Trimer hydroxylated quinone (IIIHyQ) derived from apocynin targets cysteine residues of p47phox preventing the activation of human vascular NADPH oxidase

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Authors
Mora-Pale, M.
Kwon, S.J.
Linhardt, Robert J.
Dordick, J.S.
Issue Date
2012
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Article
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ENG
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Abstract
Enzymatically derived oligophenols from apocynin can be effective inhibitors of human vascular NADPH oxidase (Nox). An isolated trimer hydroxylated quinone (IIIHyQ) has been shown to inhibit endothelial NADPH oxidase with an IC(50) ~30 nM. In vitro studies demonstrated that IIIHyQ is capable of disrupting the interaction between p47(phox) and p22(phox), thereby blocking the activation of the Nox2 isoform. Herein, we report the role of key cysteine residues in p47(phox) as targets for the IIIHyQ. Incubation of p47(phox) with IIIHyQ results in a decrease of ~80% of the protein free cysteine residues; similar results were observed using 1,2- and 1,4-naphthoquinones, whereas apocynin was unreactive. Mutants of p47(phox), in which each Cys was individually replaced by Ala (at residues 111, 196, and 378) or Gly (at residue 98), were generated to evaluate their individual importance in IIIHyQ-mediated inhibition of p47(phox) interaction with p22(phox). Specific Michael addition on Cys196, within the N-SH3 domain, by the IIIHyQ is critical for disrupting the p47(phox)-p22(phox) interaction. When a C196A mutation was tested, the IIIHyQ was unable to disrupt the p47(phox)-p22(phox) interaction. However, the IIIHyQ was effective at disrupting this interaction with the other mutants, displaying IC(50) values (4.9, 21.0, and 2.3μM for the C111A, C378A, and C98G mutants, respectively) comparable to that of wild-type p47(phox).
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Free Radical Biology and Medicine, 52, 962-969
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Trimer hydroxylated quinone (IIIHyQ) derived from apocynin targets cysteine residues of p47phox preventing the activation of human vascular NADPH oxidase, M.Mora-Pale, S.J. Kwon, R. J. Linhardt, J. S. Dordick, Free Radical Biology and Medicine, 52, 962-969, 2012.
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Elsevier
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