Figure 2: Hydrogen sulfide enhances parkin E3 ligase activity via sulfhydration.

(a) Parkin E3 ligase activity in HEK293 cells is augmented by GYY4137 in a dose-dependent manner, but not by ‘old’ GYY4137, which was exposed to air overnight in PBS, and is subsequently unable to donate H₂S. (b) Parkin activity, measured by target ubiquitination of AIMP2, is stimulated by GYY4137 (100 μM) and by GST–CBS. (c) Ubiquitination by parkin of synphilin-1 is enhanced by treatment with GYY4137 or GST–CBS. (d) Parkin E3 ligase activity in vitro is increased by the addition of 100 μM NaHS and decreased by GSNO. DTT, a reducing agent, returns activity to baseline by reversing sulfhydration or nitrosylation. (e) MPTP influences parkin sulfhydration. Parkin sulfhydration was determined by the maleimide technique in wild-type (WT), nNOS^−/− and iNOS^−/− mice injected with saline or MPTP, and killed at 2, 4, 24 and 48 h after MPTP injection. Following MPTP treatment, parkin sulfhydration in WT mice increases by almost twofold at 4 h before returning to baseline. Sulfhydration levels are substantially greater at 2–4 h in iNOS and nNOS mice (n=3 mice for each data point). Statistical significance is as noted **P<0.01 and *P<0.05 by the ANOVA analysis, with Tukey HSD post-hoc test. All data expressed as mean±s.e.m.