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

定义热休克因子的基本功能及其与年龄相关的下降的后果

• 批准号: 9050618
• 负责人: Vladimir Denic
• 金额: $ 25.35万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9050618
• 关键词: Ablation; Age; Aging; Aging-Related Process; Animal Model; Attenuated; Cell Aging; Cell Nucleus; Cell Survival; Cells; Cytoplasm; Cytosol; Data; Disease; Engineering; Essential Genes; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Enhancement; Genetic Screening; Genomics; Goals; Health; Heat shock factor; Heat shock proteins; Heat-Shock Response; Homeostasis; Human; Incidence; Knowledge; Light; Link; Longevity; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Methods; Molecular; Molecular Chaperones; Monitor; Mothers; Mus; Nematoda; Organism; Pathway interactions; Pharmacotherapy; Phenotype; Proteins; Rejuvenation; Reporter; Role; Saccharomyces cerevisiae; Saccharomycetales; Sequence Analysis; Signal Pathway; Stress; System; Temperature; Testing; Therapeutic; Transcript; Transcriptional Regulation; Work; Yeasts; activating transcription factor; age effect; aged; cell age; chemical genetics; clinically relevant; dietary restriction; fly; genetic approach; heat shock transcription factor; inhibitor/antagonist; insight; mortality; mutant; programs; protein aggregate; protein folding; protein misfolding; research study; response; senescence; small molecule; targeted treatment; thermal stress; tool; tumorigenesis

 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.

 描述（由申请人提供）：拟议研究的长期目标是更好地了解热休克因子（Hsf）在蛋白质折叠稳态中的作用。Hsf作为蛋白质折叠应激诱导的转录调节因子的作用已经研究了几十年。最近，对衰老模型生物的研究表明，Hsf功能随着年龄的增长而下降，从而限制了健康寿命和寿命。然而，我们仍然有一个初步的了解之间的连接Hsf和特定的蛋白质折叠途径是如何切断在老细胞。这些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稳态的负面影响至关重要。从酵母到人类Hsf都是保守的。酵母有强大的工具来研究基因表达，并经历一种被称为复制性细胞衰老的细胞衰老形式，这种细胞衰老是在保守的衰老机制的控制下进行的。然而，Hsf一直难以在酵母中进行研究，因为即使在没有蛋白质折叠应激的情况下（即Hsf细胞死亡），它也是一种必需蛋白质。现有的方法来有条件地抑制Hsf是缓慢的，从而引起关注，他们诱导二级基因表达的影响无关的Hsf的基本功能。此外，它是不知道是否Hsf活性下降，在酵母细胞复制衰老。我们开发了一种化学遗传学方法，通过将Hsf从细胞核（通常存在于酵母中）错误定位到细胞质来快速有效地抑制Hsf。我们的计划是将联合收割机这一工具与新生转录本的基因组分析相结合，以确定Hsf失活对基因表达的直接影响，并利用这一知识设计可行的Hsf细胞（目标1）。我们还确定了Hsf活性随着复制酵母细胞年龄的变化而下降。令人惊讶的是，我们发现一种替代的转录途径通过将蛋白质的折叠转化为蛋白质的折叠来维持衰老细胞中的蛋白质折叠。 在许多但不是所有的Hsf基因靶点上。我们的数据还表明，在衰老细胞中不维持的蛋白质折叠途径之一是Hsp90蛋白质折叠途径，其包含许多严格的Hsf基因靶点。我们将使用多种Hsp90功能的单细胞报告者来测试Hsf与Hsp90的连接是否在老细胞中被切断（目的2）。拟议的研究将确定蛋白质折叠途径如何从旧细胞中的Hsf转录控制中切断，并使Hsf效应物的靶向治疗能够延长健康寿命和寿命。