Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation.
Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.
Berthier
Sylvie
S
Groupe de Recherche et d'Etude du Processus Inflammatoire, Laboratoire d'Enzymologie, Centre Hospitalier Universitaire de Grenoble, BP 217, 38043 Grenoble, France.
Paclet
Marie-Helene
MH
Lerouge
Sandra
S
Roux
Florence
F
Vergnaud
Sabrina
S
Coleman
Anthony W
AW
Morel
Francoise
F
D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D040501:Calgranulin A / Q000302:isolation & purification / Q000378:metabolism*; D040502:Calgranulin B / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010586:Phagocytes / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D040501:Calgranulin A; D040502:Calgranulin B; D003573:Cytochrome b Group; D018076:DNA, Complementary; D010750:Phosphoproteins; D011994:Recombinant Proteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D016718:Arachidonic Acid; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D014357:Trypsin; D010100:Oxygen