A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins.
Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.
Townsend
Danyelle M
DM
Department of Pharmaceutical Science, Medical University of South Carolina, Charleston, 29425, USA.
Findlay
Victoria J
VJ
Fazilev
Farit
F
Ogle
Molly
M
Fraser
Jacob
J
Saavedra
Joseph E
JE
Ji
Xinhua
X
Keefer
Larry K
LK
Tew
Kenneth D
KD
D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism*; D051549:Glutathione S-Transferase pi / Q000378:metabolism*; D006801:Humans; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D000090243:Nucleophosmin; D011355:Prodrugs / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D062366:para-Aminobenzoates
D001391:Azo Compounds; C000715570:Npm1 protein, mouse; C485370:O(2)-(2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate; D011355:Prodrugs; D011506:Proteins; D062366:para-Aminobenzoates; D000090243:Nucleophosmin; D009569:Nitric Oxide; D051549:Glutathione S-Transferase pi; D020929:Mitogen-Activated Protein Kinase Kinases; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516609:Ptpn1 protein, mouse; D005978:Glutathione; D010129:4-Aminobenzoic Acid