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High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants.

Lauri Vanharanta1,2, Johan Peränen1,2, Simon G Pfisterer1

  • 1Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.

Traffic (Copenhagen, Denmark)
|March 8, 2020
PubMed
Summary

The common NPC1 wild-type variant (WT-V) used in research has a defect (L472P) that impairs lysosomal cholesterol export. This finding clarifies subtle Niemann-Pick C1 protein functional differences.

Keywords:
Niemann-Pick C1cholesterol transportgene variantslate endosomeslipid dropletslysosomal storage diseases

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Area of Science:

  • Cell Biology
  • Genetics
  • Biochemistry

Background:

  • The human Niemann-Pick C1 (NPC1) gene is crucial for cellular cholesterol homeostasis.
  • Genetic variants in NPC1 can cause Niemann-Pick disease, but the function of many rare variants is unknown.
  • A widely used NPC1 wild-type variant (WT-V) differs from the reference sequence, potentially impacting experimental results.

Purpose of the Study:

  • To investigate the functional impact of amino acid changes in the NPC1 WT-V construct.
  • To quantitatively assess the effect of NPC1 variants on lysosomal cholesterol transport and lipid droplet formation.
  • To elucidate subtle differences in NPC1 protein function using advanced cellular and computational methods.

Main Methods:

  • Engineered NPC1-null human cells using CRISPR/Cas9 gene editing.
  • Generated stable cell lines expressing NPC1 variants from the AAVS1 safe-harbor locus.
  • Employed high-content imaging and automated image analysis to quantify lysosomal cholesterol and lipid droplets.
  • Utilized all-atom molecular dynamics simulations to model protein structural changes.

Main Results:

  • The L472P change in NPC1 WT-V was identified as compromising NPC1 functionality.
  • This variant significantly impaired lysosomal cholesterol export in quantitative imaging assays.
  • Molecular dynamics simulations revealed that L472P disrupts the NPC1 cholesterol efflux tunnel by altering domain positioning.

Conclusions:

  • The NPC1 WT-V construct contains functional defects that impact experimental outcomes.
  • Stable expression systems combined with quantitative imaging and simulations are powerful tools for dissecting subtle protein functional differences.
  • This study clarifies the functional consequences of specific NPC1 variants, improving the accuracy of Niemann-Pick disease research.