title: An Unbiased Mass Spectrometry Approach Identifies Glypican-3 as an Interactor of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR) in Hepatocellular Carcinoma Cells.
abstract: The mechanism of LDL receptor (LDLR) degradation mediated by the proprotein convertase subtilisin/kexin type 9 (PCSK9) has been extensively studied; however, many steps within this process remain unclear and still require characterization. Recent studies have shown that PCSK9 lacking its Cys/His-rich domain can still promote LDLR internalization, but the complex does not reach the lysosome suggesting the presence of an additional interaction partner(s). In this study we carried out an unbiased screening approach to identify PCSK9-interacting proteins in the HepG2 cells' secretome using co-immunoprecipitation combined with mass spectrometry analyses. Several interacting proteins were identified, including glypican-3 (GPC3), phospholipid transfer protein, matrilin-3, tissue factor pathway inhibitor, fibrinogen-like 1, and plasminogen activator inhibitor-1. We then validated these interactions by co-immunoprecipitation and Western blotting. Furthermore, functional validation was examined by silencing each candidate protein in HepG2 cells using short hairpin RNAs to determine their effect on LDL uptake and LDLR levels. Only GPC3 and phospholipid transfer protein silencing in HepG2 cells significantly increased LDL uptake in these cells and displayed higher total LDLR protein levels compared with control cells. Moreover, our study provides the first evidence that GPC3 can modulate the PCSK9 extracellular activity as a competitive binding partner to the LDLR in HepG2 cells.
authors.affiliation: From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and.
authors.forename: Kévin
authors.initials: K
authors.lastname: Ly
authors.affiliation: the Institut de Recherches Cliniques de Montréal, Affiliated with Université de Montréal, Montréal, Quebec H2W 1R7, Canada.
authors.forename: Rachid
authors.initials: R
authors.lastname: Essalmani
authors.affiliation: From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and.
authors.forename: Roxane
authors.initials: R
authors.lastname: Desjardins
authors.affiliation: the Institut de Recherches Cliniques de Montréal, Affiliated with Université de Montréal, Montréal, Quebec H2W 1R7, Canada.
authors.forename: Nabil G
authors.initials: NG
authors.lastname: Seidah
authors.affiliation: From the Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4 and. Electronic address: robert.day@usherbrooke.ca.
authors.forename: Robert
authors.initials: R
authors.lastname: Day
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D053673:Glypicans / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008077:Lipoproteins, LDL / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D064235:Matrilin Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071449:Proprotein Convertase 9 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*
chemical_list: C105937:GPC3 protein, human; D053673:Glypicans; C573412:LDLR protein, human; D008077:Lipoproteins, LDL; D064235:Matrilin Proteins; D009363:Neoplasm Proteins; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9
keywords: HepG2 cells; LDL receptor (LDLR); glypican-3; knockdown; lentivirus; low density lipoprotein (LDL); mass spectrometry (MS); proprotein convertase subtilisin/kexin type 9 (PCSK9); secretome; short hairpin RNA (shRNA)