title: Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway.
abstract: Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.
authors.affiliation: Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Rita
authors.initials: R
authors.lastname: Kohen-Avramoglu
authors.forename: Fariborz
authors.initials: F
authors.lastname: Rashid-Kolvear
authors.forename: Crystal S
authors.initials: CS
authors.lastname: Au
authors.forename: Taryne M
authors.initials: TM
authors.lastname: Chong
authors.forename: Gary F
authors.initials: GF
authors.lastname: Lewis
authors.forename: Denny K Y
authors.initials: DK
authors.lastname: Trinh
authors.forename: Richard C
authors.initials: RC
authors.lastname: Austin
authors.forename: Reiko
authors.initials: R
authors.lastname: Urade
authors.forename: Khosrow
authors.initials: K
authors.lastname: Adeli
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002729:Chloromercuribenzoates / Q000494:pharmacology*; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014161:Transduction, Genetic
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D002729:Chloromercuribenzoates; D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases; C077733:ER-60 protease