NHS · Roles of Hydrogen Sulfide and its Metabolites in Neutrophil Function and Redox Signaling

Roles of Hydrogen Sulfide and its Metabolites in Neutrophil Function and Redox Signaling

contact: peter.nagy@oncol.hu Hydrogen sulfide biology was an emerging field when this grant was awarded. Ever since groundbreaking discoveries made it clear that sulfide is a master regulator in a plethora of physiological processes in all forms of life. Using a mechanistic chemical approach this project investigated the roles of sulfide in inflammatory processes (Objective 1) and in Cys protein regulation (Objective 2). Objective 1: By addressing key features of sulfide solution chemistry we established rigorous protocols to make and handle sulfide solutions. We established a number of sulfide measurement methods in our laboratory and conducted a comprehensive literature review of sulfide detection methodologies. As a result of our analyses we introduced the concept that in biological systems most of the sulfide is bound to biomolecules by a variety of chemical interactions. This biomolecule-bound sulfide pool can serve as an endogenous sulfide buffer/donor system that can liberate sulfide with tightly regulated kinetics. We proposed a role for this sulfide bioavailability in signaling. Systematic kinetic analyses revealed favorable redox reactions between sulfide and a number of Reacting Oxygen Species (ROS). However, due to the low abundance of sulfide compared to protein thiols and glutathione we proposed that the observed antioxidant properties of sulfide are most likely not due to direct ROS scavenging. On the other hand, our kinetic studies on the reactions of sulfide with highly oxidizing metalloenzyme intermediate species revealed that these reactions represent a likely route of sulfide's antioxidant mechanisms. The observed tight regulations of these pathways and the generation of biologically potent inorganic polysulfides suggest that metalloproteins may also play a role in mediating sulfide-signaling. We described the chemical foundations of the cross talk between sulfide and nitric oxide (NO) signaling. We have characterized the three main products of the interactions of sulfide with NO and NO donor molecules and demonstrated their different biological chemical properties in in vitro and in vivo model systems. Finally we managed to show in rat and human neutrophil lysates as well as in inflamed rat colon homogenates that sulfide could potentially work as an anti-inflammatory molecule via a reversible inhibition of myeloperoxidase (MPO) activity. In addition, we could also show the MPO inhibiting potential of sulfide when MPO and its substrate peroxide were produced and excreted upon stimulation of live neutrophils. Objective 2: We have shown that protein Cys sulfhydration can occur via polysulfide-mediated oxidation of Cys residues. Using PTEN and roGFP we revealed that trace amounts of polysulfide contaminations in sulfide solutions can efficiently inhibit the enzymatic activity of thiol proteins via sulfhydrating their functional or regulatory Cys residues. Furthermore, our comprehensive kinetic analyses potentiated the disulfide reducing potential of sulfide in a variety of biological situations. We also developed a novel protocol, which is the first one that can detect protein persulfides in intact cells. We conducted a detailed mechanistic study using purified enzyme systems as well as in live cells to reveal a persulfide reducing enzyme cascade that we believe has a major role in maintaining cellular sulfane-sulfur homeostasis.

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