Protein S-sulfhydration as a major sources of H 2 S bioactivity Guangdong

The physiological and biomedical importance of hydrogen sulfide (H2S) has been extensively studied in our body. H2S can be endogenously produced in a variety of cells and tissues by cystathionine γ-lyase, cystathionine β-synthase, and/or 3-mercaptopyruvate sulfurtransferase, and is involved in the regulation of vascular function, cell growth, insulin secretion, neurotransmission, myocardial contractility, inflammation, and nociception, etc.H2S posttranslation ally modifies proteins by yielding a hydropersulfide moiety (–SSH) in specific cysteine residue(s), termed as S-sulfhydration. It is becoming increasingly recognized that S-sulfhydration is a major sources of H2S bioactivity. In this research highlight, we discuss our latest published findings which demonstrate the S-sulfhydration regulation of proteins by H2S and their importance in aging and cancer protection.

In 2002, Wang first created and defined the word "gasotransmitter", and described the criteria for being a gasotransmitter [1] .Hydrogen sulfide (H2S), joining with nitric oxide (NO) and carbon monoxide, is important member in gas transmitter family [1,2] .Not only from environment and industry activity, H2S can also be endogenously produced in our body by specific enzymes, including cystathionine γ-lyase, cystathionine β-synthase, and/or 3-mercaptopyruvate sulfurtransferase, which are expressed or can be induced in most of cell types [3][4][5] .H2S can freely penetrate into cell membrane independent of any cognate membrane receptors or other transportation machineries [1] .Accumulated evidence demonstrated that H2S is almost involved in all life functions, including cell growth, glucose metabolism, insulin secretion, neurotransmission, myocardial contractility, inflammation, energy generation, and redox balance, etc [2]   .Despite a wealth of recent publications suggest the important path physiological roles for H2S, its molecular mechanisms of action remains poorly defined.It is proposed that the major signaling mechanism of H2S is through the S-sulfhdyration of reactive cysteine residues on target proteins by yielding a hydropersulfide moiety (-SSH), with the potential to confer a functional changes, which is analogous to S-nitrosylation of proteins by NO [5] .
Multiple cellular functions are controlled through the post-translational modification of proteins, including phosphorylation, glycosylation, ubiquitination, methylation, and S-nitrosylation, etc. Post-translational modifications are key mechanisms to increase proteomic diversity and serve to sense and transduce cellular signals in a precisely coordinated manner [6] .The amino acid cysteine in protein is quite reactive, and oxidative modification of cysteine residues is an important mechanism that regulates protein structure and ultimately functions, including S-nitrosylation, S-glutathionylation, and S-sulfenylation, etc [6] .Since the first paper describing H2S S-sulfhyration of protein in 2009 [5] , there have been a dozen of proteins observed to be modified by H2S through S-sulfhydration, as demonstrated by modified biotin switch assay and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Table 1) [3][4][5] .It is predicted that, by change in electrostatic environment, hydrophobicity, contiguity and orientation of aromatic side chains, and proximity of target thiols to transition metals or redox centres, S-sulfhydration can alter protein conformation and the final function and activity of target proteins [16,21] .

RESEARCH HIGHLIGHT
It is well known that H2S at physiological relevant concentrations suppresses oxidative stress and protects from cell death.The relative low concentration and the small molecular weight of H2S make it hard to directly scavenge reactive oxygen species.Our recent paper discovered that H2SS-sulfhydrates Keap1, which subsequently stimulates Nrf2 nuclear translocation and induces anti-oxidant gene transcription and glutathione production.Deficiency of H2S enhanced oxidative stress and promoted cellular aging [12] .Nrf2 is a master transcription factor that regulates the expressions of a group of antioxidant genes.Nrf2 activation can be attributed to the dissociation from Keap1 through posttranslational modulation of crucial cysteine residues in Keap1 protein.In normal condition, Keap1 is basically Ssulfhydated, and exogenously applied H2Sincreases but removal of endogenous H2S reduces Keap1 Ssulfhydration.The cysteine residues 151 in Keap1 BTB domain is required for H2S-mediated S-sulfhydration.H2S interacted with cysteine residues 151 leading Keap1 conformational change, which triggerNrf2 release from Keap1 following its nuclear translocation and anti-oxidant gene transcription.H2S-induced activation of Nrf2 is not caused by increased Nrf2 protein expression, Nrf2 phosphorylation, or Nrf2 S-sulfhydration.
More recently, we made another discovery that H2S effectively inhibits androgen receptor (AR) transactivation by S-sulfhydrating both cysteine 611 and 614 located in the DNA binding domain (DBD) of AR [16] .AR signaling is essential for the normal development and functions of prostate as well as the initiation and progression of prostate cancer.AR is a nuclear receptor, which is often activated as DBDs bind as dimmers to two hexameric sequences orientated as direct or inverted repeats.The interaction of H2S and AR through both cysteine611 and 614 may destroy zinc-sulfur cluster and cause a structural change in AR-DBD, triggering abnormal AR dimerization and DNA binding ability.It is still unclear how H2S S-sulfhydration of cysteine-611/614 alters AR zinc-finger structure and dimerization.H2S suppressing AR downstream gene expression were not due to the alteration of AR interaction with heat shock proteins and AR nuclear localization (Figure 1).We further observed that glucocorticoid receptor and estrogen receptor α, another two hormone receptors containinghighly identical DBD as AR, are not S-sulfhydrated by H2S, indicating the specificity of cysteine S-sulfhydration by H2S in target proteins.Maintenance of sufficient level of H2S could effectively inhibit ant androgen-resistant growth of prostate cancer cells.Based on these discoveries, H2S can not only serve as a valuable prognosis indicator but also an effective therapeutic target for treatment of both early state of prostate cancer and castration-resistant prostate cancer.
In summary, our studies reveal that protein Ssulfhydration serves an important role in a wide range of H2S-mediated signaling pathways, including protein activity, localization, stability, and interaction, and stress response.Compared with the large number of Snitrosylated proteins which have been identified, the observed S-sulfhydrated proteins and the specificity of target cysteine are incompletely understood.Protein desulfhydration/trans-sulfhydration, the removal or transfer of SH group from cysteine thiol side in proteins, is very important but never reported so far.It is also not clear whether S-sulfhydration or de-sulfhydration/ transsulfhydration is spontaneous and unregulated, or catalyzed by enzymes and other cellular constituents [21] .Similar to S-nitrosylation, the formation or removal of an individual S-sulfhydration can depend on many factors including the reactivity of the individual cysteine residues, its surrounding environment, and the composition of the local redox-environment, etc. Advances in the detection of Ssulfhydration will facilitate research directed at the identification of S-sulfhydrated proteins in both health and diseases.There is a long way to fully understand the stories of S-sulfhydrationand de-sulfhydration/transsulfhydration, but I believe we do have a bright future.

Figure 1 .
Figure 1.S-sulfhydration regulation of androgen receptor (AR) by H2S.H2S represses AR transactivation by S-sulfhydrating cysteine residues 611/614 located in the DNA binding domain (DBD).AR S-sulfhydration leads to reduced AR dimerization following lower AR binding with the promoters of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes, while AR Ssulfhydration does not affect heat shock protein (HSP) 70 and 90 interaction with AR and AR nuclear translocation.

Table 1 . S-sulfhydrated proteins and their functional changes S-sulfhydrated proteins/ electrophiles Tissue/cell types Outcome Targeted cysteine residues Detection methods
Note: BSA, biotin switch assay; TSA, tag-switch assay; MS, mass spectrometry