Identification of Protein Succination as a Novel Modification of Tubulin

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). We demonstrate that both alpha (a) and beta (b) 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 dimethylfumarate (DMF, 500 μM) inhibited polymerization up to 35% and 59%, respectively. Using mass spectrometry we identified Cys347a, Cys376a, Cys12b and Cys303b as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteines increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-a-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose vs. normal glucose also had reduced reactivity with the anti-atubulin antibody; suggesting that succination may interfere with tubulin:protein interactions. DMF reacted rapidly with 11 of the 20 cysteines in the ab tubulin dimer, decreased the number of free sulfhydryls and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggests 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...
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Cell Source Type: research