Studies on degradative mechanisms mediating post-translational fragmentation of apolipoprotein B and the generation of the 70-kDa fragment.
It has been well established that the biogenesis of apoB is mediated co-translationally by the cytosolic proteasome. Here, however, we investigated the role of both the cytosolic proteasome as well as non-proteasome-mediated degradation systems in the post-translational degradation of apoB. In pulse-chase labeling experiments, co-translational (0-h chase) apoB degradation in both intact and permeabilized cells was sensitive to proteasome inhibitors. Interestingly, turnover of apoB in intact cells over a 2-h chase was partially inhibitable by lactacystin, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. In permeabilized cells, however, there was no post-translational protection of apoB by lactacystin. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20 S proteasomal subunits, and consequently the cells exhibited no detectable lactacystin-inhibitable activity. Thus, apoB fragmentation and the generation of the 70-kDa apoB degradation fragment, characteristic of permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. Similar results were observed when we used a derivative of lactacystin, clastolactacystin beta-lactone, which represents the active species of the inhibitor. Interestingly, however, the abundance of the 70-kDa fragment could be modulated by the microsomal triglyceride transfer protein inhibitor, BMS-197636, as well as by pretreatment of the permeabilized cells with dithiothreitol. These data thus suggest that although the cytosolic proteasome appears to be involved in the post-translational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70-kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome.
Cavallo
D
D
Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
Rudy
D
D
Mohammadi
A
A
Macri
J
J
Adeli
K
K
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005449:Fluorenes / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D054833:Isoindoles / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational
D001055:Apolipoproteins B; C000621959:BMS 197636; D002352:Carrier Proteins; D005449:Fluorenes; D054833:Isoindoles; D007783:Lactones; D009097:Multienzyme Complexes; D010446:Peptide Fragments; C106092:microsomal triglyceride transfer protein; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004229:Dithiothreitol; D000111:Acetylcysteine