Adenovirus-mediated overexpression of microsomal triglyceride transfer protein (MTP): mechanistic studies on the role of MTP in apolipoprotein B-100 biogenesis.
The intracellular concentration of the microsomal triglyceride transfer protein large subunit (lMTP), the abetalipoproteinemia gene product, is tightly controlled. To date, attempts at overexpressinglMTP in vivo or in vitro have been unsuccessful. We successfully overexpressed lMTP in HepG2 cells using an adenoviral vector containing an lMTP cDNA, AdMTP. AdMTP-transduced HepG2 cells overexpressed MTP activity. They secreted increased amounts of apoB-100 lipoproteins with LDL and HDL density into the medium. lMTP overexpression alone minimally changed the density profile of apoB-containing lipoproteins, but addition of oleic acid shifted the profile toward lower densities. Oleic acid had a greater stimulatory effect on apoB-100 secretion in control HepG2 cells than in AdMTP-transduced cells, because (i) adenoviral transduction per se suppressed protein synthesis, affecting apoB-100 and albumin equally, and (ii) adenoviral transduction partially attenuated the increase in triglyceride synthesis in response to oleic acid supplementation. AdMTP treatment greatly diminished the intracellular degradation of apoB-100, but in comparison with recombinant virus containing luciferase cDNA (AdLuc), it caused no change in its biosynthetic rate. It greatly reduced, but did not eliminate, its proteasomal degradation. Our study constitutes the initial demonstration that adenovirus-mediated transfer of lMTP markedly stimulates MTP expression which in turn stimulates apoB-100 production. The mechanism involves a downregulation of ubiquitin-proteasome-mediated degradation without any change in synthetic rate.
Liao
W
W
Department of Medicine, Baylor College of Medicine, Texas Medical Center, Houston 77030, USA.
Kobayashi
K
K
Chan
L
L
D000260:Adenoviruses, Human / Q000235:genetics*; D000418:Albumins / Q000378:metabolism; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002499:Centrifugation, Density Gradient; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000502:physiology; D018076:DNA, Complementary / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000502:physiology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008074:Lipoproteins / Q000096:biosynthesis; D008861:Microsomes / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000502:physiology; D009829:Oleic Acids / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000235:genetics; D014161:Transduction, Genetic; D014280:Triglycerides / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000502:physiology
D000418:Albumins; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D002352:Carrier Proteins; D003470:Culture Media; D018076:DNA, Complementary; D008074:Lipoproteins; D009097:Multienzyme Complexes; D009829:Oleic Acids; D011994:Recombinant Proteins; D014280:Triglycerides; D014452:Ubiquitins; C106092:microsomal triglyceride transfer protein; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex