Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells

The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.
Nature Methods (Impact Factor: 32.07). 02/2013; 10(3). DOI: 10.1038/nmeth.2368
Source: PubMed


Cholesterol is an essential structural component of cellular membranes and serves as a precursor for several classes of signaling molecules. Cholesterol exerts its effects and is, itself, regulated in large part by engagement in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown. Here we describe a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells. We identified over 250 cholesterol-binding proteins, including receptors, channels and enzymes involved in many established and previously unreported interactions. Prominent among the newly identified interacting proteins were enzymes that regulate sugars, glycerolipids and cholesterol itself as well as proteins involved in vesicular transport and protein glycosylation and degradation, pointing to key nodes in biochemical pathways that may couple sterol concentrations to the control of other metabolites and protein localization and modification.

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Available from: Micah J Niphakis, Jul 02, 2014
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    • "Upon completion of membrane fusion, a tyrosine-based sorting motif (YGRL, position 140–143) located between the N-terminal domain and the SNARE motif is activated, which results in its re-sorting to the TGN (Jahn and Scheller, 2006; Jung et al., 2012). Importantly, STX6 binds to cholesterol (Hulce et al., 2013) and has been previously implicated in cholesterol transport; it contributes to the delivery of late-endosome-derived cholesterol to the ER via the TGN (Urano et al., 2008) and of lipids and proteins that are required for caveolae endocytosis to the plasma membrane (Choudhury et al., 2006). This work has shown that inhibition of STX6 leads to a reduction of caveolin-1 and caveolae at the cell surface, lending further support for a model by which STX6 regulates secretory pathways in a cholesterol-sensitive manner. "
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    • "Given that most proteomics screens involve TAG-enriched LDs, these proteins may represent unique LD-binding proteins that have a preference for CE-enriched LDs, or may be unique to LDs formed in granulosa cells; further analysis of these unique proteins is required to determine their function on the LD. A recent study using photoreactive sterol probes in combination with quantitative mass spectrometry to map cholesterol-protein interactions identified 250-cholesterol-binding proteins, which included receptors, channels and enzymes [26]. Our screen detected 34 similar proteins on CE-enriched LDs, including Vdac 1 and 2, 17-β hydroxysteroid dehydrogenase, Vapa, Scarb1, and solute carrier family 27 (Slc27). "
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