PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

<div><p>The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the <i>in trans</i> presence of the PCSK9 prosegment would interfere with PCSK9's activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular <i>in vitro</i> co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln<b>₁₅₂</b> of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.</p>