Project Summary Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that impacts protein function by modifying cysteine residues through the formation of a persulfide bond by a process termed sulfhydration (persulfidation). It is produced enzymatically by three enzymes in the cysteine biosynthesis pathway: cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfutransferase (3- MST). Numerous studies have reported a protective role for H2S in experimental models of acute myocardial ischemia-reperfusion injury and heart failure. While these studies and others have established a cytoprotective role for H2S, there are still unresolved questions regarding the biology of H2S. For instance, there is a lack of understanding in how the endogenous production of H2S is regulated. Additionally, there is a need to identify protein targets of H2S in response to different stimuli to unravel the mechanism by with H2S impacts adaptation to stress. This proposal aims to offer new insights into the regulation of the H2S-producing activity of CSE. We present new data that AMP-activated protein kinase (AMPK) increases CSE-derived H2S production via phosphorylation of serine 126. Further studies identified perilipin 5 (Plin5) – a protein that promotes the association of lipid droplets with mitochondria - as a protein modified by sulfhydration following nutrient deprivation and AMPK activation. Our data also shows that the interaction of lipid droplets with mitochondria following nutrient deprivation is dependent on CSE. Based on this evidence, we hypothesize that in response to nutrient stress, AMPK induces the H2S-producing activity of CSE to impart adaptive cellular mechanisms. Specifically, we hypothesize that CSE-derived H2S maintains energy metabolism during nutrient stress by regulating the mobilization of lipid droplets to the mitochondria, in part, by altering the sulfhydration of Plin5. In Aim 1, we will investigate the impact of serine 126 phosphorylation on the H2S-producing activity of CSE. In Aim 2, we will investigate the Impact of CSE-derived H2S on maintaining Lipid Droplet-fueled β-oxidation during nutrient deprivation. In Aim 3, we will determine the impact of sulfhydration on Plin5 during nutrient deprivation. This project breaks new ground in defining a mechanism by which the H2S-producing activity of CSE is regulated. As such, it has the potential to lead to the development of novel therapies aimed at harnessing the physiological effects of H2S to combat cardiovascular disease.