S-Nitrosoglutathione inactivation of the mitochondrial and cytosolic BCAT proteins: S-nitrosation and S-thiolation.
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.
Coles
Steven J
SJ
Faculty of Health and Life Sciences, University of the West of England, Coldharbor Lane, Bristol BS16 1QY, UK.
Easton
Peter
P
Sharrod
Hayley
H
Hutson
Susan M
SM
Hancock
John
J
Patel
Vinood B
VB
Conway
Myra E
ME
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054477:Glutaredoxins / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009599:Nitroprusside / Q000494:pharmacology; D015538:Nitrosation / Q000187:drug effects; D011257:Pregnancy Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000494:pharmacology*; D013997:Time Factors; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D054477:Glutaredoxins; D015778:Minor Histocompatibility Antigens; D011257:Pregnancy Proteins; D009599:Nitroprusside; D026422:S-Nitrosoglutathione; D005980:Glutathione Reductase; C490590:BCAT1 protein, human; D000637:Transaminases; C424463:BCAT2 protein, human; D003545:Cysteine