Protein Persulfidation Research Articles

Persulfidation (S-sulfhydration) and H2S.

The past decade has witnessed the discovery of hydrogen sulfide (H2S) as a new signalling molecule. Its ability to act as a neurotransmitter, regulator of blood pressure, immunomodulator or anti-apoptotic agent, together with its great pharmacological potential, is now well established. Notwithstanding the growing body of evidence showing the biological roles of H2S, the gap between the macroscopic descriptions and the actual mechanism(s) behind these processes is getting larger. The reactivity towards reactive oxygen and nitrogen species and/or metal centres cannot explain this plethora of biological effects. Therefore, a mechanism involving modification of protein cysteine residues to form protein persulfides is proposed. It is alternatively called S-sulfhydration. Persulfides are not particularly stable and show increased reactivity when compared to free thiols. Detection of protein persulfides is still facing methodological limitations, and mechanisms by which H2S causes this modification are still largely scarce. Persulfidation of protein such as KATP could contribute to H2S-induced vasodilation, while S-sulfhydration of GAPDH and NF-κB inhibits apoptosis. H2S regulates endoplasmic reticulum stress by causing persulfidation of PTP-1B. Several other proteins have been found to be regulated by this posttranslational modification of cysteine. This review article provides a critical overview of the current state of the literature addressing protein S-sulfhydration, with particular emphasis on the challenges and future research directions in this particular field.

S7-5 The mechanism(s) of protein persulfide formation: Detection of protein S-sulfhydration by a tag-switch assay

Hydrogen sulfide (H 2 S) has been recently classified as a critical cell-signaling molecule. Literature published in the past few years increasingly suggests that H 2 S is a mediator of many physiological and/or pathological processes. Some of these effects are ascribed to the formation of protein persulfides, or protein S -sulfhydration (i.e. conversion of cysteine residues -SH to persulfides -S-SH). Two different approaches have been widely used to detect protein persulfides, but the selectivity of these methods have been challenged, as they do not stand on solid chemical ground. In addition, the underlying mechanisms of S -sulfhydration mediated by H 2 S are still unclear. In this study we report the first selective, “tag-switch” method, which can directly label protein S -sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H 2 S alone cannot lead to S -sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers, which would facilitate the oxidation of H 2 S to HS . Persulfides are predominantly located in endoplasmic reticulum and mitochondria (the organelles richest in PSOH and metal centers, respectively) and their formation appears to be highly dependant on MST enzymatic activity. While the clean Na 2 S solution has no effect on protein structure, polysulfides, commercially available NaHS or Na 2 S solutions supplemented with metal ions have dramatic effects on protein structure, leading to cleavage of disulfide bonds with concomitant persulfide formation. Physiological consequences of these reactions will be discussed.

Physiological function of hydrogen sulfide and beyond

The relatively high concentrations of endogenous sulfide in the mammalian brain were measured in 1989, suggesting that hydrogen sulfide (H2S) might have a physiological function. In 1996 we demonstrated that cystathionine β-synthase (CBS) is a H2S producing enzyme in the brain and that H2S facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of N-methyl d -aspartate (NMDA) receptors. The following year we demonstrated that another H2S producing enzyme, cystathionine γ-lyase (CSE) is found in tissues including vasculature and that H2S relaxes them. Based on these observations we proposed that H2S is a neuromodulator and a smooth muscle relaxant. In addition to the function as a signaling molecule, we and others found a cytoprotective effect of this molecule; H2S protects neurons from oxidative stress. This finding led to the identification of the protection of various organs including the heart, pancreas, retina, and the kidney against ischemia–reperfusion injury. From our finding that the brains of CBS knockout mice was still able to produce H2S, we found another pathway; 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT). 3MST produces H2S in the presence of thioredoxin or dihydrolipoic acid (DHLA). We recently found a novel pathway to produce H2S from d -cysteine, a negative control. d -Amino acid oxidase (DAO) metabolizes d -cysteine to an achiral α–keto acid, 3-mercaptopyruvate (3MP), which is further metabolized to H2S by 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from d -cysteine is 80 times more efficient than that from l -cysteine in the kidney, and the administration of d -cysteine to mice ameliorates renal ischemia–reperfusion injury more effectively than l -cysteine. These results show a therapeutic potential of d -cysteine to the renal diseases and even to the kidney transplantation. Our additional contribution to this field is the discovery of H2S-derived polysulfides, which exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. TRPA1 channels mediate the sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, but its endogenous ligand has not been identified. The sulfane sulfur of polysulfides is reactive electrophile and readily transferred to a nucleophilic protein thiolate, to generate the protein persulfide (sulfhydration). The sulfhydration activity of polysulfides is much greater than H2S. The production of H2S and the physiological function of H2S and polysulfides will be discussed.

Persulfide Reactivity in the Detection of Protein S-Sulfhydration

Hydrogen sulfide (H2S) has emerged as a new member of the gaseous transmitter family of signaling molecules and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine S-sulfhydration may function as a mechanism for transforming the H2S signal into a biological response. However, selective detection of S-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur species, especially thiols. A modification of the biotin switch technique, using S-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration, but the underlying mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model and analogue of MMTS, S-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights into protein S-sulfhydryl chemistry, which may be exploited in future detection strategies.