Hydrogen sulphide (H2S) started emerging as important signalling molecule, which regulates numerous physiological functions. One of the main signaling mechanisms of H2S is persulfidation, oxidative post-translational modification of cysteine residues. H2S cannot react directly with cysteine residues so the mechanism(s) by which persulfidation can occur are still puzzling. Tristetraprolin (TTP or NUP475) is a non-classical zinc finger protein that regulates the production of cytokines during inflammation, by binding to specific mRNA sequences. TTP contains two Cys3His metal binding domains that can coordinate zinc. The role of H2S in inflammation is still controversial with more studies suggesting its pro-inflammatory effects. Therefore, in this study we addressed the reaction of H2S with Zn2TTP peptide. Aerobic reaction of Zn2TTP with H2S, followed by ultra-high resolution mass spectrometry, led to fast oxidation of cysteine residues with a loss of Zn2+, in a similar fashion like the treatment with H2O2 would do. Surprisingly, the same reaction performed under anaerobic conditions, showed no difference between untreated and H2S-treated Zn2TTP, suggesting important role of oxygen. Indeed when oxygen was added to anaerobic Zn2TTP/H2S mixture, an immediate transient formation of persulfides was observed in MS. In parallel, the same reaction led to superoxide generation and significant conformational change in peptide structure, as detected by fluorescence spectroscopy. We propose that Zn2+ serves as a wire for, otherwise thermodynamically unfavourable, electron-transfer from coordinated HS- to oxygen, leading to transient formation of superoxide and HS. radical. HS. radical is powerful persulfidating agent, while superoxide and its dismutation product H2O2 will lead to cysteine oxidation. The importance of H2S coordination is further confirmed by the observation that aerobic reaction of H2S with RNA-bound Zn2TTP led to no changes when compared to the control. Finally the mRNA levels of TNF-alpha in activated macrophages from control and CSE knockout mice showed higher level of TNF-alpha mRNA levels in control mice, suggesting that endogenous H2S production inhibits TTP in the cells. Physiological and pharmacological importance of this reaction is discussed, as well as general importance of this mechanism for the persulfidation of other Zn2+-metalloproteins.

Persulfidation of tristetraproline by hydrogen sulphide

251st National Meeting (2016)

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