Proteostasis sensors to assess the cellular protein folding capacity

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术和具体的挑战主题，06-AG-108用于从复杂组织中的单个活细胞获得基因组、蛋白质组和代谢组数据的技术，以及06-DK-105使能技术用于细胞生物学和大分子分析。所有后生动物细胞的长期健康与蛋白质质量控制密不可分。这是通过蛋白质平衡实现的，蛋白质平衡是一个复杂的分子相互作用网络，决定了蛋白质组的健康。蛋白质平衡平衡蛋白质的生物合成、折叠、转位、组装/拆解和清除，以应对环境或生理应激造成的挑战，从而导致一系列错误折叠和受损的蛋白质。如果不加注意，体内平衡的失衡可能会对细胞造成严重的分子损伤，导致关键组织的失调导致病理，并容易患上衰老疾病。适应和生存需要感知受损蛋白质的能力，并协调诱导保护性应激反应通路、伴侣和清除网络。尽管伴侣蛋白和蛋白平衡网络的其他组件具有丰富和明显的能力来恢复折叠平衡，但细胞对慢性蛋白毒性应激的适应能力很差，当某些易于聚集的蛋白在代谢性疾病、癌症和神经退行性疾病中表达时就会发生这种情况。这种挑战蛋白质组完整性的修复活动的下降受到控制衰老的基因的强烈影响，从而将应激生物学、新陈代谢和蛋白质动态平衡与有机体的健康和寿命联系在一起。这项提议结合了索尔克研究所的迪林实验室和西北大学的森本实验室这两个小组的互补优势，以开发和测试一套新的分子工具，该工具将报告蛋白质组的健康状况。这些“蛋白质平衡传感器”旨在实时评估细胞每个隔间的蛋白质折叠质量控制能力，并评估蛋白质损伤、细胞应激、衰老和蛋白质构象疾病的后果。这些工具最初将被开发用于线虫，以获得对假说和使用遗传方法评估功能能力的快速测试，然后扩展到哺乳动物组织培养细胞，并最终扩展到转基因小鼠。这些研究的影响非常广泛，涉及生物学和医学的所有领域，因为蛋白质质量控制对真核生物所有细胞和组织中蛋白质组的健康和保护至关重要。我们开发的工具将在出版后根据要求向所有其他研究人员提供。 公共卫生相关性：受损蛋白质的表达与数百种与衰老有关的人类疾病有关。蛋白质在细胞的一个隔室中的错误折叠如何影响另一个隔室以及组织之间的可变性是尚未解决的关键问题。这项提议是开发一个折叠传感器的分子工具箱，提供实时活细胞成像，以量化面对急性应激、受损蛋白质的慢性表达和衰老时蛋白质组的健康状况。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topics, 06-AG-108 Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues, and 06-DK-105 Enabling technologies for cell biology and macromolecular analyses. The long-term health of all metazoan cells is inextricably linked to protein quality control. This is achieved by proteostasis, a complex network of molecular interactions that determines the health of the proteome. Proteostasis balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance with the challenges imposed by environmental or physiological stress that results in a flux of misfolded and damaged proteins. An imbalance in homeostasis, if left unattended can result in severe molecular damage to the cell, dysregulation of key tissues leading to pathology, and susceptibility to diseases of aging. Adaptation and survival requires an ability to sense damaged proteins and to coordinate induction of protective stress response pathways, chaperone, and clearance networks. Despite the abundance and apparent capacity of chaperones and other components of the proteostasis network to restore folding equilibrium, the cell is poorly adapted for chronic proteotoxic stress as occurs when certain aggregation-prone proteins are expressed in metabolic disease, cancer, and neurodegenerative disease. This decline in repair activities that challenges the integrity of the proteome is influenced strongly by genes that control aging thus linking stress biology, metabolism, and protein homeostasis with the health and lifespan of the organism. This proposal brings together the complementary strengths of two groups, the Dillin laboratory at the Salk Institute and the Morimoto laboratory at Northwestern University to develop and test a new set of molecular tools that will report on the health of the proteome. These "proteostasis sensors" are designed to provide real-time assessment of the capabilities of protein folding quality control in each compartment of the cell and to assess the consequences of protein damage, cell stress, aging, and diseases of protein conformation. These tools will be initially developed for use in C. elegans to obtain a rapid test of hypothesis and the ability to assess functional capacities using genetic approaches and then extended to mammalian tissue culture cells and eventually in transgenic mice. The impact of these studies is very broad and extends across all areas of biology and medicine, for protein quality control is fundamental to the health and protection of the proteome in all cells and tissues of eukaryotes. The tools that we develop will be made available to all other researchers upon request upon publication. PUBLIC HEALTH RELEVANCE: The expression of damaged proteins is associated with hundreds of human diseases associated with aging. How protein misfolding in one compartment of the cell affects another and variability among tissues represents key questions that have not been resolved. This proposal is to develop a molecular toolbox of folding sensors that provide real-time living cell imaging to quantify the health of the proteome in the face of acute stress, chronic expression of damaged proteins, and aging.