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A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes

Nature Chemical Biology volume 17, pages 1271–1280 (2021)Cite this article

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Abstract

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC–Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

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Fig. 1: Design, synthesis and evaluation of 20(S)-OHC chemoproteomics probe 2.
Fig. 2: Probe 2 labels membrane proteins in live NIH-3T3 cells.
Fig. 3: Protein targets of probe 2 converge in distinct gene ontologies and disease signatures.
Fig. 4: 20(S)-OHC identifies enriched and competable probe 2 target proteins.
Fig. 5: Tmem97 is a selective protein target of probe 2.
Fig. 6: 20(S)-OHC enhances the interaction between Tmem97 and NPC1.

Data availability

Raw MS data for this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD027787. Source data are provided with this paper.

Code availability

Code used for MS data analysis is available on GitHub at https://github.com/alex-ondrus/mass_spec_data_imputation.

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Acknowledgements

The authors thank A. Li for synthesizing 20(R)-OHC intermediates, the Beckman Institute/Caltech Flow Cytometry Cell Sorting Facility for cell sorting and A.K. Menon and F.R. Maxfield for helpful discussions. This work was funded by the Margaret Early Foundation Research Trust (A.E.O.), a John Stauffer Charitable Trust SURF Fellowship (T.Z.), a William N. Lacy SURF fellowship (S.P.) and NIH training grant NIH GM07616 (G.C.Z.). The Proteome Exploration Laboratory was supported by NIH OD010788, NIH OD020013, the Betty and Gordon Moore Foundation through grant GBMF775 and the Beckman Institute at Caltech.

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Authors and Affiliations

  1. Department of Chemistry, Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    Yu-Shiuan Cheng, Tianyi Zhang, Xiang Ma, Sarida Pratuangtham, Grace C. Zhang, Amirhossein Mafi & Alison E. Ondrus

  2. Mathematics Department, Northern Alberta Institute of Technology, Edmonton, Alberta, Canada

    Alexander A. Ondrus

  3. Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA

    Brett Lomenick & Jeffrey J. Jones

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Contributions

Y.-S.C. performed all gel- and MS-based chemoproteomics experiments, MS data processing, cloning, gene editing and coimmunoprecipitation experiments. T.Z., X.M., Y.-S.C. and G.C.Z. performed chemical synthesis. S.P. performed homology modeling and molecular docking experiments. G.C.Z. performed Shh-LIGHT2 signaling assays. A.A.O. performed MS data analysis and visualization. A.M. performed molecular dynamics simulations. B.L. participated in experimental design, provided new reagents/analytical tools and participated in data analysis. J.J.J. participated in data analysis. A.E.O., Y.-S.C., T.Z. and S.P. wrote the manuscript.

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Correspondence to Alison E. Ondrus.

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Peer review information Nature Chemical Biology thanks Anant Menon and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Endogenous Smoothened protein is inefficiently resolubilized after acetone precipitation.

a Western blot of Smoothened protein eluted from streptavidin beads after live-cell photocrosslinking to 2, biotinylation, acetone precipitation (to remove excess biotin), and streptavidin enrichment. Analysis of precipitate removed before incubation with streptavidin resin shows that Smoothened is largely insoluble in incubation buffer (1% SDS and 1% IGEPEL CA-630 in PBS) after acetone precipitation. The experiment was repeated five times independently with similar results.

Source data

Extended Data Fig. 2 The enrichment profile of 2 is independent of quantification method and mRNA transcript abundance.

a Volcano plot of proteins enriched by 2 versus DMSO alone, performed as in Fig. 3a but quantified by label-free quantification (LFQ) instead of TMT analysis. The top 12 enriched proteins identified by TMT analysis (labeled in Fig. 3b) are highlighted in red. Data represent two biological replicates consisting of matched experiments with 2 and DMSO. Statistical significance for each protein was calculated using a two-tailed paired t-test. b Plot of enrichment values from label-free versus TMT quantification, showing a positive correlation. Statistical significance, assessed by linear regression in GraphPad Prism, is shown (R2 = 0.2725, p < 0.0001). c Plot of RNA transcript abundances in NIH-3T3 cells (ref. 16) versus enrichment of the corresponding proteins by TMT quantification, showing no significant correlation. Statistical significance, assessed by linear regression in GraphPad Prism, is shown (R2 = 0.002232, p = 0.0503).

