Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant. Currently, pyridoxal-5'-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) is thought to be the major H(2)S-producing enzyme in the brain. We recently found that brain homogenates of CBS-knockout mice, even in the absence of PLP, produce H(2)S at levels similar to those of wild-type mice, suggesting the presence of another H(2)S-producing enzyme. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) produces H(2)S from cysteine. In addition, 3MST is localized to neurons, and the levels of bound sulfane sulfur, the precursor of H(2)S, are greatly increased in the cells expressing 3MST and CAT but not increased in cells expressing functionally defective mutant enzymes. These data present a new perspective on H(2)S production and storage in the brain.

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine b-synthase, cystathionine g-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase. Unlike the L-cysteine pathway, this D-cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D-cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues.

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Significance: Accumulating evidence shows that hydrogen sulfide may function as a signaling molecule in processes such as neuromodulation in the brain and smooth muscle relaxation in the vascular system. It also has a cytoprotective effect, since it can protect neurons and cardiac muscle from oxidative stress and ischemiareperfusion injury, respectively. Hydrogen sulfide can also modulate inflammation, insulin release, and angiogenesis. Recent Advances: The regulation of the activity of 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT), one of the H 2 S producing pathways, has been demonstrated. The production of H 2 S by the pathway, which is regulated by Ca 2+ and facilitated by thioredoxin and dihydrolipoic acid, is also involved in H 2 S signaling as well as cytoprotection. Sulfur hydration of proteins by H 2 S has been proposed to modulate protein functions. H 2 S-sensitive fluorescent probes, which enable us to measure the localization of H 2 S in real time, have been developed. Critical Issues: The basal concentrations of H 2 S have recently been measured and found to be much lower than those initially reported. However, the concentration of H 2 S reached in stimulated cells, as well as the regulation of H 2 S producing enzymes is not well understood. It has been proposed that some of the effects of H 2 S on the regulation of enzymes and receptors might be explained through the properties of sulfane sulfur (S 0 ), another form of active sulfur. Future Directions: The determination of H 2 S concentrations in activated cells using new methods including H 2 S-sensitive fluorescent probes, as well as the investigation of the effects of H 2 S using specific inhibitors, may provide better understanding of the physiological function of this molecule. Clarifying mechanisms of H 2 S activity may also facilitate the development of new therapeutic compounds. Antioxid. Redox Signal. 17, 45-57.

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem-loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA(Phe) or its anticodon stem- loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.