TY - JOUR
T1 - Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection
AU - Lu, Feiran
AU - Liang, Qiren
AU - Abi-Mosleh, Lina
AU - Das, Akash
AU - de Brabander, Jef K.
AU - Goldstein, Joseph L.
AU - Brown, Michael S.
N1 - Publisher Copyright:
© Lu et al.
PY - 2015/12/8
Y1 - 2015/12/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84955259695&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84955259695&partnerID=8YFLogxK
U2 - 10.7554/eLife.12177
DO - 10.7554/eLife.12177
M3 - Article
C2 - 26646182
AN - SCOPUS:84955259695
SN - 2050-084X
VL - 4
JO - eLife
JF - eLife
IS - December2015
M1 - e12177
ER -