Fig. 3.

S-nitrosylation of TDP-43 at Cys173 or Cys175. (A) SH-SY5Y cells transfected with V5-tagged WT TDP-43 or Cys to Ala mutants (C173A, C175A, C173A/C175A, C198A, and C244A) were exposed to SNOC. Control cells were exposed to old SNOC, from which NO had been dissipated. After 30 min, SNO-TDP-43 was assessed by biotin-switch assay. Left: Immunoblotting with anti-V5 antibody. Right: Quantification of SNO-TDP-43 levels (n = 4). Graph indicates mean + SEM; *P = 0.0066 by one-way ANOVA, followed by Bonferroni’s post hoc test. (B) SNOC increases accumulation of insoluble TDP-43 aggregates. SH-SY5Y cells were transfected with V5-tagged WT TDP-43 or TDP-43(C173A/C175A) mutant, and exposed to SNOC (200 μM). RIPA insoluble TDP-43 was detected by immunoblot with anti-V5 antibody. (C) Domain structure of human TDP-43. The positions of Cys173 and Cys175 are shown within the RRM1 RNA recognition motif. (D) Structure of TDP-43 RRM domains. Left: Crystallographic structure of TDP-43 RRM domains (PDB ID: 4BS2) with position of Cys residues 173 and 175 (magenta). Right: Snapshot of the TDP-43 RRM structure containing an intramolecular disulfide bond between Cys173 and Cys175 after molecular dynamics simulation.