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J Biol Chem. 2014 Jun 20;289(25):17732-46. doi: 10.1074/jbc.M113.541094. Epub 2014 May 7.

Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9.

Author information

1
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.
2
the Laboratories of Biochemical Neuroendocrinology and.
3
the Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H8M5, Canada.
4
Neuropeptides Structure and Metabolism, Institut de Recherches Cliniques de Montréal, Université de Montréal, Montreal H2W1R7, Quebec, and.
5
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.

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.

KEYWORDS:

Annexin; Cycloheximide; Drosophila; HepG2 Cells; Huh7 Cells; LDLR; Low Density Lipoprotein (LDL); Proprotein Convertase PCSK9; Short Hairpin RNA (shRNA); Translation Regulation

PMID:
24808179
PMCID:
PMC4067207
DOI:
10.1074/jbc.M113.541094
[Indexed for MEDLINE]
Free PMC Article

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