Fig. 3

Parkin mediates the recycling of oxidized to reduced glutathione, resulting in its own S-glutathionylation. a Silver staining of recombinantly expressed maltose binding protein (MBP; tag only) and MBP-tagged, human parkin proteins separated on SDS/PAGE under reducing conditions. b–d Fluorescence-based quantification of eosin (E)-labelled GSH following incubation of full-length (FL) MBP-parkin (b), MBP-IBR-RING2-parkin (c), or untagged, recombinant (r-) human parkin (d) with 20 mM Di-E-GSSG, monitored over 10 min; (n = 2 runs (a, b, c) in triplicate wells). e Quantification of free GSH levels, as measured in the monochlorobimane assay, following incubation of indicated levels of untagged glutathione (mM) at various GSH:GSSG ratios in the presence of 1 mM of untagged, full-length, human r-parkin (n = 3 ± SEM). A 1-way ANOVA with Dunnett’s post-hoc test was used to compare all values to r-parkin incubated with 10 μM GSH, where *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. f In vitro S-glutathionylation studies of recombinant parkin, where preparations of 10 mM MBP-IBR-RING2 parkin were treated with 20 mM Di-E-GSSG followed by SDS/PAGE (lanes 1 and 2; both panels). Deglutathionylation studies in the presence of 5 mM DTT alone or in the presence of either 1 mM glutaredoxin 1 (Grx1) or Grx2 (together with: 1 mM NADPH; 5 mM GSH; 0.1 mM glutathione reductase), as indicated (lanes 3–5). Left panel shows a transilluminated gel; the right panel a Coomassie-stained gel. Results are representative of three independent experiments. g (upper) Schema of streptavidin-based enrichment of cellular biotin-labelled S-glutathionylated myc-parkin following treatment of cells with biotin-tagged GSSG (BioGEE) where S-glutathionylated proteins elute from the streptavidin beads in the presence of DTT and are then resolved by SDS-PAGE. (lower) Western blot of S-glutathionylated myc-parkin isolated from CHO-parkin cells either untreated (−) or treated with (+) BioGEE (20 µM; 3 h). Both were exposed to 1 mM H₂O₂ for 10 min prior to lysis. A fraction of input lysate is shown for each condition; high (5 µg) and low (2 µg). h,i Examples of LC–MS/MS-generated spectra following trypsin digestion of MBP-parkin proteins incubated with Di-E-GSSG showing S-glutathionylation (as in f) are shown at two residues: (h) identification of residue Cys59 within human parkin peptide aa 52–75, and in (i) of Cys95 within human parkin peptide aa 90–104