PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

Yascara Grisel Luna Saavedra,Jianbing Zhang,Nabil G Seidah,Maria Cristina Vinci

doi:10.1371/journal.pone.0072113

Abstract

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 in trans 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 in vitro 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 Gln152 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.

Highlights

The mammalian proprotein convertases (PCs) [1] are members of a secretory serine protease family composed of nine members related to bacterial subtilisin and yeast kexin
After screening transformants, testing protein production, scaling-up and purification of the His-tagged PCSK9 prosegment, we end up with a very poor production level after dialysis and concentration of 10 L batches, with a total yield of,65 mg of pure protein/L. To circumvent this poor yield, we fused the Fc fragment of human immunoglobulin IgG1 with the N-terminus of the hPCSK9 prosegment, resulting in an fragment such chimeric PCSK9 prosegment (Fcpro) chimera ending at natural C-terminus Gln152 of the prosegment (Figure 1A)
Since the validation by genetic studies that PCSK9 has a clear role in the regulation of cholesterol homeostasis, many efforts have been made to develop an inhibitor of this attractive therapeutic target for the treatment of hypercholesterolemia [1]

Summary

Introduction

The mammalian proprotein convertases (PCs) [1] are members of a secretory serine protease family composed of nine members related to bacterial subtilisin and yeast kexin. Seven of these (PC1/ 3, PC2, Furin, PC4, PC5/6, PACE4 and PC7) exhibit homology of their catalytic domain to that of yeast kexin, and are known to cleave after basic residues. These convertases are synthesized as inactive zymogens Their prosegment located at their N-terminus is implicated in the productive folding of the enzyme and in its stabilization as an inactive form, like a natural inhibitor, until one or more cleavages occur followed by the release of the active enzyme dissociated from its prosegment [2]

Methods

Results

Conclusion