RESUMO
Bromodomains (BRDs) are a family of evolutionarily conserved domains that are the main readers of acetylated lysine (Kac) residues on proteins. Recently, numerous BRD-containing proteins have been proven essential for transcriptional regulation in numerous contexts. This is exemplified by the multi-subunit mSWI/SNF chromatin remodeling complexes, which incorporate up to 10 BRDs within five distinct subunits, allowing for extensive integration of Kac signaling to inform transcriptional regulation. As dysregulated transcription promotes oncogenesis, we sought to characterize how BRD-containing subunits contribute molecularly to mSWI/SNF functions. By combining genome editing, functional proteomics, and cellular biology, we found that loss of any single BRD-containing mSWI/SNF subunit altered but did not fully disrupt the various mSWI/SNF complexes. In addition, we report that the downregulation of BRD7 is common in invasive lobular carcinoma and modulates the interactome of its homologue, BRD9. We show that these alterations exacerbate sensitivities to inhibitors targeting epigenetic regulatorsânotably, inhibitors targeting the BRDs of non-mSWI/SNF proteins. Our results highlight the interconnections between distinct mSWI/SNF complexes and their far-reaching impacts on transcriptional regulation in human health and disease. The mass spectrometry data generated have been deposited to MassIVE and ProteomeXchange and assigned the identifiers MSV000089357, MSV000089362, and PXD033572.
Assuntos
Proteínas Cromossômicas não Histona , Fatores de Transcrição , Humanos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Domínios ProteicosRESUMO
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.
Assuntos
Hormônios , Receptores Citoplasmáticos e Nucleares , Animais , Biotinilação , Mamíferos , Mapeamento de Interação de Proteínas/métodos , Reprodutibilidade dos TestesRESUMO
Triple negative breast cancer (TNBC) is one of the most aggressive form of breast cancer (BC) with the highest mortality due to high rate of relapse, resistance, and lack of an effective treatment. Various molecular approaches have been used to target TNBC but with little success. Here, using machine learning algorithms, we analyzed the available BC data from the Cancer Genome Atlas Network (TCGA) and have identified two potential genes, TBC1D9 (TBC1 domain family member 9) and MFGE8 (Milk Fat Globule-EGF Factor 8 Protein), that could successfully differentiate TNBC from non-TNBC, irrespective of their heterogeneity. TBC1D9 is under-expressed in TNBC as compared to non-TNBC patients, while MFGE8 is over-expressed. Overexpression of TBC1D9 has a better prognosis whereas overexpression of MFGE8 correlates with a poor prognosis. Protein-protein interaction analysis by affinity purification mass spectrometry (AP-MS) and proximity biotinylation (BioID) experiments identified a role for TBC1D9 in maintaining cellular integrity, whereas MFGE8 would be involved in various tumor survival processes. These promising genes could serve as biomarkers for TNBC and deserve further investigation as they have the potential to be developed as therapeutic targets for TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas/genética , Antígenos de Superfície/genética , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Aprendizado de Máquina , Recidiva Local de Neoplasia/genética , Prognóstico , Transcriptoma/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Steroid hormones have far-ranging biological impacts and more are continuously being uncovered. Over the last decades, proteomics approaches have become key to better understand biological processes. Due to multiple technical breakthroughs allowing for the concurrent identification and/or quantification of thousands of analytes using mass spectrometers, researchers employing proteomics tools today can now obtain truly holistic views of multiple facets of the human proteome. Here, we review how the field of proteomics has contributed to discoveries about steroid hormones, their receptors and their impact on human pathologies. In particular, the involvement of steroid receptors in cancer initiation, development, metastasis and treatment will be highlighted. Techniques at the forefront of the proteomics field will also be discussed to present how they can contribute to a better understanding of steroid hormone receptors.
