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 of p40-phox, a cytosolic component of NADPH oxidase, is expressed but its protein is hardly detected in myeloid cells such as promyelocytic HL-60 cells and neutrophils. Here, we have examined the 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 fractions. The degradation of variant protein by the granule fraction was observed using sonicated but not intact granules, suggesting that the variant protein is unlikely to be degraded by the granules in intact cells. To identify the enzyme(s) involved, we examined the effects of various enzyme inhibitors on the degradation of variant protein by the cytosol fraction. Degradation was completely inhibited by proline-specific serine protease (prolyl endopeptidase) inhibitors but not by proteasome, calpain, and metalloprotease inhibitors. Furthermore, the variant protein was degraded by a purified prolyl endopeptidase, and the degradation was protected by treating HL-60 cells with a cell-permeable inhibitor (S17092-1) for prolyl endopeptidase. These observations suggest that a cytosolic prolyl endopeptidase is involved in the degradation of p40-phox variant protein in myeloid cells.
Hasebe
T
T
Department of Biochemistry, Juntendo University, School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.
Hua
J
J
Someya
A
A
Morain
P
P
Checler
F
F
Nagaoka
I
I
D017398:Alternative Splicing; D003594:Cytoplasmic Granules / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D018922:HL-60 Cells / Q000201:enzymology; D006801:Humans; D022423:Myeloid Cells / Q000201:enzymology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012697:Serine Endopeptidases / Q000502:physiology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; C085072:neutrophil cytosol factor 40K; D019255:NADPH Oxidases; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases