Regulatory Dynamics of the Proteostasis Network

项目摘要/摘要 癌症和神经退行性变通常被认为在疾病谱的两端-- 前者以细胞无节制生长为特征，后者以细胞过早死亡为特征。然而，它们是联系在一起的 由于一种常见的潜在细胞状态的失调：蛋白质稳态(蛋白质稳态)。蛋白平衡指的是 到蛋白质合成、折叠和降解的动态平衡，这需要维持 绝大多数的细胞过程。蛋白平衡缺陷导致蛋白质聚集，这是 阿尔茨海默氏症、帕金森氏症、亨廷顿氏症和肌萎缩侧索硬化症等神经退行性疾病。相反，许多人 癌细胞过度表达蛋白平衡机制，以对抗高突变负荷。我的长期目标是 研究计划是确定健康细胞用来维持蛋白质平衡和 确定这一规定是如何被打破并被疾病劫持的。 蛋白质平衡网络(PN)的中央调节轴是热休克反应(HSR)，a 真核生物中转录因子HSF1调控的普遍保守的基因表达程序。 尽管它早在30年前就被发现，但控制HSF1活性从而表达HSF1的机制 高铁仍然难以捉摸。我的实验室最近使用发芽酵母进行的研究揭示了一个关键的反馈 控制HSF1活动的环路。我们发现，伴侣Hsp70在非应激细胞中抑制HSF1，并 HSF1介导的Hsp70的诱导是HSF1失活所必需的。因此，HSF1和HSP70构成了 促进自我平衡适应压力的反馈回路 这项建议建立在这一发现以及我们为回答主要问题而开发的工具和分析的基础上 关于HSF1监管的未决问题。在目标1中，我们将定义上行信令事件 激活HSF1。我们假设C末端丙氨酸/苏氨酸的核糖体-新生链状复合体 延伸(“猫尾”)和新合成的“孤儿”核糖体蛋白(ORPS)通过以下方式触发HSR 将Hsp70辅助伴侣Sis1与HSF1隔离。在目标2中，我们将定义分子机制 Hsp70通过其抑制HSF1。我们将验证Hsp70通过抑制HSF1抑制HSF1的假设 DNA结合和阻断HSF1介导的反式激活。在目标3中，我们将揭示新的反馈环路 控制HSF1。我们将系统地确定所有HSF1靶标对野生型HSF1激活的贡献 动力学。通过对HSR进行多方面的分析-结合细胞生物学、分子遗传学、 生物化学和系统生物学-我们将建立信号、调节机制和反馈回路 它支配着这个中央适应回路，它位于人类疾病的核心，如癌症和 神经退行性变。

PROJECT SUMMARY / ABSTRACT Cancer and neurodegeneration are often thought to be on opposing ends of the disease spectrum – the former characterized by unchecked cell growth and the latter by premature cell death. However, they are linked by dysregulation of a common underlying cellular state: protein homeostasis (proteostasis). Proteostasis refers to the dynamic balance of protein synthesis, folding and degradation, the maintenance of which is required for the vast majority of cellular processes. Defects in proteostasis lead to protein aggregation, a hallmark of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and ALS. Conversely, many cancer cells overexpress proteostasis machinery to counteract high mutational loads. The long-term goal of my research program is to define the mechanisms that healthy cells employ to maintain proteostasis and to establish how this regulation breaks down and is hijacked in disease. The central regulatory axis of the proteostasis network (PN) is the heat shock response (HSR), a universally conserved gene expression program under the control of transcription factor Hsf1 in eukaryotes. Despite its identification 30 year ago, the mechanisms that control Hsf1 activity and thereby expression of the HSR have remained elusive. Recent work from my laboratory using budding yeast revealed a key feedback loop that governs Hsf1 activity. We found that the chaperone Hsp70 represses Hsf1 in unstressed cells and that Hsf1-mediated induction of Hsp70 is requied to deactivate Hsf1. Thus, Hsf1 and Hsp70 constitute a feedback loop that promotes homeostatic adaptation to stress This proposal builds upon this discovery and the tools and assays we have developed to answer major outstanding questions concerning Hsf1 regulation. In Aim 1 we will define the upstream signaling events that activate Hsf1. We hypothesize that ribosome-nascent chain complexes with C-terminal alanine/threonine extensions (“CAT-tails”) and newly synthesized “orphan” ribosomal proteins (oRPs) trigger the HSR by sequestering the Hsp70 co-chaperone Sis1 away from Hsf1. In Aim 2 we will define the molecular mechanisms by which Hsp70 represses Hsf1. We will test the hypothesis that Hsp70 represses Hsf1 both by inhibiting Hsf1 DNA binding and by blocking Hsf1-mediated transactivation. In Aim 3 we will reveal novel feedback loops that control Hsf1. We will systematically determine the contribution of all Hsf1 targets to wild-type Hsf1 activation dynamics. By undertaking a multi-faceted analysis of the HSR – combining cell biology, molecular genetics, biochemistry and systems biology – we will establish the signals, regulatory mechanisms and feedback loops that govern this central adaptive circuit that lies at the heart of human diseases as diverse as cancer and neurodegeneration.