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Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells

Nature Methods volume 10, pages 259–264 (2013)Cite this article

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Abstract

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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Figure 1: Clickable photoreactive sterol probes.
Figure 2: Gel-based profiling of sterol-binding proteins in HeLa cells.
Figure 3: Mass spectrometry–based profiling of sterol-binding proteins in HeLa cells.
Figure 4: Analysis of group I proteins.

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Acknowledgements

We thank P. Baran for helpful advice on the synthesis of sterol probes, and A. Rheingold and C. Moore for X-ray crystallographic analysis. This work was supported by US National Institutes of Health (CA132630) and the Skaggs Institute for Chemical Biology.

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Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Jonathan J Hulce, Armand B Cognetta, Micah J Niphakis, Sarah E Tully & Benjamin F Cravatt

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  1. Jonathan J Hulce
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  2. Armand B Cognetta
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  3. Micah J Niphakis
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  4. Sarah E Tully
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Contributions

J.J.H., S.E.T., M.J.N. and B.F.C. designed experiments; J.J.H., A.B.C. and S.E.T. performed experiments; J.J.H. and B.F.C. analyzed data; J.J.H. and B.F.C. wrote the manuscript.

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Correspondence to Benjamin F Cravatt.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Table 5, Supplementary Note (PDF 2876 kb)

Supplementary Table 1

List of group I–IV proteins with experimental SILAC ratios. Groups I, II, III, and IV are presented on separate sheets, with the mean SILAC ratio obtained in the trans versus no–UV light, trans versus PEA-DEA, and trans versus 10 × cholesterol competition (Ch) shown. For proteins that were not detected in the no–UV light experiments but were found in the vehicle control experiments, the vehicle ratios are shown instead (colored in blue). For group I proteins, gene expression data are presented as 1 = yes; 0 = no, where ‘yes’ means that the gene expression change was > twofold at 30 min cholesterol treatment or > twofold at 12 h cholesterol treatment. All quantifications were manually filtered here. ND, not detected. See Supplementary Table 2 for a list of individual peptide sequences and ratios detected for each protein. (XLSX 152 kb)

Supplementary Table 2

Complete proteomic data for trans versus no UV light and versus vehicle. Data sets include the individual peptides detected for each protein, their SILAC ratios and charge states. SILAC ratios are presented as the mean of duplicates but are not manually filtered. (XLSX 1574 kb)

Supplementary Table 3

Data sets for each set of SILAC experiments. Mean ratios are presented on separate sheets for each set of SILAC experiments. Control experiments (no UV light and vehicle comparisons) are shown together, are unfiltered and were each run in duplicate. PEA-DA comparisons and cholesterol competition, shown together, were run in duplicate and quadruplicate, respectively, and background proteins from control runs are removed. Probe comparisons (trans versus epi, cis and trans), shown together, were run in duplicate, and background proteins are removed. All quantifications are raw and were not manually filtered. (XLSX 211 kb)

Supplementary Table 4

Complete gene expression profiles for cholesterol and probe treatments. The signal intensity for each gene in each microarray experiment is included. The three conditions tested were: (i) control/vehicle: cells were untreated, and were collected and processed in parallel with the other conditions; (ii) 30 min: cells received 20 μM cis probe + 100 μM cholesterol for 30 min before harvest; (iii) 12 h: cells received 20 μM cis probe + 100 μM cholesterol for 12 h before harvest. Total mRNA was extracted for each condition, and gene expression was analyzed by HU133 Set GeneChip from Affymetrix. (XLSX 6362 kb)

Supplementary Table 6

Unenriched versus trans-enrichment membrane proteome comparison by spectral counting. Raw data set presenting proteins in groups I–IV that were detected with >10 average spectral counts over quadruplicate trans probe–mediated enrichments (columns t1–t4) or unenriched membrane proteomes (columns u1–u4). (XLSX 108 kb)

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Hulce, J., Cognetta, A., Niphakis, M. et al. Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells. Nat Methods 10, 259–264 (2013). https://doi.org/10.1038/nmeth.2368

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