In the present study, we have demonstrated that parkin is physiologically sulfhydrated, a process that enhances its ubiquitination activity. This contrasts markedly with nitrosylation, which decreases such activity. Sulfhydration and nitrosylation of parkin appear to be reciprocal events. This may be related to the difference in chemical reactivity between the two modifications. Nitrosylated cysteines will present a distinctly different chemical group to the local environment than will sulfhydration. In patient striatum, we demonstrate major decreases in parkin sulfhydration, which are reciprocal to the increases in nitrosylation.
What might be the comparative roles of parkin nitrosylation and sulfhydration in the pathophysiology of PD? One possibility is that the cell stress of PD leads to increased generation of NO, which accounts for the augmented nitrosylation of parkin in patient brain. Cell stressors do lead to activation of both iNOS and nNOS, and increased S-nitrosylation of several proteins, such as parkin, protein-disulfide isomerase, and XIAP, has been speculated to impact PD17–19
. However, increased NO formation in PD has not been directly demonstrated, and measurements of H2
S generation in PD are lacking. Nitrosylation and sulfhydration are sensitive and specific reflections of the presence of ambient NO and H2
S respectively, implying that altered nitrosylation/sulfhydration in PD brain reflects changes in levels of the two gasotransmitters.
Our experiments also establish cytoprotective actions of H2S donors, which appear to reflect sulfhydration of parkin. Mutation of C95, a principal site of parkin sulfhydration, largely prevents the protective influences of H2S donors indicating that the donors act via parkin sulfhydration to enhance its ubiquitination activity. C182 and C59 also appear to contribute to influences of H2S donors upon parkin. We could not evaluate effects of H2S on C212 and C377, as they did not express or were unstable in our cell lines. Interestingly, C95 occurs in human but not rodent parkin, while the other sulfhydrated cysteines are conserved in rodent and human species.
The beneficial effects of H2
S donors in PD models may have therapeutic implications. H2
S donors have already been noted to be beneficial in rodent models of PD20–22
, and an H2
S donating variant of L-DOPA has shown promising effects in cellular models of PD23
. These influences had been speculated to reflect general antioxidant and anti-inflammatory actions of H2
S. However, numerous studies have failed to reveal a beneficial effect of antioxidants in PD24,25
. Our findings provide a specific molecular mechanism whereby H2
S therapy may benefit PD and can explain the ineffectiveness of generalized antioxidant treatment. Modifications of parkin and other interactors of parkin may offer promise in the therapy of PD15,26,27
so that H2
S donors selectively targeted to parkin may provide notable benefit.