Extended Data Fig. 3 Co-treatment with MβCD enhances 20(S)-OHC competition without altering 2-enrichment profiles.

a Streptavidin IRDye 800CW signal for biotinylated proteins in aliquots from mass spectrometry samples. MβCD reduces overall labeling, enhances competition of the probe-labeled 21 kDa band, and preserves equal intensity of non-competable bands in the presence and absence of 20(S)-OHC competitor. The experiments were repeated four times independently with similar results. b Left: Enrichment profiles in the presence and absence of MβCD show a uniform ~2.8-fold decrease. Right: Competition by 20(S)-OHC in the absence and presence of MβCD shows that a greater fraction of proteins are competable in the presence of MβCD. A horizonal line at 1.5-fold competition is provided for reference. c Plot of 2 enrichment values in the presence and absence of MβCD, demonstrating that enrichment profiles are not dramatically influenced by MβCD. Statistical significance, assessed by linear regression in GraphPad Prism, is shown (R2 = 0.1843, p < 0.0001).

Source data

Extended Data Fig. 4 Comparison of Western blot and mass spectrometry quantification.

a Log2(fold change) values for target protein enrichment obtained from mass spectrometry analysis versus quantitative Western blot. Statistical significance, assessed by linear regression in GraphPad Prism, is shown (R2 = 0.4230, p = 0.0808). b Log2(fold change) values for 20(S)-OHC competition obtained from mass spectrometry analysis versus quantitative Western blot. Statistical significance, assessed by linear regression in GraphPad Prism, is shown (R2 = 0.5028, p = 0.0489). For a and b, data points represent quantification of one set of Western blots, where two independent experiments were performed with similar results, plotted against average mass spectrometry values from four biological replicates.

Extended Data Fig. 5 Tmem97 is competably labeled by probe 2 but not alternative sterol probes.

a Crosslinking of probe 2 in NIH-3T3 cells, biotinylation of crosslinked proteins, streptavidin enrichment, and Western blot detection of endogenous Tmem97. Addition of 20(S)-OHC as a competitor reduces the intensity of whole-cell biotin labeling at 21 kDa and streptavidin-enriched Tmem97 in a dose-dependent manner. The experiment was repeated two times independently with similar results. b Crosslinking of probe 2 or PhotoClick Cholesterol in HEK293T cells overexpressing Tmem97-Myc-FLAG. Labeling of overexpressed Tmem97 is competable for probe 2 but not PhotoClick Cholesterol (14). Data are the average of 3 biological replicates ± s.e.m. Statistical significance, evaluated by two-tailed unpaired t-tests between indicated conditions, is shown, ***p = 0.0007; n.s. = not significant. c Quantitative fluorescence analysis of probe 2 labeling at 21 kDa by competitors shown in Fig. 5c, normalized to DMSO. Values are the average of 2 biological replicates.

Source data

Extended Data Fig. 6 Y150 mediates 20(S)-OHC:Tmem97 binding.

a Homology model of mouse Tmem97 based on the cryo-EM structure of human EBP (PDB 6OHT) embedded in a phosphatidylcholine (POPC) bilayer. 20(S)-OHC docks in a central binding pocket. b Quantitative fluorescence analysis of probe 2 labeling with wild-type and Tmem97 and Y150 mutants shown in Fig. 5e, normalized to labeling of the wild-type Tmem97 construct in the presence of DMSO alone. Values are the average of 2 biological replicates.

Source data

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Supplementary Information

Supplementary Tables 1 and 2 and Notes 1 and 2.

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Source Data Extended Data Fig. 1

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Cheng, YS., Zhang, T., Ma, X. et al. A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes. Nat Chem Biol 17, 1271–1280 (2021). https://doi.org/10.1038/s41589-021-00907-2

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