Defining the Essential Function of Heat Shock Factor and the Consequences of its Age-Associated Decline

 描述(申请人提供)：拟议研究的长期目标是更好地了解热休克因子(HSF)在蛋白质折叠动态平衡中的作用。HSF作为蛋白质折叠应激诱导的转录调节因子的作用已经被研究了几十年。最近，对衰老模型生物的研究表明，HSF的功能随着年龄的增长而下降，从而限制了健康和寿命。然而，我们仍然对HSF和特定的蛋白质折叠途径之间的连接在老年细胞中是如何被切断的有一个初步的了解。这一知识对于指导正在进行的治疗努力以改善衰老对蛋白质折叠动态平衡的负面影响至关重要。HSF从酵母到人类都是保守的。酵母拥有研究基因表达的强大工具，并经历一种被称为复制细胞衰老的细胞衰老形式，这种衰老受保守的衰老机制控制。然而，在酵母中研究Hsf一直很困难，因为它是一种必不可少的蛋白质，即使在没有蛋白质折叠压力的情况下(即Hsf细胞死亡)。现有的有条件地灭活HSF的方法很慢，因此引起了人们的担忧，即它们诱导了与HSF的基本功能无关的次要基因表达效应。此外，目前尚不清楚HSF活性在酵母细胞复制衰老过程中是否会下降。我们已经开发出一种化学遗传学方法，通过将HSF从通常位于酵母中的细胞核错位定位到细胞质，从而快速而有效地抑制HSF。我们的计划是将这一工具与对新生转录本的基因组分析相结合，以确定热休克蛋白失活对基因表达的直接影响，并利用这一知识来设计可存活的热休克蛋白细胞(目标1)。我们还证实了HSF活性随着复制酵母细胞年龄的增加而下降。令人惊讶的是，我们发现了另一种转录途径，通过将 在许多但不是所有的HSF基因靶点上。我们的数据还表明，在衰老细胞中没有维持的蛋白质折叠途径之一是Hsp90蛋白质折叠途径，它包含许多严格的HSF基因靶点。我们将使用Hsp90功能的各种单细胞报告器来测试HSF与Hsp90的连接是否在旧细胞中被切断(目标2)。拟议中的研究将确定蛋白质折叠途径如何从HSF转录控制中分离出来，并使HSF效应器的靶向治疗能够延长健康和寿命。

 DESCRIPTION (provided by applicant): The long-range goal of the proposed study is to better understand the role of Heat Shock Factor (Hsf) in protein folding homeostasis. The role of Hsf as a transcriptional regulator that is induced by protein folding stress has been studied for decades. More recently, studies of aging model organisms have revealed that Hsf function declines with age, thus limiting healthspan and lifespan. However, we still have a rudimentary understanding of how connections between Hsf and specific protein folding pathways are severed in old cells. This knowledge is critical for guiding ongoing therapeutic efforts to ameliorate the negative effects of aging on protein folding homeostasis. Hsf is conserved from yeast to humans. Yeast has powerful tools for studying gene expression and undergoes a form of cell aging known as replicative cell senescence that is under the control of conserved aging mechanisms. However, Hsf has been difficult to study in yeast because it is an essential protein even in the absence of protein folding stress (i.e. hsf cells are dead). Existing methods to conditionally inactivate Hsf are slow, thus raising concerns that they induce secondary gene expression effects unrelated to Hsf's essential function. Moreover, it is not known whether Hsf activity declines during yeast cell replicative senescence. We have developed a chemical genetics approach that rapidly and potently inhibits Hsf by mislocalizing it from the nucleus, where it normally resides in yeast, to the cytoplasm. Our plan is to combine this tool with genomic analysis of nascent transcripts to define the immediate effects of Hsf inactivation on gene expression and use this knowledge to engineer viable hsf cells (Aim 1). We have also established that Hsf activity declines as a function of replicative yeast cell age. Surprisingly, w found that an alternative transcriptional pathway maintains protein folding in aged cell by turning on many but not all Hsf gene targets. Our data also suggest that one of the protein folding pathways that is not maintained in aged cells is the Hsp90 protein folding pathway, which comprises many strict Hsf gene targets. We will test if the Hsf connection with Hsp90 is severed in old cells using a variety of single-cell reporters of Hsp90 function (Aim 2). The proposed studies will define how protein folding pathways are severed from Hsf transcriptional control in old cells and enable targeted therapies of Hsf effectors that extend healthspan and lifespan.