TY - JOUR
T1 - NADPH oxidase 4 function as a hydrogen peroxide sensor
AU - Nisimoto, Yukio
AU - Ogawa, Hisamitsu
AU - Kadokawa, Yuzo
AU - Qiao, Shanlou
N1 - Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
PY - 2018/6/1
Y1 - 2018/6/1
N2 - Nox4, a member of the NADPH- and oxygen-dependent oxidoreductases that generate reactive oxygen species (ROS), is widely expressed and constitutively active. To understand better its function and regulation, specific mutations in the Nox4 dehydrogenase (DH) domain were examined for effects on Nox4 oxidase activity. Transfection of His6-tagged Nox4 increased the amount of p22phox subunit in HEK293 cells, and a higher level of the heterodimer was observed in the nucleus- enriched fraction (NEF). NEF from Nox4-expressing HEK293 cells exhibited oxygen and H2O2 concentration-dependent NADPH oxidation rate. In Nox4-expressing cells, NEF and its partially purified form, the Nox4(P437H) mutant almost completely lost its oxidase activity, while Nox4(C546S), Nox4(C546L) or/and (C547L) had a significantly decreased rate of ROS production. The NADPH-dependent reduction of cytochrome c or cytochrome b5 by purified Nox4 DH domain was found regulated by the H2O2 concentration, and C546L and C547L mutants showed lower rates of the hemeprotein reduction. These conserved Cys residues in the DH domain respond to the cytosolic H2O2 concentration to regulate Nox4 activity.
AB - Nox4, a member of the NADPH- and oxygen-dependent oxidoreductases that generate reactive oxygen species (ROS), is widely expressed and constitutively active. To understand better its function and regulation, specific mutations in the Nox4 dehydrogenase (DH) domain were examined for effects on Nox4 oxidase activity. Transfection of His6-tagged Nox4 increased the amount of p22phox subunit in HEK293 cells, and a higher level of the heterodimer was observed in the nucleus- enriched fraction (NEF). NEF from Nox4-expressing HEK293 cells exhibited oxygen and H2O2 concentration-dependent NADPH oxidation rate. In Nox4-expressing cells, NEF and its partially purified form, the Nox4(P437H) mutant almost completely lost its oxidase activity, while Nox4(C546S), Nox4(C546L) or/and (C547L) had a significantly decreased rate of ROS production. The NADPH-dependent reduction of cytochrome c or cytochrome b5 by purified Nox4 DH domain was found regulated by the H2O2 concentration, and C546L and C547L mutants showed lower rates of the hemeprotein reduction. These conserved Cys residues in the DH domain respond to the cytosolic H2O2 concentration to regulate Nox4 activity.
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U2 - 10.1093/jb/mvy014
DO - 10.1093/jb/mvy014
M3 - Article
C2 - 29365138
AN - SCOPUS:85045423126
SN - 0021-924X
VL - 163
SP - 489
EP - 501
JO - Journal of Biochemistry
JF - Journal of Biochemistry
IS - 6
ER -