title: Regulated intracellular degradation of apolipoprotein B in semipermeable HepG2 cells.
abstract: A digitonin-permeabilized HepG2 cell system has been developed and used to characterize the pathway responsible for apolipoprotein B100 (apoB) degradation. Degradation of 35S-labeled apoB occurred after an initial lag period and resulted in the loss of approximately 75.8% of apoB after 2 h of chase. The degradation rate in permeabilized cells was slower than that in intact cells, but approximately the same percentage of apoB was lost over 2 h of chase. The loss of intact apoB in permeabilized cells coincided with the appearance of a number of degradation fragments, including 335- and 70-kDa fragments. The detection of a 70-kDa fragment was a sensitive indicator of degradation. ApoB degradation was inhibited at temperatures below 37 degrees C and maximally activated at 42 degrees C. Degradation was also pH-dependent, with inhibition at pH > or = 7.5. ApoB decay was stimulated by ATP supplementation but not GTP and was inhibited in the presence of energy inhibitors. Degradation was not significantly affected by cycloheximide. However, the stability of the 70-kDa fragment was prolonged with cycloheximide. Preincubation of cells with brefeldin A and nocodazole, as well as monensin, did not diminish the degradation of intact apoB or the appearance of the 70-kDa fragment, suggesting a lack of requirement for intracellular transport from the endoplasmic reticulum. Among the various protease inhibitors tested, degradation was most sensitive to N-acetylleucylleucylnorleucinal (ALLN), which abolished the generation of the 70-kDa fragment in a dose-dependent manner. ALLN-sensitive degradation of apoB was unaffected by the calcium ionophore, A23187. Interestingly, degradation of unglycosylated apoB, detected in tunicamycin-pretreated cells, occurred earlier, resulted in generation of additional fragments, and was largely uninhibited by ALLN. In summary, apoB degradation occurs in permeabilized HepG2 cells by a temperature- and pH-sensitive, pre-Golgi degradation system and is catalyzed by a calcium-independent, ALLN-sensitive protease. Specificity of the initial apoB cleavage may require the proper N-linked glycosylation of the protein in the endoplasmic reticulum.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Adeli
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001055:Apolipoproteins B / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D000001:Calcimycin / Q000494:pharmacology; D002463:Cell Membrane Permeability; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008985:Monensin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013696:Temperature; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D001055:Apolipoproteins B; D003517:Cyclopentanes; D011480:Protease Inhibitors; D014415:Tunicamycin; D020126:Brefeldin A; D000001:Calcimycin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008985:Monensin; D003513:Cycloheximide; D004072:Digitonin; D015739:Nocodazole