title: Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.
abstract: Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9's C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human 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.
authors.forename: Kévin
authors.initials: K
authors.lastname: Ly
authors.affiliation: the Laboratories of Biochemical Neuroendocrinology and.
authors.forename: Yascara Grisel Luna
authors.initials: YG
authors.lastname: Saavedra
authors.affiliation: the Laboratories of Biochemical Neuroendocrinology and.
authors.forename: Maryssa
authors.initials: M
authors.lastname: Canuel
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.
authors.forename: Sophie
authors.initials: S
authors.lastname: Routhier
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.
authors.forename: Roxane
authors.initials: R
authors.lastname: Desjardins
authors.affiliation: the Laboratories of Biochemical Neuroendocrinology and.
authors.forename: Josée
authors.initials: J
authors.lastname: Hamelin
authors.affiliation: the Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H8M5, Canada.
authors.forename: Janice
authors.initials: J
authors.lastname: Mayne
authors.affiliation: Neuropeptides Structure and Metabolism, Institut de Recherches Cliniques de Montréal, Université de Montréal, Montreal H2W1R7, Quebec, and.
authors.forename: Claude
authors.initials: C
authors.lastname: Lazure
authors.affiliation: the Laboratories of Biochemical Neuroendocrinology and.
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, Robert.day@usherbrooke.ca.
authors.forename: Robert
authors.initials: R
authors.lastname: Day
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014176:Protein Biosynthesis / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: C472822:ANXA2 protein, human; D017306:Annexin A2; D011994:Recombinant Proteins; C472125:PCSK9 protein, human; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: Annexin; Cycloheximide; Drosophila; HepG2 Cells; Huh7 Cells; LDLR; Low Density Lipoprotein (LDL); Proprotein Convertase PCSK9; Short Hairpin RNA (shRNA); Translation Regulation