title: A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Science, Medical University of South Carolina, Charleston, 29425, USA.
authors.forename: Danyelle M
authors.initials: DM
authors.lastname: Townsend
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Findlay
authors.forename: Farit
authors.initials: F
authors.lastname: Fazilev
authors.forename: Molly
authors.initials: M
authors.lastname: Ogle
authors.forename: Jacob
authors.initials: J
authors.lastname: Fraser
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Tew
mesh_terms: 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
chemical_list: 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