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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    • "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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    ABSTRACT: Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.
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