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Crucial Role of Two Potential Cytosolic Regions of Nox2, 191 TSSTKTIRRS 200 and 484 DESQANHFAVHHDEEKD 500 , on NADPH Oxidase Activation *

Journal of Biological Chemistry, ISSN: 0021-9258, Vol: 280, Issue: 15, Page: 14962-14973
2005
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Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191 TSSTKTIRRS 200 and the C-terminal 484 DESQANHFAVHHDEEKD 500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47 phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop Nox4 -Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the α-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47 phox membrane translocation. This suggests that the α-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.

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