Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity.
The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.
GREPI AGIM FRE CNRS-UJF, Université Joseph Fourier, Grenoble, France. mvchuong@yahoo.fr
Minh Vu Chuong
MV
Nguyen
Leilei
L
Zhang
Stanislas
S
Lhomme
Nicolas
N
Mouz
Jean-Luc
JL
Lenormand
Bernard
B
Lardy
Françoise
F
Morel
D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D009245:NADH Dehydrogenase