Mechanisms of cytosolic proteostasis in yeast

近50种主要疾病，从糖尿病到神经退行性疾病 包括阿尔茨海默氏症(AD)、帕金森氏症(PD)和亨廷顿氏病(HD)在内的 与蛋白质错误折叠和聚集有关。细胞在压力下生长和增殖 内源性、外源性活性氧(ROS)的持续危害威胁 氧化剂和活性亲电剂。胞浆蛋白半胱氨酸几乎是唯一的 维持在还原状态，氧化应激引起的半胱氨酸氧化 预计会导致严重的错误折叠和聚集。然而，相对较少的是 了解氧化还原失衡对蛋白质动态平衡的影响 (蛋白质平衡)。此外，细胞还原-氧化(氧化还原)缓冲的作用 维持胞质的途径，包括硫氧还蛋白和谷胱甘肽系统 蛋白质代谢还没有被很好地理解。我们试图了解两者之间的相互作用 细胞保护性应激反应途径及其维持机制 蛋白质平衡。提案中详细介绍的已公布和初步结果使我们能够 假设氧化还原诱导细胞保护性热休克反应(HSR) 失衡部分是由主要蛋白伴侣中的半胱氨酸开关调节的 HSP70(芽殖酵母中的SSA1)和硫醇氧化还原缓冲在 维持胞质蛋白的稳定。此续订申请的主要目标 是为了确定硫醇反应胁迫对细胞质蛋白生物发生的影响 和蛋白质质量控制，并阐明氧化剂之间的调节相互作用 并通过三条不同的研究路线展开蛋白质反应。具体而言 目的1确定关键分子伴侣Ssa1/Hsp70的作用机制 通过主热调节转录活性来调节HSR 休克转录因子HSF1。特定目标2将调查硫醇的后果- 反应性应激对Ssa1/Hsp70活性和细胞功能的影响，包括 SSA1/Hsp70氧化还原开关调节HSR，并决定HSR的作用 保守的氧化还原缓冲系统在调节硫醇反应应激中的作用。在具体目标3中， 我们将确定蛋白质硫醇修饰对一般胞浆的影响 使用蛋白质组学和遗传学方法的蛋白质平衡。这项提案中概述的工作 将揭示细胞氧化还原和蛋白质质量控制之间的机制联系 通过开发易处理的酵母模型系统来实现对网络的支持。这些结果反过来将指导 针对ROS和蛋白质质量的治疗干预的未来发展 以控制为基础的障碍。

Nearly 50 major diseases ranging from diabetes to neurodegenerative disorders including Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s (HD) diseases have been linked to protein misfolding and aggregation. Cells grow and proliferate under the constant threat of damage from endogenous reactive oxygen species (ROS), exogenous oxidants and reactive electrophiles. Cytosolic protein cysteines are almost exclusively maintained in the reduced state, and cysteine oxidation caused by oxidative stress is predicted to result in significant misfolding and aggregation. However, relatively little is known about the consequences of redox imbalance on protein homeostasis (proteostasis). Furthermore, the roles of cellular reduction-oxidation (redox) buffering pathways, including the thioredoxin and glutathione systems, in maintaining cytosolic proteostasis are not well understood. We seek to understand the interplay between cytoprotective stress response pathways and the machinery employed to maintain proteostasis. Published and preliminary results detailed in the proposal lead us to hypothesize that induction of the cytoprotective heat shock response (HSR) by redox imbalance is mediated in part by a cysteine switch in the principal protein chaperone Hsp70 (Ssa1 in budding yeast) and that thiol redox buffering plays a significant role in maintenance of cytosolic proteostasis. The primary objectives of this renewal application are to determine the impacts of thiol-reactive stress on cytoplasmic protein biogenesis and protein quality control and to elucidate the regulatory interactions between oxidant and unfolded protein responses, through three distinct lines of investigation. In Specific Aim 1 we will define the mechanism by which the key molecular chaperone Ssa1/Hsp70 regulates the HSR through modulation of transcriptional activity by the master heat shock transcription factor Hsf1. Specific Aim 2 will investigate the consequences of thiol- reactive stress on Ssa1/Hsp70 activity and cellular functions, including how the Ssa1/Hsp70 redox switch regulates the HSR, and determine the roles of the highly conserved redox buffering systems in mediating thiol-reactive stress. In Specific Aim 3, we will determine the impacts of protein thiol modification on general cytosolic proteostasis using proteomic and genetic approaches. The work outlined in this proposal will reveal the mechanistic connections between cellular redox and protein quality control networks by exploiting the tractable yeast model system. These results in turn will guide future development of therapeutic interventions targeting ROS- and protein quality control-based disorders.