title: Peroxiredoxin-6 and NADPH oxidase activity.
abstract: Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2. Peroxiredoxin 6 (Prdx6), a protein belonging to the class of 1-cys Prdxs, was identified in polymorphonuclear leukocytes or neutrophils, defined by amino acid sequence and activity, and found associated with a component of the NADPH oxidase (Nox2), p67(phox). Prdx6 plays an important role in neutrophil function and supports the optimal activity of Nox2. In this chapter, methods are described for determining the Prdx activity of Prdx6. In addition, the approach for assessing the effect of Prdx6 on Nox2 in the SDS-activated, cell-free system of NADPH oxidase activity is presented. Finally, the techniques for suppressing Prdx6 expression in phox-competent K562 cells and cultured myeloid cells with siRNA and shRNA methods are described. With these approaches, the role of Prdx6 in Nox2 activity can be explored with intact cells. The biochemical mechanisms of the Prdx6 effect on the NADPH oxidase can be investigated with the experimental strategies described.
authors.affiliation: Department of Pediatrics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA. daniel.ambruso@ucdenver.edu
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Ambruso
mesh_terms: D002474:Cell-Free System; D057075:Enzyme Assays; D055785:Gene Knockdown Techniques; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D020014:K562 Cells; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000502:physiology; D010084:Oxidation-Reduction; D054465:Peroxiredoxin VI / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D013481:Superoxides; D006861:Hydrogen Peroxide; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D005974:Glutamate-Ammonia Ligase
keywords: Hydrogen peroxide; NADPH oxidase; Neutrophil; Nox2; Peroxidase; Peroxiredoxin 6; Phagocyte; Reactive oxygen species; Respiratory burst; Superoxide anion