The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR.
The low-density lipoprotein receptor (LDLR) mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins (LDLs). Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafilomycin A1 or addition of the irreversible serine protease inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1- positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.
Tveten
Kristian
K
Medical Genetics Laboratory, Department of Medical Genetics, Rikshospitalet University Hospital, N-0027 Oslo, Norway.
Ranheim
Trine
T
Berge
Knut Erik
KE
Leren
Trond P
TP
Kulseth
Mari Ann
MA
D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D049934:Isocoumarins; D018942:Macrolides / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids; D011480:Protease Inhibitors / Q000378:metabolism; D011973:Receptors, LDL / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D013450:Sulfones / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D003374:Coumarins; D004791:Enzyme Inhibitors; D049934:Isocoumarins; D018942:Macrolides; D011480:Protease Inhibitors; D011973:Receptors, LDL; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; C046235:3,4-dichloroisocoumarin; C040929:bafilomycin A1