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Comprehensive Interactome Mapping of Nuclear Receptors Using Proximity Biotinylation

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Proteomics in Systems Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2456))

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.

Author information

Authors and Affiliations

  1. Department of Molecular Medicine, Cancer Research Center and Big Data Research Center, Université Laval, Québec, QC, Canada

    Lynda Agbo, Pata-Eting Kougnassoukou Tchara & Jean-Philippe Lambert

  2. Endocrinology and Nephrology Division, CHU de Québec-Université Laval Research Center, Québec, QC, Canada

    Lynda Agbo, Pata-Eting Kougnassoukou Tchara & Jean-Philippe Lambert

  3. Oncology Division, CHU de Québec-Université Laval Research Center, Québec, QC, Canada

    Sophie Anne Blanchet & Amélie Fradet-Turcotte

  4. Department of Molecular Biology, Medical Biochemistry and Pathology, Cancer Research Center, Université Laval, Québec, QC, Canada

    Sophie Anne Blanchet & Amélie Fradet-Turcotte

Authors
  1. Lynda Agbo
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  2. Sophie Anne Blanchet
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  3. Pata-Eting Kougnassoukou Tchara
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  4. Amélie Fradet-Turcotte
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  5. Jean-Philippe Lambert
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Corresponding authors

Correspondence to Amélie Fradet-Turcotte or Jean-Philippe Lambert .

Editor information

Editors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

    Jennifer Geddes-McAlister

1 Electronic Supplementary Material

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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  • DOI: https://doi.org/10.1007/978-1-0716-2124-0_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2123-3

  • Online ISBN: 978-1-0716-2124-0

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