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

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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.

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Feiran Lu

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.
Qiren Liang

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.
Lina Abi-Mosleh

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.
Akash Das

Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.
Jef K De Brabander

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.
Joseph L Goldstein

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States

For correspondence
joe.goldstein@utsouthwestern.edu

Competing interests
The authors declare that no competing interests exist.
Michael S Brown

Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, United States

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The authors declare that no competing interests exist.

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