Mitochondrion is considered as the major source of intracellular reactive oxygen species (ROS). in SH-SY5Y cells or in the mice cortex. H2S also decreased mitochondrial ROS production and protected neuronal cells against stress-induced senescence. PKCβII and PP2A are the two key proteins to regulate p66Shc phosphorylation. Although H2S failed to affect the activities of these two proteins it disrupted their association. Cysteine-59 resides in proximity to serine-36 the phosphorylation site of p66Shc. The C59S mutant attenuated the above-described biological function of H2S. We revealed a novel mechanism for the antioxidant effect of H2S and its role in oxidative stress-related diseases. H2S inhibits mitochondrial ROS production the sulfhydration of Cys-59 residue which in turn prevents the phosphorylation of p66Shc. a p66Shc-dependent mechanism. H2S sulfhydrated p66Shc at cysteine-59 which resides in proximity to the phosphorylation site serine-36. Sulfhydration of p66Shc further impaired the association of PKCβII and p66Shc and attenuated H2O2-induced p66Shc phosphorylation a critical step in p66Shc-mediated mitochondrial ROS generation. This was further confirmed in the D-galactose-induced aging model. Thus we revealed in the present study a novel mechanism for the antioxidant effect of H2S and its role in PF-04217903 oxidative stress-related diseases. An emerging aspect of H2S signaling is the pathway mediated by protein sulfhydration. This H2S-induced posttranslational modification has been confirmed to regulate the function of a large number of proteins such as the potassium channels (like KATP IKca and SKca) (19) PTP1B (10) NF-κB (27) and Keap1 (38). It was believed that the conserved cysteine residue at the key PF-04217903 point holds the key (23) to the sulfhydration. Structure analysis revealed that p66Shc also contains a unique conserved cysteine residue which locates at position 59 (Cys-59) in the CH2 domain (5). We thereby hypothesized that the conserved Cys-59 was also subject to S-sulfhydration by H2S and this modification would provide a mechanism for the regulation of H2S on p66Shc function. The study presented here was designed to examine the effect of H2S on p66Shc and its role in mitochondrial ROS production. Results H2S alleviates H2O2-induced mitochondrial ROS production in SH-SY5Y neuroblastoma cells The first step of our experiments is to confirm the effect of H2O2 on mitochondrial oxidative stress. We measured the mitochondrial ROS generation using a selective fluorescence indicator MitoSOX? Red mitochondrial superoxide indicator (Molecular Probes). As shown in Figure 1A treatment of SH-SY5Y neuroblastoma cells with different concentrations of H2O2 (0-200?μincreased the mitochondrial ROS level in a time-dependent manner (Fig. 1B). In contrast pretreatment with NaHS (an H2S donor 100 binding to mitochondrial complex III. Interestingly NaHS failed to affect antimycin (10?μH2O2 for 20?min significantly increased PF-04217903 the level of p66Shc Ser-36 phosphorylation. This effect was concentration dependently reversed by exogenous application of NaHS (1-100?μinduced p66Shc sulfhydration in a concentration-dependent manner (Fig. 3A). This effect was almost completely abolished by 2?midoacetamine a sulfhydryl-reactive alkylating reagent which binds covalently with the thiol group in the cysteine residues to prevent disulfide bond formation (Fig. 3A). The similar effect was also observed in CBS overexpressed SH-SY5Y cells (Fig. 3B). FIG. 3. H2S-induced p66Shc sulfhydration at cysteine-59 and its effects on mitochondrial ROS generation. (A) NaHS concentration dependently induced p66Shc sulfhydration in SH-SY5Y cells. This was largely abolished by idoacetamine (IA) a sulfhydryl-reactive alkylating … To identify the sulfhydrated cysteine PF-04217903 residue of p66Shc the conserved cysteine-59 was mutated to serine (C59S) (Fig. 3C). It was found that the Cys-59 mutation markedly attenuated the sulfhydration of p66Shc induced by NaHS PF-04217903 (Fig. 3D) suggesting CD127 the critical role of Cys-59 in H2S-induced p66Shc sulfhydration. Meanwhile the C59S mutation also significantly eliminated the inhibitory effect of H2S on H2O2-induced p66Shc phosphorylation (Fig. 3E). These data showed that H2S-induced sulfhydration contributes to its inhibitory effect on p66Shc phosphorylation. To link the Cys-59 sulfhydration of p66Shc to its function on mitochondrial oxidative stress we thereby examined the effect of H2S.