Inhibitory action of NoxA1 on dual oxidase activity in airway cells.
Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.
Pacquelet
Sandrine
S
Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California 92037, USA.
Lehmann
Mandy
M
Luxen
Sylvia
S
Regazzoni
Karine
K
Frausto
Monika
M
Noack
Deborah
D
Knaus
Ulla G
UG
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000074623:Dual Oxidases; D004789:Enzyme Activation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D008954:Models, Biological; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology*; D015290:Second Messenger Systems / Q000502:physiology; D015854:Up-Regulation / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476420:NOXA1 protein, human; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431513:DUOX1 protein, human; D002118:Calcium