Regulation of the C. elegans heat shock response by LST-3

 描述(由申请人提供)：由于蛋白平衡缺陷是许多人类衰老疾病的核心，因此需要制定策略来调节这一过程。热休克转录因子HSF1是热休克反应(HSR)和分子伴侣表达的主要调节因子，因此是一个关键的治疗靶点。PI已经证明哺乳动物HSF1受衰老因子和脱乙酰酶SIRT1的调节，并且这种调节可以被SIRT1抑制剂DBC1调节。我们的主要假设是LST-3，一种与DBC1同源的线虫蛋白，抑制了蠕虫中HSR的激活，从而导致对蛋白平衡、健康寿命和寿命的负面影响。因此，抑制LST-3活性预计会增强HSR，并导致更健康的衰老。这项研究的基本原理是，更好地了解sirtuins和sirtuin调节器如何调节HSR，将允许通过新的机制来调节这种反应。将追求以下两个具体目标：1)确定LST-3是否与SIR-2.1和HSF-1活性共定位并调节SIR-2.1和HSF-1活性；2)表征LST-3对线虫热休克反应生理读数的影响。在第一个目标下，我们将检验LST-3通过钝化SIR-2.1活性来抑制HSF-1活性的假设。我们将首先使用CRISPR技术产生的三重荧光菌株来确定LST-3是否与SIR-2.1和HSF-1共存。接下来，我们将通过体外和体内实验来检测LST-3是否能够抑制SIR-2.1的活性。最后，将通过对HSF-1靶基因的影响以及HSF-1：：GFP与HSP-70启动子结合的能力来测试LST-3抑制HSF-1活性的能力。因此，这一目标的结果有望在线虫中提供关于推测的sirtuin调制器Lst-3如何调节SIR-2.1和HSF-1的详细信息，这两个同源基因在预防人类衰老疾病方面发挥着关键作用。在第二个目标下，我们将检验这样的假设，即LST-3通过抑制SIR-2.1活性从而抑制HSF-1活性，钝化HSR激活的生理读数。LST-3的表达将发生改变，并将评估对细胞保护、蛋白稳定、寿命和健康的影响。因此，在这一目标中取得的结果将提供信息，可用于确定促进人类蛋白质稳定、寿命和健康的新方法。这项工作的预期结果是，将更好地理解sirtuins和假定的sirtuin调节剂LST-3是如何调节HSR的。这项研究意义重大，因为获得关于sirtuins和sirtuin调节剂如何调节HSR的信息将有助于开发新的方法来调节这种反应，用于治疗目的。提出的这项研究具有创新性，因为它专注于一种完全不同的方法来理解HSF1的调节，通过SIRT1调节器，并使用遗传易驯化的模式生物秀丽线虫作为基础，以发现可能参与调节蛋白质平衡和健康寿命的新基因。此外，由于这项研究是由研究生和本科生推动的，未来科学家的培训是一个额外的好处。

 DESCRIPTION (provided by applicant): As defects in proteostasis are central to many human diseases of aging, strategies are needed to regulate this process. The heat shock transcription factor HSF1 is a master regulator of the heat shock response (HSR) and molecular chaperone expression, and thus is a key therapeutic target. It has previously been shown by the PI that mammalian HSF1 is regulated by the aging factor and deacetylase SIRT1, and that this regulation can be modulated by the SIRT1 inhibitor DBC1. Our overarching hypothesis is that LST-3, a C. elegans protein with homology to DBC1, inhibits activation of the HSR in the worm, thus leading to negative effects on proteostasis, healthspan and longevity. Inhibition of LST-3 activity would thus be predicted to enhance the HSR and lead to healthier aging. The rationale for this research is that a better understanding of how sirtuins and sirtuin modulators regulate the HSR will allow the regulation of this response by new mechanisms. The following two specific aims will be pursued: 1) Determine whether LST-3 colocalizes with and regulates SIR-2.1 and HSF-1 activity, and 2) Characterize the effects of LST-3 on physiological readouts of the C. elegans heat shock response. Under the first aim, we will test the hypothesis that LST-3 can inhibit HSF-1 activity by blunting SIR-2.1 activity. We will first determine whether LST-3 colocalizes with SIR-2.1 and HSF-1 using a triple-fluorescent strain generated using CRISPR technology. Next, we will assay whether LST-3 can inhibit SIR-2.1 activity using both in vitro and in vivo assays. Finally, the ability of LST-3 to inhibit HSF-1 activity will be tested via effects n HSF-1 target genes and the ability of HSF-1::GFP to bind to the hsp-70 promoter. The results of this aim are thus expected to provide detailed information in C. elegans about how the putative sirtuin modulator LST-3 regulates SIR-2.1 and HSF-1, the homologs of which play critical roles in preventing human diseases of aging. Under the second aim, we will test the hypothesis that LST-3, through inhibiting SIR-2.1 activity and thus HSF-1 activity, blunts the physiological readouts of HSR activation. LST-3 expression will be altered, and effects on cytoprotection, proteostasis, lifespan and healthspan will be assessed. The results obtained in this aim will thus provide information that can be used to identify new ways to promote human proteostasis, lifespan and healthspan. The expected outcome of this work is that a better understanding of how sirtuins and the putative sirtuin modulator LST-3 regulate the HSR will be established. This study is significant because gaining information on how sirtuins and sirtuin modulators regulate the HSR will allow the development of new methods to regulate this response for therapeutic purposes. The research proposed is innovative because it focuses on an entirely different approach to understanding the regulation of HSF1, through SIRT1 modulators, and uses the genetically tractable model organism C. elegans as a basis for uncovering new genes that may be involved in regulating proteostasis and healthspan. Additionally, as this study is driven by graduate and undergraduate students, the training of future scientists is a fringe benefit.