Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation -: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O-2(.-) 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-p47(phox) 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 p67(phox) for cytochrome b(558) synergistically with p47(phox). Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b(558) as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b(558), which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67(phox) and p47(phox). Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.