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

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 unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. In this Resource, we present a structurally precise chemoproteomics probe for the osteogenic 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 (σ2 receptor), enabling molecular reconstruction of the Tmem97:20(S)-OHC binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of OHC activity and identify actionable targets for molecular therapy.

Mass spectrometry‐based proteomics analysis of human globus pallidus from progressive supranuclear palsy patients discovers multiple disease pathways

“…These results are likely due to sample preparation differences between proteomics and Western blot experiments. Although quantifications by MS and Western blot did not confirm all three proteins, it is well‐known that mass spectrometry‐based quantification data often shows a poor correlation with Western blot‐based quantification 39,40 …”

Identification of unusual oxysterols biosynthesised in human pregnancy by charge-tagging and liquid chromatography - mass spectrometry

“…20S-HC also inhibits the processing of SREBP-2 (sterol regulatory element-binding protein 2) to its active form as the master transcription factor regulating cholesterol biosynthesis (35, 36), presumably by binding to INSIG (insulin induced gene) in a manner similar to other side-chain hydroxycholesterols (37). Recently, 20S-HC has been identified as a ligand to the sigma 2 (s2) receptor (38), also known as transmembrane protein 97 (Tmem97), which is expressed in the central nervous system (39), and has been suggested to be a chaperone protein for NPC1 (Niemann Pick C1), the lysosomal cholesterol transport protein (38). The enzyme required to biosynthesise 20S-HC has not been identified, although CYP11A1 has been reported to generate both 20-hydroxyvitamin D 3 and 20,22dihydroxyvitamin D 3 or 20,23-dihydroxyvitamin D 3 from vitamin D 3 (40,41).…”

Identification of Unusual Oxysterols Biosynthesised in Human Pregnancy by Charge-Tagging and Liquid Chromatography - Mass Spectrometry

“…20S-HC also inhibits the processing of SREBP-2 (sterol regulatory element-binding protein 2) to its active form as the master transcription factor regulating cholesterol biosynthesis (34, 35), presumably by binding to INSIG (insulin induced gene) in a manner similar to other side-chain hydroxycholesterols (36). Recently, 20S-HC has been identified as a ligand to the sigma 2 (σ2) receptor (37), also known as transmembrane protein 97 (Tmem97), which is expressed in the central nervous system (38), and has been suggested to be a chaperone protein for NPC1 (Niemann Pick C1), the lysosomal cholesterol transport protein (37). The enzyme required to biosynthesise 20S-HC has not been identified, although CYP11A1 has been reported to generate both 20-hydroxyvitamin D 3 and 20,22-dihydroxyvitamin D 3 or 20,23-dihydroxyvitamin D 3 from vitamin D 3 (39, 40).…”