的荧光报告监测细胞表型的筛选。总之，这些创新将使上下文相关的，多表型，增益和功能丧失的遗传相互作用地图的生成。我们的长期目标是利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稳态网络，并利用其治疗潜力。在本申请中，我们将使用我们的技术来解决对蛋白质稳态领域具有根本重要性以及医学意义的三个问题：（A）Hsp 70分子伴侣和辅助分子伴侣在不同的细胞环境中如何功能性地相互作用以维持蛋白质稳态和存活？我们将确定不同癌细胞对不同Hsp 70抑制剂易感性的遗传决定因素。这些结果是重要的，因为它们可以指导未来的治疗用途的Hsp 70抑制剂作为抗癌药物。(B)VCP/p97是蛋白质稳态网络的一个中央多效性节点，它如何协调不同的细胞过程，以及它在疾病中的功能是如何重新连接的？CB-5083是目前作为抗癌药物进行临床试验的一种VCP抑制剂，它的筛选将揭示癌细胞对VCP抑制的脆弱性的决定因素。在表达与包含体肌病相关的VCP突变等位基因的细胞中的遗传相互作用图谱，该包含体肌病与骨和额颞叶痴呆的佩吉特病（IBMPFD）相关，预期将揭示在IBMPFD中被破坏的VCP的细胞作用。(C)蛋白质稳态网络如何控制与神经退行性疾病相关的蛋白质聚集，毒性聚集体又如何重新连接蛋白质稳态网络？研究结果将指出疾病机制和潜在的治疗靶点。我们建议在这里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，这些领域的进展一直受到缺乏阐明大型上下文依赖性细胞通路的方法的阻碍。

项目成果

Elucidating drug targets and mechanisms of action by genetic screens in mammalian cells.

• DOI: 10.1039/c7cc02349a
• 发表时间: 2017-06-29
• 期刊: Chemical communications (Cambridge, England)
• 作者: Kampmann M

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.

• DOI: 10.1083/jcb.202006180
• 发表时间: 2021-02-01
• 期刊: The Journal of cell biology
• 作者: Kanfer G;Sarraf SA;Maman Y;Baldwin H;Dominguez-Martin E;Johnson KR;Ward ME;Kampmann M;Lippincott-Schwartz J;Youle RJ
• 通讯作者: Youle RJ

CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine.

• DOI: 10.1021/acschembio.7b00657
• 发表时间: 2018-02-16
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: Kampmann M

An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.

E3 连接酶网络与 GCN1 结合，促进停滞核​​糖体上翻译因子的降解。

• DOI: 10.1016/j.cell.2022.12.025
• 发表时间: 2023
• 期刊: Cell
• 影响因子: 64.5
• 作者: Oltion,Keely;Carelli,JordanD;Yang,Tangpo;See,StephanieK;Wang,Hao-Yuan;Kampmann,Martin;Taunton,Jack
• 通讯作者: Taunton,Jack