Degradation of methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF) in cultured myotubes and HepG2 cells is proteasome- and calpain/calcium-dependent.
During recent years, it has become increasingly clear that the ubiquitin-proteasome proteolytic pathway regulates intracellular protein degradation in various physiological and pathophysiological conditions. Substrates specifically degraded by the proteasome are important tools to assess the involvement of the proteasome in cellular proteolysis. It was recently proposed that the membrane permeable substrate methoxysuccinyl-phenylalanine-leucine-phenylalanine-7-amido-4- trifluoromethyl coumarin (FLF) is degraded specifically by the proteasome. The role of other proteolytic pathways in the degradation of FLF, however, is not fully understood. In the present study, we tested the role of different proteolytic pathways in the degradation of FLF in cultured myotubes and HepG2 cells by treating the cells with inhibitors of lysosomal, calpain and proteasome activity. In addition, we tested the hypothesis that insulin blocks proteasome-dependent degradation of FLF in myotubes and HepG2 cells. Results suggest that degradation of FLF in both myotubes and HepG2 cells is regulated by proteasome and calpain activity but not by lysosomal activity. Insulin inhibited proteasome-dependent but not calpain-dependent degradation of FLF in both myotubes and HepG2 cells. The results are important because they suggest that FLF degradation does not specifically reflect proteasome activity.
Li
B G
BG
Department of Surgery, University of Cincinnati, and Shriners Hospital for Children, Cincinnati, USA.
Fang
C H
CH
Hasselgren
P
P
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D007328:Insulin; D007783:Lactones; D009097:Multienzyme Complexes; D009842:Oligopeptides; C411815:methoxysuccinyl-phenylalanyl-leucyl-phenylalanyl-7-amido-4-trifluoromethylcoumarin; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium