TY - JOUR
T1 - Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation
AU - Qian, Jin
AU - Chen, Feng
AU - Kovalenkov, Yevgeniy
AU - Pandey, Deepesh Raj
AU - Moseley, M. Arthur
AU - Foster, Matthew W.
AU - Black, Stephen Matthew
AU - Venema, Richard C
AU - Stepp, David W.
AU - Fulton, David J.R.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants R01 HL085827 (D.J.R.F., R.C.V.), R01HL092446 (D.J.R.F., D.W.S.), and P01 HL101902-01A1 (D.J.R.F., S.M.B.) and an Established Investigator Award from the American Heart Association (D.J.R.F.).
PY - 2012/5/1
Y1 - 2012/5/1
N2 - The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.
AB - The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.
KW - Free radicals
KW - NADPH oxidase
KW - Nitric oxide
KW - Nox5
KW - Reactive oxygen species
KW - S-nitrosylation
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U2 - 10.1016/j.freeradbiomed.2012.02.029
DO - 10.1016/j.freeradbiomed.2012.02.029
M3 - Article
C2 - 22387196
AN - SCOPUS:84859057754
SN - 0891-5849
VL - 52
SP - 1806
EP - 1819
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 9
ER -