Cisztein származékok poliszulfidációjának biológiailag releváns mechanizmusai

Abstract

The topic of my PhD research was the mechanistic investigation of signaling pathways mediated by hydrogen sulfide. For a long time, hydrogen sulfide was considered as a noxious gas due to the inhibition of mitochondrial energetics via blocking cytochrome–c–oxidase. Surprisingly, then it was found to be produced in vivo via enzymatic and non enzymatic pathways and accumulating evidence indicated its regulating role regarding biochemical pathways. These discoveries corroborated that sulfide plays a key role in numerous physiological and patophysiological processes due to mediating intracellular signaling pathways, resulting that now sulfide is regarded as a gasotransmitter, like NO or CO. During my research work I studied the per- and polysulfidation reactions of cysteine residues, focusing on their detection, which is still quite challenging. A comprehensive study was focused on the recent detection techniques, considering their advantages, disadvantages and limitations. The impact of the nature of the applied electrophile agent and experimental conditions like concentration and incubation time was thorougly studied with respect to three commonly used, cell permeable alkylating agents, and these parameters were found to have a significant effect on the detected speciation due to shifting the dynamic redox equilibrium reactions of reactive sulfur species as well as due to cleavage of longer chain polysulfides. Polysulfides as signaling molecules are also produced during the cross-talk of signaling events mediated by sulfide and nitric–oxide, due to the formation and decomposition of a hybrid S/N intermediate called nitrosopersulfide (SSNO‒). Experiments were carried out focusing on the chemical characterization of this species, as well as studying its decomposition reactions and its biochemical effects on several biologically relevant models. Kinetic data suggested that nitrosopersulfide is resistent towards the reducing capacity of the thioredoxin and glutahione dependent eznymatic systems and can function as a sustained polysulfide donor, inducing slow per- and polysulfidation reactions, thus playing a key role in signaling processes. Experiments on reduced glutathione, human serum albumine and HEK293 cells corroborated the delayed polysulfidating effects of SSNO‒ via a slow production of HSx̶ during its decomposition. Flow cytometry experiments indicated that transient receptor potential ankyrin 1 (TRPA1) receptors are also activated by inorganic polysulfides, which are released during the slow decomposition of nitrosopersulfide. Regarding the results mentioned above, I believe that my doctoral research work contributed to the deeper understanding of the molecular mechanisms of sulfide mediated signaling pathways.