Identification of protein succination as a novel modification of tubulin

Abstract

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 (α) and beta (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 dimethylfumarate (DMF, 500 μM) inhibited polymerization up to 35% and 59%, respectively. Using mass spectrometry we identified Cys347α, Cys376α, Cys12β and Cys303β 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-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose vs. normal glucose also had reduced reactivity with the anti-αtubulin antibody; suggesting that succination may interfere with tubulin:protein interactions. DMF reacted rapidly with 11 of the 20 cysteines in the αβ 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 that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

“…One-dimensional PAGE and Western Blotting-Western blotting to probe for protein succination (2SC), Ndufs4, HNE, VDAC1, VDAC2, and tubulin was performed as described previously, after separation of the proteins by SDS-PAGE (2,12). For glutathione (GSH)-modified proteins, reducing agents were omitted during sample preparation and gel electrophoresis.…”

“…Two-dimensional Gel Electrophoresis-Isoelectric focusing on pI 4 -7 11 cm strips and two-dimensional (2D) gel electrophoresis was performed as described previously (2,12). The 2D gels were stained for protein using Coomassie brilliant blue (ProtoSafe Blue; National Diagnostics, Atlanta, GA) or transferred onto PVDF membranes to detect protein succination, VDAC1, VDAC2, DJ-1 and tubulin by Western blotting.…”

“…At the end of this period, the remaining cells were expanded and differentiated into neurons in the presence of 2% FBS, 1.25% DMSO in DMEM in addition to 1 g/ml puromycin for 5 days. The cells were harvested in RIPA as described previously (12) and the levels of fumarase (to assess efficiency of shRNA knockdown) and protein succination were determined by immunoblotting (described below). In a separate experiment the levels of fumarate were determined by GC-MS (described below).…”

A computational analysis of S-(2-succino)cysteine sites in proteins

“…Furthermore, although the prediction/identification of 2SC is a crucial step in the effort of deciphering the pathophysiological role of protein succination, functional data should be also collected in future studies. Indeed, to date only in few cases the effects of protein succination have been established [12,13,29,48]. Functional data could not only give an insight into the role of protein succination as a new thiol-switch mechanism [49,50], but also reveal unknown functional sites to regulate protein function, and functional protein systems [4] targeted by FA.…”

Insulin Age-Dependently Modulates Synaptic Transmission and AMPA Receptor Trafficking in Region CA1 of the Rat Hippocampus

“…NC membranes were then washed with TBST and developed using enhanced chemiluminescence reagents (GE Healthcare, Pittsburgh, PA) as described by the manufacturer. After the initial probing, NC membranes were stripped as previously described [25] and later probed with an antibody for actin (or total Akt for blots probed with pAkt antibodies), for normalization purposes. Computer assisted microdensitometry of autoradiographic images was determined using the MCID image analysis system (Imaging Research, INC., St. Catherines, Canada), as previously described [22].…”