Tampere University of Technology

TUTCRIS Research Portal

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

Research output: Contribution to journalArticleScientificpeer-review

Standard

High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants. / Vanharanta, Lauri; Peränen, Johan; Pfisterer, Simon G.; Enkavi, Giray; Vattulainen, Ilpo; Ikonen, Elina.

In: Traffic, Vol. 21, No. 5, 2020, p. 386-397.

Research output: Contribution to journalArticleScientificpeer-review

Harvard

Vanharanta, L, Peränen, J, Pfisterer, SG, 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

APA

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, 21(5), 386-397. https://doi.org/10.1111/tra.12727

Vancouver

Vanharanta L, Peränen J, Pfisterer SG, Enkavi G, Vattulainen I, Ikonen E. High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants. Traffic. 2020;21(5):386-397. https://doi.org/10.1111/tra.12727

Author

Vanharanta, Lauri ; Peränen, Johan ; Pfisterer, Simon G. ; Enkavi, Giray ; Vattulainen, Ilpo ; Ikonen, Elina. / High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants. In: Traffic. 2020 ; Vol. 21, No. 5. pp. 386-397.

Bibtex - Download

@article{5ceca4e25f0640cfa9482ac40f3ad58c,
title = "High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants",
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.",
keywords = "cholesterol transport, gene variants, late endosomes, lipid droplets, lysosomal storage diseases, Niemann-Pick C1",
author = "Lauri Vanharanta and Johan Per{\"a}nen and Pfisterer, {Simon G.} and Giray Enkavi and Ilpo Vattulainen and Elina Ikonen",
year = "2020",
doi = "10.1111/tra.12727",
language = "English",
volume = "21",
pages = "386--397",
journal = "Traffic",
issn = "1398-9219",
publisher = "Wiley",
number = "5",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - High-content imaging and structure-based predictions reveal functional differences between Niemann-Pick C1 variants

AU - Vanharanta, Lauri

AU - Peränen, Johan

AU - Pfisterer, Simon G.

AU - Enkavi, Giray

AU - Vattulainen, Ilpo

AU - Ikonen, Elina

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - cholesterol transport

KW - gene variants

KW - late endosomes

KW - lipid droplets

KW - lysosomal storage diseases

KW - Niemann-Pick C1

U2 - 10.1111/tra.12727

DO - 10.1111/tra.12727

M3 - Article

VL - 21

SP - 386

EP - 397

JO - Traffic

JF - Traffic

SN - 1398-9219

IS - 5

ER -