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Updated: Aug 24, 2025
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
Effects of PCSK9 missense variants on molecular conformation and biological activity in transfected HEK293FT cells
Bruna Los1, Glaucio Monteiro Ferreira2, Jéssica Bassani Borges3
1Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Abstract:
PCSK9 gain-of-function (GOF) variants increase degradation of low-density lipoprotein receptor (LDLR) and are potentially associated with Familial Hypercholesterolemia (FH). This study aimed to explore the effects of PCSK9 missense variants on protein structure and interactions with LDLR using molecular modeling analyses and in vitro functional studies. Variants in FH-related genes were identified in a Brazilian FH cohort using an exon-target gene sequencing strategy. Eight PCSK9 missense variants in pro- [p.(E32K) and p.(E57K)], catalytic [p.(R237W), p.(P279T) and p.(A443T)], and C-terminal histidine-cysteine rich (CHR) [p.(R469W), p.(Q619P) and p.(R680Q)] domains were identified. Molecular dynamics analyses revealed that GOF variants p.(E32K) and p.(R469W) increased extreme motions in PCSK9 amino acid backbone fluctuations and affected Hbond and water bridge interactions between the pro-domain and CM1 region of the CHR domain. HEK293FT cells transfected with plasmids carrying p.(E32K) and p.(R469W) variants reduced LDLR expression (8.7 % and 14.8 %, respectively) compared to wild type (p < 0.05) but these GOF variants did not affect PCSK9 expression and secretion. The missense variants p.(P279T) and p.(Q619P) also reduced protein stability and altered Hbond interactions. In conclusion, PCSK9 p.(E32K), p.(R469W), p.(P279T) and p.(Q619P) variants disrupt intramolecular interactions that are essential for PCSK9 structural conformation and biological activity and may have a potential role in FH pathogenesis.
Insights
Certain PCSK9 variants, like p.(E32K) and p.(R469W), disrupt protein structure, reduce low-density lipoprotein receptor (LDLR) levels, and may contribute to Familial Hypercholesterolemia (FH).
Area of Science:
- Genetics and Molecular Biology
- Cardiovascular Research
- Biochemistry
Background:
- Gain-of-function (GOF) variants in PCSK9 are linked to increased low-density lipoprotein receptor (LDLR) degradation and Familial Hypercholesterolemia (FH).
- Understanding the structural and functional impact of PCSK9 missense variants is crucial for FH pathogenesis research.
Purpose of the Study:
- To investigate the effects of identified PCSK9 missense variants on protein structure, LDLR interaction, and potential association with FH.
- To analyze the molecular mechanisms underlying PCSK9 variant function using computational and in vitro approaches.
Main Methods:
- Exon-target gene sequencing in a Brazilian FH cohort to identify PCSK9 variants.
- Molecular dynamics simulations to analyze variant effects on PCSK9 protein structure and interactions.
- In vitro functional studies using HEK293FT cells to assess LDLR expression and PCSK9 secretion.
Main Results:
- Eight PCSK9 missense variants (p.(E32K), p.(E57K), p.(R237W), p.(P279T), p.(A443T), p.(R469W), p.(Q619P), p.(R680Q)) were identified.
- GOF variants p.(E32K) and p.(R469W) altered PCSK9 intramolecular interactions and significantly reduced LDLR expression in cells.
- Variants p.(P279T) and p.(Q619P) also impacted protein stability and interactions, suggesting potential roles in FH.
Conclusions:
- PCSK9 variants p.(E32K), p.(R469W), p.(P279T), and p.(Q619P) disrupt critical intramolecular interactions, affecting PCSK9 conformation and biological activity.
- These variants may play a significant role in the pathogenesis of Familial Hypercholesterolemia.
