Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9 [Protein Synthesis and Degradation]

May 7th, 2014 by Ly, K., Saavedra, Y. G. L., Canuel, M., Routhier, S., Desjardins, R., Hamelin, J., Mayne, J., Lazure, C., Seidah, N. G., Day, R.

Annexin A2 was reported as an extracellular endogenous inhibitor of PCSK9 activity on cell-surface LDLR 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 (CHRD) 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 CHRD and AnxA2. Finally, we produced and purified recombinant human and mouse PCSK9, which were characterized and used to perform DiI-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.
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