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.
As HSF1 is the master regulator of protein homeostasis, increased knowledge of how HSF1 is controlled by sirtuins and sirtuin modulators will allow the development of new strategies to treat diseases of aging. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help us to reduce the burden of a vast array of human diseases.