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A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
08:14

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Published on: August 28, 2018

Loss- and gain-of-function PCSK9 variants: cleavage specificity, dominant negative effects, and low density

Suzanne Benjannet1, Josée Hamelin, Michel Chrétien

  • 1Laboratory of Biochemical Neuroendocrinology, University of Montreal, Montreal, Quebec, Canada.

The Journal of Biological Chemistry
|August 10, 2012
PubMed

Insights

Proprotein convertase PCSK9 processing has specific P1 site requirements. Mutations preventing PCSK9 exit from the endoplasmic reticulum cause dominant negative effects, offering new therapeutic targets for hypercholesterolemia.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Proprotein convertase PCSK9 (PCSK9) is a key regulator of LDL receptor (LDLR) levels, crucial for managing hypercholesterolemia.
  • PCSK9 functions by binding to the LDLR and promoting its degradation, thereby increasing circulating cholesterol.
  • Autocatalytic cleavage within the endoplasmic reticulum is essential for PCSK9 secretion and activity.

Purpose of the Study:

  • To investigate the substrate specificity of the PCSK9 autocatalytic cleavage site.
  • To characterize the cellular fate and functional consequences of PCSK9 mutants with altered cleavage.
  • To explore the impact of mutations at residue 374 on PCSK9's LDLR degradation activity.

Main Methods:

  • Site-directed mutagenesis of the PCSK9 P1 cleavage site (Gln152).
  • Analysis of PCSK9 mutant expression, secretion, and intracellular localization using various assays.
  • Evaluation of LDLR degradation activity for wild-type and mutant PCSK9 proteins, including gain-of-function variants.

Main Results:

  • PCSK9 exhibits specific P1 residue recognition: Gln > Met > Ala > Ser > Thr ≈ Asn.
  • Mutations at the P1 site, other than those recognized, result in unprocessed zymogen formation.
  • Unprocessed PCSK9 mutants are retained in the endoplasmic reticulum, exhibiting dominant-negative effects on native PCSK9.
  • Mutations at Asp374 generally enhance LDLR degradation, with notable exceptions like D374E, D374G, D374N, and D374P.

Conclusions:

  • The endoplasmic reticulum possesses stringent quality control mechanisms for PCSK9 processing.
  • PCSK9 autocatalytic cleavage specificity offers potential for developing targeted small molecule inhibitors.
  • Understanding PCSK9 processing and gain-of-function mutations provides insights into hypercholesterolemia pathogenesis and therapeutic strategies.

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