S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-Nitrosation and S-Thiolation

Abstract

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Biosynthetic and Iron Metabolism Is Regulated by Thiol Proteome Changes Dependent on Glutaredoxin-2 and Mitochondrial Peroxiredoxin-1 in Saccharomyces cerevisiae

“…Although this would have to be demonstrated for each enzyme, a number of them have already been reported in the literature as redox-sensitive, reinforcing the validity of our thiol proteome screening results. For instance, the human BCATs, analogous to Bat1p and Bat2p, are inactivated by both S-nitrosation and S-glutathionylation, where activity in the latter can be recovered by glutaredoxin/glutathione reductase system (27). Gln1p Cys 160 forms part of the regulatory citrate binding site (28), and we find this Cys as a specific Prx1p target.…”

“…"Other redox-modified proteins" are either "doubly specific" or "synergic specific" proteins. The function of the following proteins has been shown to be redox-sensitive in other studies (references included): Aro4p (31); Bat1p and Bat2p (27); Dys1p (29); Gln1p (28); Ilv3p, Leu1p, and Lys4p (30); Hem12p (32); Sam1p (33, 34); Rpe1p and Tkl1p (34). Proteins identified were as follows: Aah1p (adenine deaminase), Ade17p (Enzyme of "de novo" purine biosynthesis), Adi1p (acireductone dehydrogenase), Aro4p (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase), Asn2p (glutamine-dependent asparagine synthetase), Bat1p (mitochondrial amino acid transferase), Bat2p (cytosolic branched-chain amino acid aminotransferase), Dys1p (deoxyhypusine synthase), Fcy1p (cytosine deaminase), Fsh1p (serine hydrolase 1), Gdh1p (NADP-dependent glutamate dehydrogenase 1), Gln1p (glutamine synthetase), Hem12p (uroporphyrinogen decarboxylase), Hom6p (homoserine dehydrogenase), Ilv3p (dihydroxydehydratase), Ilv5p (acetohydroxyreductoisomers), Ilv6p (regulatory subunit acetolactate synthase), Leu1p (isopropylmalate dehydrogenase), Lys4p (homoaconitase), Lys9p (saccharopine dehydrogenase), Lys20p (homocitrate synthase), Met6p (cobalamin-independent methionine synthase), Prs3p (5-phospho-ribosyl-1(␣)-pyrophosphate synthetase), Rib3p (3,4-dihydroxy-2-butanone-4-phosphate synthase synthase), Rpe1p (D-ribulose-5-phosphate 3-epimerase), Sam1p (S-adenosylmethionine synthetase), Thr4p (threonine synthase), Tkl1p (transketolase), Trp5p (tryptophan synthase), Ura1p (dihydroorotate), Ura4p (dihydroorotase), and Ura5p (orotate phosphoribosyltransferase isozyme).…”

Causes and Consequences of Cysteine S-Glutathionylation

“…High intracellular GSH concentrations can shift the equilibrium of the reaction with NO to GSSG, but during nitrosative stress, certain cell compartments can harbor high levels of GSNO. Under such conditions, GSNO may contribute to protein nitrosylation and/or S-glutathionylation (9,10). The specificity of GSNO-mediated post-translational modifications depends on the microenvironment of the target cysteine within protein tertiary and quaternary structures.…”

The redox switch that regulates molecular chaperones

“…Although they share the same substrate specificity, they have subtle catalytic differences and are differentially regulated through changes in the redox environment, through their peroxide-sensitive redox switch, which is ~10 Å from the active site (9,11,(79)(80)(81). Through biochemical and X-ray crystallography investigations, it was determined that reduction and oxidation of the -CXXC-motif represented the active and inactive forms of the proteins respectively.…”

“…X-ray crystallography studies and kinetic analysis demonstrated that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction, where disruption of the CXXC centre results in altered substrate orientation and an unprotonated PMP amino group, thus rendering the enzyme catalytically inactive (83). The reactive cysteines of both isoforms are also targets for the S-nitrosylating agent, S-nitroso glutathione (GSNO), where a transition between S-nitrosation and S-glutathionylation was reported dependent on the level of RNS exposure (81). The glutaredoxin/glutathione system reversed this inactive form, supporting a role for hBCAT in cellular redox control.…”