S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-nitrosation and S-thiolation

Coles, Steven ORCID: https://orcid.org/0000-0002-1109-6971, Easton, P., Sharrod, H., Hutson, S.M., Hancock, J.T., Patel, V.B. and Conway, M.E. (2009) S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-nitrosation and S-thiolation. Biochemistry, 48 (3). pp. 645-656. ISSN Print: 0006-2960 Online: 1520-4995

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.

Actions (login required)

View Item