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The Elucidation of Hydrogen Sulfide Signalling Through Persulfidation

Abstract : Hydrogen sulfide (H2S), originally considered a toxic gas, is now a recognised gasotransmitter. Numerous studies have revealed the role of H2S as a redox signalling molecule that controls important physiological/pathophysiological functions. The underlying mechanism postulated to serve as an explanation of these effects is protein persulfidation (P-SSH, also known as S-sulfhydration), an oxidative posttranslational modification of cysteine thiols. Protein persulfidation has remained understudied due to its instability and chemical reactivity similar to other cysteine modifications, making it very difficult to selectively label. Herein, we developed a novel, versatile, two-step chemoselective method for the detection and labelling of protein persulfides, called the Dimedone-switch method. We confirmed the method’s kinetics and selectivity, and showed that protein persulfidation is an evolutionarily conserved modification controlled by H¬2S generated by transsulfuration pathway and cysteine catabolism. We adapted the method for direct in-gel detection in different model organisms, fluorescence microscopy, antibody microarray approach and proteomic analysis by mass spectroscopy.Next, we studied the role of H¬2S in redox signaling through persulfidation. To do this we investigated the interconnection between P-SSH and the sequential modifications of cysteine thiols, sulfenylation (P-SOH), sulfinylation (P-SO2H) and sulfonylation (P-SO3H), formed when exposed to oxidative stress (reactive oxygen species). Our studies showed a direct correlation between P-SSH and these modifications in a time- and dose- dependent manner. We observed a clear phase shifted response between the two cysteine modifications, P-SSH and P-SOH, revealing the presence of ‘protective waves’ of protein persulfidation. Coupled with mechanistic studies showing the efficient reduction of P-SSH by the thioredoxin system, these results suggest that protein persulfidation is the main pathway by which sulfenic acids are resolved under oxidative stress. As such, we proposed a general mechanism (potentially an evolutionary remnant of the times when life emerged and flourished in a H2S environment) in which persulfidation represents a rescue loop from cysteine overoxidation and subsequent oxidative cellular damage.Furthermore, in order to shed light on the biological relevance of this protective nature of persulfides, we explored a possible correlation between persulfide levels and aging. This was explored through a range of studies, from the persulfide’s chemical ability to scavenge the build-up of oxidants, to gaining a better understanding of the role of transsulfuration pathway in stress resistance and lifespan. We observed a direct correlation between ability to make persulfides and oxidative stress resistance, and a decrease in persulfidation with aging, in C. elegans, rats and human cells.
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Emilia Kouroussis. The Elucidation of Hydrogen Sulfide Signalling Through Persulfidation. Other. Université de Bordeaux, 2019. English. ⟨NNT : 2019BORD0435⟩. ⟨tel-02872217⟩

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