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

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. In this manuscript, 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 catalogue 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 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.