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.
Highlights
Cholesterol is essential for normal cellular functions as it modulates the properties of cell membranes and affects the functions of membraneassociated proteins
All-atom molecular dynamics (MD) simulations suggest that this mutation can disrupt the conformation of luminal domains and the cholesterol efflux tunnel of Niemann-Pick C1 (NPC1)
To investigate the function of NPC1 variants, we first disrupted the endogenous NPC1 locus using CRISPR/Cas9-mediated editing in human A431 cells that are well suited for genetic engineering and imaging (Figure 1A)
Summary
Cholesterol is essential for normal cellular functions as it modulates the properties of cell membranes and affects the functions of membraneassociated proteins. According to the prevailing view, the MLD of NPC1 recruits NPC2 to deliver luminal cholesterol to the sterol binding NTD of NPC1.6 Recent findings suggest the existence of an NPC1 intramolecular tunnel through which cholesterol passes the lysosomal glycocalyx prior to insertion to the lysosomal limiting membrane.[10]. Loss of function mutations in the NPC1 gene lead to the build-up of cholesterol in the endo-lysosomal compartment and a progressive neurodegenerative disorder, NPC disease, which leads to premature death.[11] Genetic testing together with staining of patient cells with filipin, a fluorescent cholesterol-binding compound, are used to confirm the disease diagnosis. Our data indicate that the L472P change affects the subcellular distribution of NPC1 and compromises the cholesterol export function of the protein. All-atom molecular dynamics (MD) simulations suggest that this mutation can disrupt the conformation of luminal domains and the cholesterol efflux tunnel of NPC1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
