title: Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe.
abstract: BACKGROUND
  Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe.
  
  
  RESULTS
  Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe.
  
  
  CONCLUSIONS
  This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.
authors.affiliation: Division of Endocrinology, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada. Electronic address: morris.schweitzer@mcgill.ca.
authors.forename: Morris
authors.initials: M
authors.lastname: Schweitzer
authors.affiliation: Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada.
authors.forename: Sandra
authors.initials: S
authors.lastname: Makhoul
authors.affiliation: Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, H3A 1A3, QC, Canada.
authors.forename: Miltiadis
authors.initials: M
authors.lastname: Paliouras
authors.affiliation: Division of Endocrinology, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada.
authors.forename: Lenore K
authors.initials: LK
authors.lastname: Beitel
authors.affiliation: Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada.
authors.forename: Bruce
authors.initials: B
authors.lastname: Gottlieb
authors.affiliation: Division of Endocrinology, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, H3A 1A3, QC, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Trifiro
authors.affiliation: Biotechnology Research Institute - National Research Council, 6100 Royalmount, Montreal, H4P 2R2, QC, Canada.
authors.forename: Shafinaz F
authors.initials: SF
authors.lastname: Chowdhury
authors.affiliation: Biotechnology Research Institute - National Research Council, 6100 Royalmount, Montreal, H4P 2R2, QC, Canada.
authors.forename: Naif M
authors.initials: NM
authors.lastname: Zaman
authors.affiliation: Biotechnology Research Institute - National Research Council, 6100 Royalmount, Montreal, H4P 2R2, QC, Canada.
authors.forename: Edwin
authors.initials: E
authors.lastname: Wang
authors.affiliation: CVPath Institute Inc., 19 Firstfield Road, Gaithersburg, MD, 20878, USA.
authors.forename: Harry
authors.initials: H
authors.lastname: Davis
authors.affiliation: Division of Endocrinology, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin Cote Ste Catherine, Montreal, H3T 1E2, QC, Canada; Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, H3A 1A3, QC, Canada.
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Chalifour
mesh_terms: D000924:Anticholesteremic Agents / Q000627:therapeutic use*; D015415:Biomarkers / Q000097:blood; D008078:Cholesterol, LDL / Q000097:blood*; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D000069438:Ezetimibe / Q000627:therapeutic use*; D005260:Female; D005819:Genetic Markers; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods; D014162:Transfection; D016896:Treatment Outcome
chemical_list: D000924:Anticholesteremic Agents; D015415:Biomarkers; D008078:Cholesterol, LDL; D005819:Genetic Markers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C410800:NPC1L1 protein, human; D000069438:Ezetimibe
keywords: Cholesterol; Cholesterol/absorption; Cholesterol/cell and tissue; Drug therapy/hypolipidemic drugs; Genetics; Proteomics