Oxidative stimuli induce an interaction between TRX1-CS and ATM.
(A) Schematic representation of the reaction mechanism of the TRX1-mediated reduction of a disulfide, in which WT TRX1 forms a transient mixed disulfide with a target that becomes rapidly resolved, resulting in the reduction of the target protein and TRX1 oxidation. In contrast, the TRX1 trapping mutant (CS), in which the resolving cysteine (C) is mutated to serine (S), cannot dissolve the mixed disulfide resulting in the stabilization of the TRX1-target intermediate. (B) HEK 293T cells expressing TRX1-CC, TRX1-CS or TRX1-SS were subjected to 10 mM H2O2 for 15 minutes or left untreated. Lysates were prepared in the presence of NEM and TRX1 and proteins were enriched using streptavidin-coated beads. Bound proteins were analyzed by SDS-PAGE and Western blotting. Blots were probed for ATM, PRDX1 and His-tagged TRX1. Vinculin served as loading control. Representative blots of two independent experiments are shown. (C) HEK 293T cells expressing TRX1-CS were subjected to KU55933 (10 μM, 1 hour pre-treatment), Auranofin (4 μM, 4 hours) or H2O2 (10 mM, 15 minutes) or left untreated. Experimental procedures were similar as described in (B). Vinculin and GAPDH served as loading controls. Representative blots of two independent experiments are shown.