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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 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.

SUBMITTER: Cheng YS

PROVIDER: S-EPMC8607797 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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

Cheng Yu-Shiuan YS Zhang Tianyi T Ma Xiang X Pratuangtham Sarida S Zhang Grace C GC Ondrus Alexander A AA Mafi Amirhossein A Lomenick Brett B Jones Jeffrey J JJ Ondrus Alison E AE

Nature chemical biology 20211119 12

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 l ...[more]

PMID: 34799735

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Project description:BackgroundThe sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5.MethodsTo gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells.ResultsWe identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl--co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25.ConclusionsResults support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.

Dataset Information

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

Publications

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

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