Shingo Kasamatsu

Supersulfides are emerging sulfur-containing signaling molecules involved in redox regulation, mitochondrial function, and protein S-sulfhydration. However, their dynamic behavior in living mammalian systems remains poorly understood because existing analytical methods require destructive sample preparation or lack sufficient intracellular applicability. Here, we developed a genetically encoded supersulfide-dependent translocation reporter (SuTR) for mammalian cells and in vivo imaging. Although the previously reported probe psGFP failed to respond to supersulfides in mammalian cells, fusion of psGFP with the sulfide-responsive transcriptional repressor (SqrR) generated SuTR, a novel reporter that exhibited supersulfide-dependent translocation from the nucleus to the cytoplasm. Na₂S₂ and Na₂S₃ induced dose-dependent cytosolic translocation of SuTR, whereas Na₂S showed no effect. Fluorescence recovery after photobleaching (FRAP) analysis revealed accelerated fluorescence recovery shortly after supersulfide stimulation, and overexpression of the endogenous supersulfide-producing enzyme Cysteinyl-tRNA Synthetase 2 (CARS2) similarly altered reporter dynamics. Mutational analyses demonstrated that reporter responsiveness depends on the DNA-binding activity of SqrR. Furthermore, SuTR successfully detected supersulfide induction in mouse liver in vivo following Na₂S₃ administration. These findings establish SuTR as a genetically encoded reporter for monitoring supersulfide dynamics in mammalian cells and tissues.

Cystathionine γ-lyase (CSE) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the final step of the transsulfuration pathway, converting cystathionine into cysteine. Additionally, CSE is also essential for the formation of cysteine hydropolysulfide (Cys-S-(S)n-H), known as supersulfides, by metabolizing cystine under pathological conditions. We previously reported that, during cystine metabolism, CSE undergoes self-inactivation through polysulfidation at the Cys136 residue. Here, contrary to the anticipated role of L-S-nitrosocysteine (L-CysNO) as a nitric oxide (NO) donor, we demonstrate that it serves as a substrate for CSE and that its metabolites inhibit the activity of the enzyme during L-CysNO metabolism. The in vitro incubation of CSE—but not the Cys136/171Val mutant—with L-CysNO resulted in the dose-dependent inhibition of supersulfide production, which was not reversed by the reducing agents. Notably, CSE activity remained unchanged upon preincubation with other NO donors, such as S-nitrosoglutathione or D-CysNO, but was inhibited when coincubated with cysteine. Furthermore, when PLP was removed from the CSE/L-CysNO premix, L-CysNO no longer inhibited CSE activity, suggesting that CSE metabolizes L-CysNO and that its metabolites contribute to enzyme inactivation. Indeed, we identified thionitrous acid and pyruvate as the primary CSE/L-CysNO reaction products. Thus, we establish L-CysNO as a CSE substrate and demonstrate that its metabolites act as enzyme inhibitors through a novel irreversible modification at the Cys136/171 residues.

Cystathionine γ-lyase (CSE) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. Additionally, CSE also plays a crucial role in the production of cysteine hydropolysulfide (Cys-S-(S)n-H), known as supersulfides, by metabolizing cystine under pathological conditions. We previously reported that, during cystine metabolism, CSE undergoes self-inactivation through polysulfidation at the Cys136 residue. Here, contrary to the anticipated role of L-S-nitrosocysteine (L-CysNO) as a nitric oxide (NO) donor, we demonstrate that it serves as a substrate for CSE and that its metabolites inhibit its activity. The in vitro incubation of CSE—but not the Cys136/171Val mutant—with L-CysNO resulted in the dose-dependent inhibition of supersulfide production, which was not reversed by the reducing agents. Notably, CSE activity remained unchanged upon pre-incubation with other NO donors, such as S-nitrosoglutathione or D-CysNO, but was inhibited when co-incubated with cysteine. Furthermore, when PLP was removed from the CSE/L-CysNO premix, L-CysNO no longer inhibited CSE activity, suggesting that CSE metabolizes L-CysNO and that its metabolites contribute to enzyme inactivation. Indeed, we identified thionitrous acid and pyruvate as the primary CSE/L-CysNO reaction products. Thus, we establish L-CysNO as a CSE substrate and demonstrate that its metabolites act as enzyme inhibitors through a novel irreversible modification at the Cys136/171 residues.

