Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and β-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db / db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 μM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin–protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αβ-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. Abbreviations: C2SC, cysteine succination by fumarate; CID, collision-induced dissociation; CTT, C-terminal tail of tubulin; DMF, dimethylfumarate; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTPA, diethylenetriaminepenta-acetic acid; GI, gastrointestinal; 4-HNE, 4-hydroxynonenal; MAP, microtubule-associated protein; NEM, N-ethylmaleimide; PE, pyridylethylation; PTM, post-translational modification; RIPA, radioimmunoprecipitation assay; 2SC, S-2-(succino)cysteine; TFA, trifluoroacetic acid; TOF, time-of-flight
