S-Nitrosoglutathione inactivation of the mitochondrial and cytosolic BCAT proteins: S-nitrosation and S-thiolation

Coles, Steven J.; Easton, Peter; Sharrod, Hayley; Hutson, Susan M.; Hancock, John T.; Patel, VB; Conway, Myra E.

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.

• Journal Article Type: Article
• Publication Date: Jan 27, 2009
• Journal: Biochemistry
• Print ISSN: 0006-2960
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 48
• Issue: 3
• Pages: 645-656
• DOI: https://doi.org/10.1021/bi801805h
• Keywords: S-Nitrosoglutathione, inactivation, mitochondrial, cytosolic, BCAT proteins, S-nitrosation, S-thiolation
• Public URL: https://uwe-repository.worktribe.com/output/999199
• Publisher URL: http://dx.doi.org/10.1021/bi801805h