TY - JOUR
T1 - Triazoles inhibit cholesterol export from lysosomes by binding to NPC1
AU - Trinha, Michael N.
AU - Lua, Feiran
AU - Lib, Xiaochun
AU - Dasa, Akash
AU - Liangd, Qiren
AU - De Brabanderd, Jef K.
AU - Browna, Michael S.
AU - Goldsteina, Joseph L.
N1 - Funding Information:
We thank our colleagues Yansong Gao, Donald Anderson, and Ting Han for helpful suggestions; Lisa Beatty, Shomanike Head, and Lucie Batte for invaluable help with tissue culture; and Jessica Proulx for excellent technical assistance. This work was supported by grants from the National Institutes of Health (HL20948, to J.L.G. and M.S.B.) and the Robert A. Welch Foundation (I-1422, to J.K.D.B.). X.L. is supported by funds from Howard Hughes Medical Institute (Günter Blobel, Investigator), and is a Gordon and Betty Moore Foundation Fellow of Life Sciences Research Foundation.
PY - 2017/1/3
Y1 - 2017/1/3
N2 - Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also crosslinked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterolsensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.
AB - Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also crosslinked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterolsensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.
KW - Cholesterol transport
KW - Lipid nanodiscs
KW - Niemann-Pick C disease
KW - Photoactivatable cross-linking
KW - Sterol-sensing domain
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U2 - 10.1073/pnas.1619571114
DO - 10.1073/pnas.1619571114
M3 - Article
C2 - 27994139
AN - SCOPUS:85008155368
SN - 0027-8424
VL - 114
SP - 89
EP - 94
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 1
ER -