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

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Vanharanta , L , Peränen , J , Pfisterer , S G , Enkavi , G , Vattulainen , I & Ikonen , E 2020 , ' High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants ' , Traffic , vol. 21 , no. 5 , pp. 386-397 . https://doi.org/10.1111/tra.12727

Title: High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants
Author: Vanharanta, Lauri; Peränen, Johan; Pfisterer, Simon G.; Enkavi, Giray; Vattulainen, Ilpo; Ikonen, Elina
Contributor: University of Helsinki, Medicum
University of Helsinki, Institute of Biotechnology
University of Helsinki, Department of Anatomy
University of Helsinki, Materials Physics
University of Helsinki, Department of Physics
University of Helsinki, STEMM - Stem Cells and Metabolism Research Program
Date: 2020-05
Language: eng
Number of pages: 12
Belongs to series: Traffic
ISSN: 1398-9219
URI: http://hdl.handle.net/10138/314545
Abstract: Abstract The human Niemann-Pick C1 (NPC1) gene encoding a 1278 amino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild-type variant (WT-V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe-harbor locus on an NPC1-null background engineered by CRISPR/Cas9 editing. We then employed high-content imaging with automated image analysis to quantitatively assess LDL-induced, time-dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT-V compromises NPC1 functionality in lysosomal cholesterol export. All-atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C-terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT-V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function.
Subject: cholesterol transport
gene variants
late endosomes
lipid droplets
lysosomal storage diseases
Niemann-Pick C1
3111 Biomedicine
1182 Biochemistry, cell and molecular biology
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