Methods
Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking[S]

https://doi.org/10.1194/jlr.D091470Get rights and content
Under a Creative Commons license
open access

Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, trans-sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.

lipids
lipoproteins
metabolism
biorthogonal probes
regulation

Cited by (0)

This work was supported by National Institutes of Health Grants R01 HL067773 (D.F.C., D.S.O., J.E.S.), T32 HL134635 (M.F.), and F31 HL142167 (M.F.) and the Taylor Family Institute for Innovative Psychiatric Research (D.F.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

    Abbreviations:

    acLDL

    acetylated LDL

    CHO

    Chinese hamster ovary

    HA

    human influenza hemagglutinin

    HMGCS

    HMG-CoA synthase

    LAL

    lysosomal acid lipase

    LALi

    lalistat

    LPDM

    lipoprotein-deficient media

    NPC

    Niemann-Pick C

    qPCR

    quantitative PCR

    Rplp0

    ribosomal protein lateral stalk subunit P0

    SRA

    scavenger receptor A

    THPTA

    tris-hydroxypropyltriazolylmethylamine

    VAMP7

    vesicle-associated membrane protein 7

[S]

The online version of this article (available at http://www.jlr.org) contains a supplement.