title: Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway.
abstract: Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.
authors.affiliation: Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Fisher
authors.affiliation: Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5.
authors.forename: Neeraj A
authors.initials: NA
authors.lastname: Khanna
authors.affiliation: Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5. Electronic address: rmcleod2@dal.ca.
authors.forename: Roger S
authors.initials: RS
authors.lastname: McLeod
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053299:Apolipoprotein B-100 / Q000096:biosynthesis / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D015243:Cholesterol, VLDL / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014280:Triglycerides / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D053299:Apolipoprotein B-100; D002070:Butadienes; D015243:Cholesterol, VLDL; D004791:Enzyme Inhibitors; D009570:Nitriles; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D018121:Receptors, Cytokine; D014280:Triglycerides; C113580:U 0126; D025801:Ubiquitin; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D020929:Mitogen-Activated Protein Kinase Kinases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C404942:p97 ATPase