Abstract 12654: The Dual Roles of the PCSK9 Active Site: Evidence for an Intermolecular Proteolytic Mechanism and a Separate Secretion Signal Encoded After Proteolytic Maturation
Background: The protease proprotein subtilisin kexin type 9 (PCSK9) raises serum LDL cholesterol (LDL-C) by binding to the LDL receptor (LDL-R) on the cell surface and targeting it for degradation, thus accelerating heart disease. Antibody-based inhibitors of the PCSK9:LDL-R interaction have shown remarkable efficacy in lowering LDL-C in early trials. Given that PCSK9 self-cleavage is required for proper secretion, targeting its proteolytic activity is an attractive option for developing small molecule therapeutics. However, the active site remains auto-inhibited after cleavage due to persistent binding of the prodomain, hindering further study of proteolytic function.
Methods and Results: To evaluate for an expanded role of the PCSK9 active site, and specifically whether PCSK9 can perform intermolecular cleavage, we designed a mammalian cell culture system in which PCSK9 fragment constructs engineered to assemble in trans were co-expressed with full-length PCSK9 constructs devoid of proteolytic activity. Immunoblots of both cell lysates and media show that the “protease-dead” construct is present in cleaved form only when co-expressed with our “protease-active” in trans constructs. To test the importance of C-terminal prodomain residues near the PCSK9 active site in permitting secretion after self-cleavage, we co-expressed in trans PCSK9 constructs engineered with C-terminal prodomain mutations and deletions. Immunoblots of media reveal that secreted PCSK9 is permitted only for certain mutations and not permitted for any C-terminal deletions. Furthermore, certain mutations which do not allow for self-cleavage do permit secretion of our in trans constructs.
Conclusions: We conclude from our results that the PCSK9 catalytic domain is capable of intermolecular cleavage. Moreover, our results show that PCSK9 proteolysis is necessary but not sufficient for proper PCSK9 secretion, suggesting that a second signal must be present for secretion to occur. Our intermolecular cleavage system indicates that access to the PCSK9 active site, a theoretically “druggable” pocket, is possible. In addition, our findings suggest that the active site plays a second and previously unknown role, beyond self-cleavage, in mediating PCSK9 secretion.
- © 2013 by American Heart Association, Inc.