CHIP: A link between the chaperone and proteasome system

描述(申请人提供)：70 kDa和90 kDa热休克蛋白(HSPs)最为人所知的是它们作为分子伴侣的作用，参与新生蛋白质的折叠、成熟和适当的亚细胞靶向，或者相反，参与错误折叠或以其他方式被细胞应激破坏的蛋白质的功能恢复。然而，热休克蛋白参与了各种过程--细胞内信号事件、增殖、分化和蛋白质的靶向降解--这些过程似乎只间接地与蛋白质折叠有关，或者根本与之无关。与此同时，越来越明显的是，分子伴侣并不代表细胞应激反应的总和，除了蛋白质复性外，还需要一系列事件来维持生存能力，并在不利环境中保护细胞内环境。1999年，我们在应激反应基因筛查中发现了CHIP(Hsc70相互作用蛋白的羧基末端)。从那时起，我们发现CHIP与伴侣Hsc70、Hsp70和Hsp90相互作用，并对它们的功能产生复杂和协调的影响。此外，CHIP还具有泛素连接酶活性，在调节细胞质内的蛋白质质量控制中起着关键作用。最后，我们在体外和体内都发现，芯片是细胞和组织应激反应的中央调节因子。令人惊讶的是，这些效应中至少有一些似乎与CHIP对转录、信号和代谢反应的特定影响有关。这项资助的目的是作为我们对参与分子伴侣事件的蛋白质的初始筛选的合乎逻辑的扩展。为了实现我们的目标，我们制定了一种新的高度集成的方法，使用体外和体内测试。这项建议的目的是：(1)。通过结合转录组/蛋白质组分析，研究CHIP在细胞应激背景下对全球mRNA和蛋白质表达变化的影响；(2)表征CHIP在细胞信号和代谢反应中的作用；以及(3)评估CHIP在体内病理生理应激中的综合作用。这项提议的范围旨在利用最先进的分子生物学技术解决相关的生物学和生理学问题。从这一建议中获得的知识应该提供关于这些不同的通路如何调节泛素/蛋白酶体系统控制的生理和病理过程的关键信息，并可能为涉及该系统的疾病的治疗提供潜在的治疗靶点。 与公众健康相关：适当的蛋白质折叠对于最佳的蛋白质性能和正常的细胞功能是必不可少的。在新蛋白质的合成和变性蛋白质的重折叠过程中，由于某些蛋白质不能自发地获得正确的三级构象，细胞的分子伴侣与其降解机制之间必须发生合作。相互排斥的折叠和降解途径构成了细胞的蛋白质质量控制系统。伴侣和泛素-蛋白酶体系统在调节应激反应信号和健康和疾病的细胞保护机制中发挥着关键作用。我们的总体目标是确定CHIP的分子和生理功能是如何协调的，以协调其应激反应能力。这些研究将帮助我们开发关于细胞质质量控制机制和细胞应激反应之间关系的新模型。

DESCRIPTION (provided by applicant): 70- and 90-kDa heat shock proteins (Hsps) are best known by their roles as molecular chaperones, participating in the folding, maturation, and proper subcellular targeting of nascent proteins or, conversely, the recovery of function of proteins that have been misfolded or otherwise damaged by cellular stressors. However, Hsps participate in a variety of processes- intracellular signaling events, proliferation, differentiation, and targeted degradation of proteins- that seem related to protein folding only indirectly, or not at all. At the same time, it is becoming increasingly apparent that molecular chaperones do not represent the sum total of the cell stress response, and that a range of events other than protein refolding are required to maintain viability and protect the intracellular environment in adverse circumstances. We identified CHIP (carboxyl-terminus of Hsc70-interacting protein) in a screen for stress-responsive genes in 1999. Since then, we have found that CHIP interacts with the chaperones Hsc70, Hsp70, and Hsp90 and has complex and coordinated effects on their functions. In addition, CHIP has ubiquitin ligase activity and plays a critical role in regulating protein quality control within the cytoplasm. Finally, we have found using both in vitro and in vivo approaches that CHIP is a central regulator of cellular and organismal stress responses. Surprisingly, at least some of these effects seem to be related to specific effects of CHIP on transcriptional, signaling, and metabolic responses. The aims of this grant are intended as a logical extension of our initial screen for proteins participating in molecular chaperone events. To accomplish our aims, we have formulated a novel and highly integrated approach using both in vitro and in vivo assays. The aims of this proposal are to: (1). Examine the consequences of CHIP on global changes in mRNA and protein expression in the context of cellular stress using a combined transcriptome/proteome analysis; (2) Characterize the role of CHIP on cell signaling and metabolic responses and (3) Evaluate the integrated effects of CHIP on pathophysiologic stress in vivo. The scope of this proposal is intended to address relevant biological and physiological questions using state of the art molecular biology techniques. Knowledge gained from this proposal should provide crucial information about how these various pathways regulate physiologic and pathologic processes that are under control of the ubiquitin/proteasome system and may provide potential therapeutic targets for treatment of diseases where this system is involved. PUBLIC HEALTH RELEVANCE: Proper protein folding is essential for optimum protein performance and normal cellular function. During synthesis of new proteins and refolding of denatured proteins, cooperation between the cell's molecular chaperones and its degradation machinery must occur because some proteins cannot attain their correct tertiary conformation spontaneously. The mutually exclusive pathways of folding and degradation constitute the cell's protein quality control system. The chaperone and ubiquitin-proteasome systems play critical roles in regulating stress-responsive signaling and cellular protective mechanisms in health and disease. Our overall goal is to determine how the molecular and physiologic functions of CHIP, a molecular chaperone, are coordinated to orchestrate its stress response capabilities. These studies will help us develop new models about the relationship between cytoplasmic quality control mechanisms and the cellular stress response.