Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

Feiran Lu,Michael S Brown,Lina Abi-Mosleh,Akash Das,Jef K De Brabander,Qiren Liang,Joseph L Goldstein

doi:10.7554/elife.12177

Feiran Lu, Michael S Brown + Show 5 more

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https://doi.org/10.7554/elife.12177

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Journal: eLife	Publication Date: Dec 8, 2015
Citations: 273	License type: CC BY 4.0

Affiliation: The University of Texas Southwestern Medical Center

Abstract

Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.

Highlights

Niemann-Pick C1 (NPC1), a lysosomal membrane protein, has emerged as the culprit in two fatal diseases
We show the inhibitory constant (Ki) that represents the concentration producing a 50% inhibition of the transfer of low density lipoprotein (LDL)-derived cholesterol from lysosomes to endoplasmic reticulum (ER) as determined with the cholesterol esterification assay
The current data provide further insight into the complex transport functions of NPC1, a lysosomal membrane protein required for cellular cholesterol homeostasis as well as for susceptibility to Ebola and other filoviruses

Summary

Introduction

Niemann-Pick C1 (NPC1), a lysosomal membrane protein, has emerged as the culprit in two fatal diseases. Mutations in NPC1 underlie Niemann-Pick C disease, a devastating lysosomal storage disease in which accumulation of cholesterol in lysosomes causes abnormalities in brain, liver, lung, and other tissues, leading to death in the teenage years (Pentchev, 2004). NPC1 mediates the egress of cholesterol that has entered lysosomes through the receptor-mediated endocytosis of low density lipoprotein (LDL) (Liscum et al, 1989). When cell membranes are depleted of cholesterol, LDL receptors bind circulating LDL, internalize it, and deliver it to lysosomes where acid lipase hydrolyzes the lipoprotein’s cholesteryl esters (Brown and Goldstein, 1986). The liberated cholesterol binds to NPC2, a soluble diffusible protein in the lysosome lumen (Sleat et al, 2004). Designated a hydrophobic handoff, this transfer occurs in such a way that hydrophobic cholesterol is shielded from the aqueous environment (Kwon et al, 2009)

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