Cysteinylation of peroxiredoxin 2 upon incubation with BODIPY® FL L-Cystine
It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteostatic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knock-out mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionary conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice and humans. Cysteinylation of recombinant human Prx2 (hPrx2) Protein cysteinylation was achieved in vitro upon incubation of purified hPrx2 with BODIPY™ FL L-Cystine (Thermo Fisher, Massachusetts, USA). hPrx2 samples were pre-reduced with 1 mM DTT for 30 min at room temperature (RT), and then desalted using a Zeba™ Spin Desalting Column, 40 K MWKO, 0.5 mL (Thermo Fisher, Massachusetts, USA) and incubated with 100 µM BODIPY™ FL L-Cystine overnight at 4°C protected from light. After incubation, the remaining non-bound BODIPY™ FL L-Cystine was removed by desalting. Protein samples were loaded into a gel (Novex™ 4-20% Tris-Glycine, Invitrogen, California, USA) using non-reducing loading buffer (NuPAGE™LDS Sample Buffer, Invitrogen, California, USA) to conserve the mixed disulfide formed in the previous step. Electrophoretic separation was performed at a constant voltage of 200 V for 20-25 min while the gel was protected from light. Once resolved, the gel was developed in a UV transilluminator (ChemiDoc™ XRS+ Imaging System, Bio-Rad, California, USA) and Coomassie staining was later performed as loading control.