Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defence against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. S379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem SH3 domains, allowing the AIR to undergo phosphorylation to expose the tandem SH3 groove for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox-/- coronary microvascular cells. Compared to wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial superoxide production in response to acute PMA (a potent PKC activator) stimulation. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. S379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.
Pharmacology, Toxicology and Pharmaceutical Science
2
4%
Other
7
13%
Unknown
9
16%
Attention Score in Context
Attention Score in Context
This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 14 August 2014.
All research outputs
#17,285,668
of 25,373,627 outputs
Outputs from Journal of Biological Chemistry
#76,082
of 85,238 outputs
Outputs of similar age
#146,342
of 242,712 outputs
Outputs of similar age from Journal of Biological Chemistry
#250
of 441 outputs
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