The number of people with dementia is increasing annually worldwide. Alzheimer’s disease (AD), which accounts for the highest percentage of dementia-causing diseases, remains difficult to cure, and prevention of its onset is important. We aimed to discover new AD-preventive ingredients and investigate the inhibitory effects of ten different species of seafood digests prepared by protease treatment on β-secretase 1 (BACE1) activity. Substantial inhibition of BACE1 activity was observed in five species of seafood, and protease-digested whitebait (WPD) showed the highest inhibitory effect among the ten marine samples. We further examined the potential of WPD as an AD preventive component using a familial AD strain (5xFAD) murine model. The intraperitoneal administration of WPD for 28 days substantially decreased the insoluble amyloid β_1–42 content and the expression of glial fibrillary acidic protein, a marker of astrogliosis, in the cerebral cortex of the 5xFAD mice. These results strongly suggest that WPD is a novel functional food-derived ingredient with preventive effects against AD.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S -nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5 ^C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has β-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder whereby oxidative stress augmentation results in mitochondrial dysfunction and cell death by apoptosis. Emerging evidence indicates that reactive sulfur species (RSS), such as glutathione hydropersulfide (GSSH), is endogenously produced, functions as potent antioxidants, and regulate redox signaling through the formation of protein polysulfides. However, the relationship between RSS and AD pathogenesis is not fully understood. In this study, we analyzed endogenous RSS production in the brain tissue of a familial AD model (5xFAD) mouse using multiple RSS-omics approaches. Memory impairment, increased amyloid plaques, and neuroinflammation have been confirmed in 5xFAD mice. Quantitative RSS omics analysis revealed that the total polysulfide content was significantly decreased in the brains of 5xFAD mice, whereas there was no significant difference in the levels of glutathione, GSSH, or hydrogen sulfide between wild-type and 5xFAD mice. In contrast, a significant decline in the protein polysulfide status was observed in the brains of 5xFAD mice, suggesting that RSS production and subsequent redox signaling might be altered during the onset and progression of AD. Our findings have important implications for understanding the significance of RSS in the development of preventive and therapeutic strategies for AD.

Although hydrogen sulfide (H ₂ S) is an endogenous signaling molecule with antioxidant properties, it is also cytotoxic by potently inhibiting cytochrome c oxidase and mitochondrial respiration. Paradoxically, the primary route of H ₂ S detoxification is thought to occur inside the mitochondrial matrix via a series of relatively slow enzymatic reactions that are unlikely to compete with its rapid inhibition of cytochrome c oxidase. Therefore, alternative or complementary cellular mechanisms of H ₂ S detoxification are predicted to exist. Here, superoxide dismutase [Cu-Zn] (SOD1) is shown to be an efficient H ₂ S oxidase that has an essential role in limiting cytotoxicity from endogenous and exogenous sulfide. Decreased SOD1 expression resulted in increased sensitivity to H ₂ S toxicity in yeast and human cells, while increased SOD1 expression enhanced tolerance to H ₂ S. SOD1 rapidly converted H ₂ S to sulfate under conditions of limiting sulfide; however, when sulfide was in molar excess, SOD1 catalyzed the formation of per- and polysulfides, which induce cellular thiol oxidation. Furthermore, in SOD1-deficient cells, elevated levels of reactive oxygen species catalyzed sulfide oxidation to per- and polysulfides. These data reveal that a fundamental function of SOD1 is to regulate H ₂ S and related reactive sulfur species.