Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells

Our previous studies indicated that an alternatively spliced variant mRNA o f p40-phox, a cytosolic component of NADPH oxidase, is expressed but its pr otein is hardly detected in myeloid cells such as promyelocytic HL-60 cells and neutrophils, Here, we have examined die stability of p40-phox variant protein in undifferentiated HL-60 cells. When in vitro-translated proteins were incubated with subcellular fractions of HL-60 cells, p40-phox variant protein but not native p40-phox was degraded by the cytosol and granule fra ctions. The degradation of variant protein by the granule fraction was obse rved using sonicated but not intact granules, suggesting that the variant p rotein is unlikely to be degraded by the granules in intact cells, To ident ify the enzyme(s) involved, we examined the effects of various enzyme inhib itors oil the degradation of variant protein by the cytosol fraction, Degra dation was completely inhibited by proline-specific serine protease (prolyl endopeptidase) inhibitors but not by proteasome, calpain, and metallprotea se inhibitors. Furthermore, the variant protein was degraded by a purified prolyl endopeptidase, and the degradation was protected by treating HL-60 c ells with a cell-permeable inhibitor (S17092-1) for prolyl endopeptidase. T hese observations suggest that a cytosolic prolyl endopeptidase is involved in the degradation of p40-phox variant protein in myeloid cells.