Abstract
Recently, we demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). Because cysteine residue(s) in IDPm are susceptible to inactivation by a number of thiol-modifying reagents, we hypothesized that IDPm is likely a target for regulation by an oxidative mechanism, specifically glutathionylation. Oxidized glutathione led to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the cysteine residue(s) in IDPm, which was detected by immunoblotting with anti-GSH IgG. The inactivated IDPm was reactivated enzymatically by glutaredoxin2 in the presence of GSH, indicating that the inactivated form of IDPm is a glutathionyl mixed disulfide. Mass spectrometry and site-directed mutagenesis further confirmed that glutathionylation occurs to a Cys(269) of IDPm. The glutathionylated IDPm appeared to be significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion, suggesting that glutathionylation plays a protective role presumably through the structural alterations. HEK293 cells and intact respiring mitochondria treated with oxidants inducing GSH oxidation such as H(2)O(2) or diamide showed a decrease in IDPm activity and the accumulation of glutathionylated enzyme. Using immunoprecipitation with anti-IDPm IgG and immunoblotting with anti-GSH IgG, we were also able to purify and positively identify glutathionylated IDPm from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model for Parkinson's disease. The results of the current study indicate that IDPm activity appears to be modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.
Highlights
The initial cellular response to oxidative stress is often a reduction in the levels of GSH, which represents the major low molecular weight antioxidant in mammalian cells, and a corresponding increase of GSSG, the oxidized form of GSH [1,2,3]
The oxidation of a limited amount of GSH to GSSG can dramatically change this ratio and affect the redox status within the cell. Under these conditions of moderate oxidative stress, thiol groups of intracellular proteins can be modified by the reversible formation of mixed disulfides between protein thiols and low molecular mass thiols such as GSH, a process known as S-glutathionylation [5]
NADPH is an essential cofactor for the regeneration of GSH, the most abundant low molecular mass thiol in most organisms, by glutathione reductase in addition to its critical role for the activity of the NADPH-dependent thioredoxin system [18, 19]
Summary
Materials—Isocitrate, -NADPϩ, NADPH, and IDPm from pig heart and GSH, GSSG, cysteine, DTT, hydrogen peroxide, chymotrypsin, Pronase, trypsin, N-ethylmaleimide (NEM), diamide, rose bengal, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 8-anilino-1naphthalene sulfonic acid (ANSA) were purchased from Sigma. For glutathionylatin detection by immunoblot, IDPm was mixed with 5ϫ SDS sample buffer, without reducing reagents, supplemented with 5 mM NEM to block unreacted thiol groups, and subjected to SDS-PAGE followed by electroblotting onto nitrocellulose membranes. Supernatants were incubated with rabbit polyclonal anti-IDPm (5 g) for 12 h at 4 °C followed by protein A-Sepharose incubation for 1 h at 4 °C. Structural Analysis—For CD spectroscopy, samples of IDPm were desalted on Econo-Pac 10 DG column (Bio-Rad) equilibrated in 20 mM Tris buffer, pH 7.4, and fractions containing the protein were pooled. In order to prepare recombinant proteins, Escherichia coli transformed with pET14b containing the cDNA insert for mouse IDPm or mutant IDPm (K212T) construct was grown and lysed, and His-tagged proteins were purified on nickel-nitrilotriacetic acidagarose as described elsewhere [23]. Control Experiments—In order to evaluate artifactual intrapreparative glutathionylation, IDPm was purified from tissue/cell homogenates treated with 3 Ci of [3H]GSH
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