title: Quantitative proteomic analysis of PCSK9 gain of function in human hepatic HuH7 cells.
abstract: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.
authors.affiliation: Ottawa Institute of Systems Biology, University of Ottawa, Ontario, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Denis
authors.forename: Heather
authors.initials: H
authors.lastname: Palmer-Smith
authors.forename: Fred
authors.initials: F
authors.lastname: Elisma
authors.forename: Alia
authors.initials: A
authors.lastname: Busuttil
authors.forename: Theodore Glenn
authors.initials: TG
authors.lastname: Wright
authors.forename: Maroun
authors.initials: M
authors.lastname: Bou Khalil
authors.forename: Annik
authors.initials: A
authors.lastname: Prat
authors.forename: Nabil G
authors.initials: NG
authors.lastname: Seidah
authors.forename: Michel
authors.initials: M
authors.lastname: Chrétien
authors.forename: Janice
authors.initials: J
authors.lastname: Mayne
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016000:Cluster Analysis; D030562:Databases, Protein; D015536:Down-Regulation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000166:cytology*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000235:genetics; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D015854:Up-Regulation
chemical_list: D002352:Carrier Proteins; C485118:EHBP1 protein, human; C484942:EHD2-binding protein 1, mouse; D010455:Peptides; D020543:Proteome; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases