H2S: A Novel Gasotransmitter that Signals by Sulfhydration

Abstract

Hydrogen sulfide (H2S) is a member of the growing family of gasotransmitters. Once regarded as a noxious molecule predominantly present in the atmosphere, H2S is now known to be synthesized endogenously in mammals. H2S participates in a myriad of physiological processes ranging from regulation of blood pressure to neuroprotection. Its chemical nature precludes H2S from being stored in vesicles and acting on receptor proteins in the fashion of other chemical messengers. Thus, novel cellular mechanisms have evolved to mediate its effects. This review focuses on sulfhydration (or persulfidation), which appears to be the principal post-translational modification elicited by H2S.

Keywords: cysteine; gasotransmitter; hydrogen sulfide; sulfhydration.

Figure 1. Potential mechanisms of protein sulfhydration and its detection.(A) Mechanisms for sulfydration/persulfidation

Sulfhydration can occur by the reaction of sulfide with oxidized cysteine residues such as cysteine sulfenic acid or disulfides (Reactions 1 and 2 respectively). Sulfhydration may also occur when an existing persulfide (on either a small molecule or a protein) reacts with a cysteine thiol (Reaction 3). Reaction of H₂S₂ with cysteine thiolates may also lead to sulfhydration (Reaction 4). (B) The modified biotin switch assay. The illustration depicts a protein with unmodified cysteines (-SH), sulfhydrated cysteines (-SSH) and disulfide bonded cysteines (S-S). Purified protein, or cell or tissue lysate, is incubated with methyl methanethiosulfonate (MMTS), to block unmodified cysteines. Unreacted MMTS is then removed by acetone precipitation or by gel filtration followed by treatment of the sulfhydrated protein with biotin-HPDP, which reacts with the protein at the site of sulfhydration. The biotinylated protein is enriched using streptavidin conjugates and analyzed by western blot analysis. (C) Maleimide assay. In this assay, the protein is first immunoprecipitated and treated with a fluorescent version of maleimide, which reacts with thiols under conditions that preserve the native conformation of the protein. After removing excess maleimide, the reaction mixture is treated with dithiothreitol (DTT), which reduces the disulfide bond resulting in the removal of the maleimide and a decrease in fluorescence that can be observed by SDS-PAGE. (D) The tag switch assay. The assay is a variation of the modified biotin switch assay. The reaction mixture is treated with the thiol blocking reagent (BR): methylsulfonyl benzothiazole (MSBT), followed by treatment with a methylcyanoacetate (MCA) derivative that comprises a nucleophilic component and a biotin moiety as a reporter. The biotinylated protein is then captured using streptavidin beads and analyzed by western blotting. The modifications caused by sulfide or its derivatives are shown in red.

Figure 2. Sulfhydration regulates expression of genes involved in mitochondrial metabolism

(A) Interferon regulatory factor 1 (IRF-1) is a transcriptional repressor of the DNA methyltransferase 3a (Dnmt-3a). IRF-1 is regulated by sulfhydration. When H₂S levels and consequently sulfhydration levels are low, IRF-1 is unable to bind its site on the Dnmt-3a promoter, leading to increased expression of Dnmt-3a, which methylates its target promoters, including the mitochondrial transcription factor A (TFAM), leading to reduced mitochondrial biogenesis. (B) When H₂S production is increased, it sulfhydrates IRF-1 and enhances its interaction with the Dnmt-3a promoter to repress its expression. Consequently, methylation of the TFAM promoter is decreased, leading to a higher expression of TFAM and increased mitochondrial biogenesis.

Figure 3. Sulfhydration regulates the expression of phase II cytoprotective genes

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of a battery of genes, including the phase II genes, which respond to stressful conditions such as oxidative stress. Under basal conditions, Nrf2 is sequestered in the cytosol by kelch-like ECH-associated protein (Keap1), which targets it for proteasomal degradation involving Cul3 and E2 ubiquitin ligases. Keap1 has reactive cysteines, whose sulfhydration results in dissociation from Nrf2. Released Nrf2 translocates to the nucleus to regulate transcription of stress-responsive genes.

Figure 4. Sulfhydration in the brain

(A) Sulfhydration is dysregulated in Parkinson’s disease (PD). In normal subjects, the E3 ubiquitin ligase, Parkin, is sulfhydrated under basal conditions, which enhances its catalytic activity. Parkin mediates ubiquitylation of substrates such as α-synuclein (a component of the Lewy bodies found in PD) and targets them for degradation. In sporadic forms of PD, sulfhydration of Parkin is diminished, leading to decreased catalytic activity, which results in accumulation of toxic proteins and neurotoxicity.(B) Sulfhydration regulates synaptic function. The proinflammtory cytokine interleukin-1β (IL-1β, purple circles) plays key roles in learning and memory and is involved in promoting long term potentiation (LTP). IL-1β activates the transcription factor specificity protein 1 (SP1), which stimulates the transcription of cystathionine β-synthase (CBS), the major H₂S producing enzyme in the brain, leading to sulfhydration of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Sulfhydrated GAPDH binds to seven in absentia homolog-1 (siah1), an E3 ubiquitin ligase, which targets post-synaptic density 95 protein (PSD95) for degradation. PSD95 is a scaffolding protein that participates in synaptic functions. Degradation of PSD95 leads to spine retraction and associated cognitive deficits.

Figure 5. Reciprocity of sulfhydration and nitrosylation

Sulfhydration and nitrosylation occur on reactive cysteines and as a result frequently modify the same residue. In general, sulfhydration and nitrosylation are functionally antagonistic although there are examples where both modifications elicit the same outcome. In several instances, sulfhydration precedes nitrosylation. During inflammatory conditions (left), cystathionine γ-lyase (CSE) expression is stimulated to produce hydrogen sulfide (H₂S). H₂S sulfhydrates the p65 subunit of the transcription factor NF-κB and promotes its association with its coactivator, the ribosomal protein S3 (rps3), to enhance expression of cytoprotective genes. If the inflammatory signals persist, the cells produce nitric oxide (NO), which nitrosylates p65 at the same residue and inhibits its DNA binding activity and cytoprotective functions.