Abstract
Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein–protein interactions. They engage with specific ligands and translocate to the nucleus, where they bind the DNA and activate extensive transcriptional programs. Therefore, gaining a comprehensive overview of the protein–protein interactions they establish requires methods that function effectively throughout the cell with fast dynamics and high reproducibility. Focusing on estrogen receptor alpha (ESR1), the founding member of the nuclear receptor family, this chapter describes a new lentiviral system that allows the expression of TurboID-hemagglutinin (HA)-2 × Strep tagged proteins in mammalian cells to perform fast proximity biotinylation assays. Key validation steps for these reagents and their use in interactome mapping experiments in two distinct breast cancer cell lines are described. Our protocol enabled the quantification of ESR1 interactome generated by cellular contexts that were hormone-sensitive or not.
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Acknowledgments
This research was supported by Project Grants from the Canadian Institutes of Health Research (PJT-168969, and PJT-152948) and Leader’s Opportunity Funds from the Canada Foundation for Innovation (37454, 41426). L.A. is supported by a scholarship from the Fondation du CHU de Québec. S.A.B. is supported by a doctoral scholarship from the Fonds de Recherche du Québec - Santé (FRQS). P.-E.K.T. is supported by a Bourse Distinction Luc Bélanger from the Cancer Research Center – Université Laval and by a doctoral scholarship from the FRQS. J.-P.L. is supported by a Junior 1 salary award from the FRQS. A.F.-T. is a tier 2 Canada Research Chair in Molecular Virology and Genomic Instability and is supported by the Fondation J.-Louis Lévesque.
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Table S1
SAINTexpress results of TurboID analysis of ESR1 in MCF-7 and MDA-MB-231 cells (XLSX 232 kb)
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Agbo, L., Blanchet, S.A., Kougnassoukou Tchara, PE., Fradet-Turcotte, A., Lambert, JP. (2022). Comprehensive Interactome Mapping of Nuclear Receptors Using Proximity Biotinylation. In: Geddes-McAlister, J. (eds) Proteomics in Systems Biology. Methods in Molecular Biology, vol 2456. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2124-0_15
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