title: Selective recapitulation of conserved and nonconserved regions of putative NOXA1 protein activation domain confers isoform-specific inhibition of Nox1 oxidase and attenuation of endothelial cell migration.
abstract: Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O2·-) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O2·- generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O2·- production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.
authors.affiliation: From the Vascular Medicine Institute and.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ranayhossaini
authors.forename: Andres I
authors.initials: AI
authors.lastname: Rodriguez
authors.forename: Sanghamitra
authors.initials: S
authors.lastname: Sahoo
authors.forename: Beibei B
authors.initials: BB
authors.lastname: Chen
authors.forename: Rama K
authors.initials: RK
authors.lastname: Mallampalli
authors.forename: Eric E
authors.initials: EE
authors.lastname: Kelley
authors.forename: Gabor
authors.initials: G
authors.lastname: Csanyi
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
authors.forename: Guillermo
authors.initials: G
authors.lastname: Romero
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Pagano
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation; D019073:HT29 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism*; D011485:Protein Binding; D011651:Pulmonary Artery / Q000166:cytology; D013481:Superoxides / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D007527:Isoenzymes; C476420:NOXA1 protein, human; D013481:Superoxides; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human
keywords: Enzyme Inhibitors; Enzyme Mechanisms; NADPH Oxidase; Nox1; Peptides; Reactive Oxygen Species (ROS)