title: Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus.
abstract: Redox regulation of signaling molecules contributes critically to propagation of intracellular signals. The main source providing reactive oxygen species (ROS) for these physiological processes are activated NADPH oxidases (Nox/Duox family). In a pathophysiological context, some NADPH oxidase complexes produce large amounts of ROS either as part of the antimicrobial immune defense or as pathologic oxidative stress in many chronic diseases. Thus, understanding the switch from a dormant, inactive conformation to the active state of these enzymes will aid the development of inhibitors. As exogenously expressed Nox4 represents the only constitutively active enzyme in this family, analysis of structural determinants that permit this active conformation was undertaken. Our focus was directed toward a cell-based analysis of the first intracellular loop, the B-loop, and the C-terminus, two regions of Nox family enzymes that are essential for electron transfer. Mutagenesis of the B-loop identified several unique residues and a polybasic motif that contribute to the catalytic activity of Nox4. By using a multifaceted approach, including Nox4-Nox2 chimeras, mutagenesis, and insertion of Nox2 domains, we show here that the penultimate 22 amino acids of Nox4 are involved in constitutive ROS generation. The appropriate spacing of the C-terminal Nox4 sequence may cooperate with a discrete arginine-based interaction site in the B-loop, providing an intrinsically active interface that could not be disrupted by peptides derived from the Nox4 C-terminus. These results indicate that accessibility for a Nox4-specific peptide inhibitor might be difficult to achieve in vivo.
authors.affiliation: Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, California 92037, USA.
authors.forename: Katharina
authors.initials: K
authors.lastname: von Löhneysen
authors.forename: Deborah
authors.initials: D
authors.lastname: Noack
authors.forename: Patti
authors.initials: P
authors.lastname: Hayes
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Friedman
authors.forename: Ulla G
authors.initials: UG
authors.lastname: Knaus
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017382:Reactive Oxygen Species / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human