(A) HEK293 cells transfected with HA-SUMO1, 2 or 3 were treated with 500 µM GSNO for 4 h, lysed in Laemmli buffer, and immunoblotted with αHA or α actin. Asterisk: free SUMO1/2/3. (B) Time-dependent effect of GSNO on SUMO1 conjugates in transfected HEK293 cells, treated as in (A). In a control experiment, cells were treated with H₂O₂ (1 mM) for 1h. (C) HeLa cells were treated with increasing concentrations of GSNO for 4 h, lysed in Laemmli buffer, and immunoblotted with αSUMO1, αubiquitin or αactin. (D) RAW264.7 cells pretreated with or without SMT were stimulated with LPS for 24 h, lysed in Laemmli buffer, and immunoblotted with αSUMO1, αiNOS or αactin.

(A) S-nitrosation of HA-Ubc9 (wild-type or cysteine mutants) in HEK293 cells stimulated with 500 µM GSNO determined by biotin-switch assay. (B) HeLa cells were incubated with 500 µM H₂O₂ for 30 min or 500 µM GSNO for the indicated time, lysed in Laemmli buffer with (lower blot) or without (upper blot) DTT, and immunoblotted with αUbc9. (C) HEK293 cells were transfected with the indicated plasmids, treated with H₂O₂ (for 1h) or GSNO (for 4 h), lysed in Laemmli buffer, and immunoblotted with αp53 or αactin.

(A) HeLa cells were treated with increasing concentrations of GSNO for 4 h, lysed in Laemmli buffer and immunoblotted with the indicated antibodies. (B) Immunoblotting analysis of endogenous Pias3 and SUMO1 conjugates in HeLa cells transfected with non-specific, mismatched or Pias3-specific siRNAs.

(A) HeLa cells expressing His-Ub were stimulated with or without GSNO for 4 h and then subjected to His-Ub pull down. Both longer and shorter exposures are shown. Bracket: poly-ubiquitinated Pias3; Arrow: mono-ubiquitinated Pias3. (B) Effect of GSNO on Pias3-Trim32 interaction determined by GST-pull down assay. GST-Pias3 fusion proteins, immobilized on beads, were mixed with cell lysates with GFP-Trim32 expression in the absence or presence of 500 µM GSNO. Pull-downed and input GFP-Trim32 was analyzed by immunoblotting. (C) Confocal microscopic analysis of subcellular localization of Myc-Pias3 and GFP-Trim32 in cotransfected HEK293 cells. 4 h after transfection, cells were incubated with or without LMB for 16 h. (D) HeLa cells transfected with or without GFP-Trim32 were stimulated with or without GSNO for 4 h and then the denatured lysates were immunoprecipitated with αPias3, followed by detection for Ub. Bracket: poly-ubiquitinated Pias3; Arrow: mono-ubiquitinated Pias3; Asterisk: unmodified Pias3. (E) HEK293 cells transfected with the indicated plasmids were stimulated with or without GSNO for 1 h and then subjected to His-Ub pull down. Bracket: ubiquitinated GFP-Trim32. In panels (A), (C), (D) and (E), cells were incubated with MG132 to prevent potential protein degradation.

(A) S-nitrosation of GFP-Pias3 in HEK293 cells stimulated with 500 µM GSNO determined by biotin-switch assay. (B) Recombinant Pias3 was exposed to 100 µM GSNO and blocked with MMTS. In-gel digested Pias3 was subjected to Nano-LC-MS/MS analysis. (C) Effect of GSNO on Pias3(C459S)-Trim32 interaction determined by GST-pull down assay.

Top: In a normal redox environment, Ubc9 conjugates SUMO to the substrate with the help of E3 ligase. Middle: Oxidative stress leads to formation of a disulfide bond between the E1 subunit Uba2 and the E2 subunit Ubc9, resulting in inactivation of both E1 and E2 enzymes. Below: Under nitrosative stress, both Ubc9 and Pias3 are S-nitrosated. Whereas S-nitrosation of Ubc9 cannot interfere with its catalytic activity, S-nitrosation of Pias3 facilitates its degradation by promoting its interplay with Ub E3 ligase Trim32, thereby resulting in decrease of SUMO conjugating efficiency. Su, SUMO; S, substrate protein.