Grant: Discovery Grants Program - Individual

Hydrogen sulfide (H2S) has been known for its toxic effects. Importantly, it is also synthesized inside eukaryotes, and plays physiological roles in regulating cellular and body functions of mammals as a signaling molecule. One of the mechanisms for H2S signaling is S-sulfhydration, in which persulfide is formed between sulfhydryl groups (SH) of proteins and H2S. This mechanism, however, has been challenged due to insufficient reducing power of H2S and the fact that H2S also interacts with many SH-free proteins. We hypothesize a novel SH-independent signaling mechanism of H2S, in which sulfide binds to amino group(s) of amino acid residues by covalent bonding, namely N-sulfuration where "N" stands for the free N-tails (NH2 group) of amino acids, altering the structures and functions of the modified proteins. To this end, we have obtained significant preliminary data, demonstrating that multiple amino acids (such as arginine & glutamine) and proteins (such as insulin & glucagon) are N-sulfurated in vitro and in vivo. The Objective of this Program is to better understand the molecular mechanisms underlying the cellular signaling effects of H2S. This short-term objective aligns with the long-term objective of my research career, i.e. to elucidate the metabolism and biological importance of H2S in eukaryotes.   Aim 1: To investigate and characterize H2S-induced N-sulfuration of selective amino acids, peptides, and proteins. We will incubate purified SH-containing as well as SH-free amino acids, synthesized peptides, and proteins with NaHS (a H2S salt) or H2S gas at physiologically relevant concentrations. Proteins obtained from mouse tissue lysates in the presence or absence of endogenous H2S will be assayed.   Aim 2: To examine relative importance of N-sulfuration and S-sulfhydration of the same SH-containing proteins and their impact on each other, under the same in vitro incubation conditions.   Aim 3: To test the relative contributions of polysulfides and H2S to N-sulfuration and S-sulfhydration, we will apply inorganic polysulfides [Na2Sn (n>2)] and organic polysulfides (diallyl disulfide and diallyl trisulfide) to the amino acids, peptides and proteins as identified in Aim 1, under normoxia and hypoxia conditions.   Aim 4: To investigate the functional significance of protein N-sulfuration, we will examine the effects of N-sulfurated insulin and glucagon, in comparison with those of native insulin and glucagon, on adipogenesis and glucose uptake and utilization in 3T3-L1 adipocytes. To unveil a completely unknown molecular mechanism for H2S signaling will be a breakthrough not only for specific H2S studies but also in our general understanding of post-translational modification and signaling processes, fundamental in mammalian cell biology and physiology. The Program would help advance life science research and innovation in Canada and beyond, keeping the status of Canada as an international leading hub in this specific research area.