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

 描述（由申请人提供）：由于蛋白质稳态缺陷是许多人类衰老疾病的核心，因此需要策略来调节该过程。热休克转录因子HSF 1是热休克反应（HSR）和分子伴侣表达的主要调节因子，因此是关键的治疗靶点。PI先前已经表明，哺乳动物HSF 1受衰老因子和脱乙酰酶SIRT 1调节，并且这种调节可以由SIRT 1抑制剂DBC 1调节。我们的总体假设是LST-3，一个C。与DBC 1同源的线虫蛋白抑制蠕虫中HSR的激活，从而导致对蛋白质稳态、健康寿命和寿命的负面影响。因此，预测LST-3活性的抑制将增强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调节剂如何调节HSR将允许通过新的机制调节这种反应。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共定位并调节SIR-2.1和HSF-1活性，和2）表征LST-3对C.热休克反应在第一个目标下，我们将测试LST-3可以通过钝化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共定位。接下来，我们将使用体外和体内测定来测定LST-3是否可以抑制SIR-2.1活性。最后，LST-3抑制HSF-1活性的能力将通过对HSF-1靶基因的影响和HSF-1：：GFP结合hsp-70启动子的能力来测试。因此，这一目标的结果，预计将提供详细的信息，在C。elegans关于假定的sirtuin调节剂LST-3如何调节SIR-2.1和HSF-1，其同系物在预防人类衰老疾病中起关键作用。在第二个目标下，我们将测试LST-3通过抑制SIR-2.1活性并因此抑制HSF-1活性来钝化HSR激活的生理读数的假设。将改变LST-3表达，并评估对细胞保护、蛋白质代谢抑制、寿命和健康寿命的影响。因此，在这一目标中获得的结果将提供可用于确定促进人类蛋白质稳态、寿命和健康的新方法的信息。这项工作的预期结果是，更好地了解如何sirtuins和假定的sirtuin调制器LST-3调节HSR将建立。这项研究是重要的，因为获得关于sirtuins和sirtuin调节剂如何调节HSR的信息将允许开发用于治疗目的的调节这种反应的新方法。这项研究是创新的，因为它专注于一种完全不同的方法来理解HSF 1的调节，通过SIRT 1调节剂，并使用遗传上易于处理的模式生物C。作为发现可能参与调节蛋白质稳态和健康寿命的新基因的基础。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附带的好处。

 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.