Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice.

Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.

Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH.

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and ß-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.

Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation.

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.