title: Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70.
abstract: Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins. The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.
authors.affiliation: Department of Medicine, Cardiovascular Institute, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029, USA.
authors.forename: V
authors.initials: V
authors.lastname: Gusarova
authors.forename: A J
authors.initials: AJ
authors.lastname: Caplan
authors.forename: J L
authors.initials: JL
authors.lastname: Brodsky
authors.forename: E A
authors.initials: EA
authors.lastname: Fisher
mesh_terms: D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D014452:Ubiquitins