BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.

Proteomic profile of BxPC-3 cells after treatment with BRC4 / Viola Previtali, Samuel H. Myers, Laura Poppi, Kieran Wynne, Irene Casamassima Stefania Girotto, Giuseppina Di Stefano, Fulvia Farabegoli, Marinella Roberti, Giorgio Oliviero, Andrea Cavalli. - In: JOURNAL OF PROTEOMICS. - ISSN 1876-7737. - STAMPA. - 288:(2023), pp. 1-11.

Proteomic profile of BxPC-3 cells after treatment with BRC4

Laura Poppi;Giuseppina Di Stefano;Fulvia Farabegoli;Marinella Roberti;Andrea Cavalli
2023

Abstract

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.
2023
Proteomic profile of BxPC-3 cells after treatment with BRC4 / Viola Previtali, Samuel H. Myers, Laura Poppi, Kieran Wynne, Irene Casamassima Stefania Girotto, Giuseppina Di Stefano, Fulvia Farabegoli, Marinella Roberti, Giorgio Oliviero, Andrea Cavalli. - In: JOURNAL OF PROTEOMICS. - ISSN 1876-7737. - STAMPA. - 288:(2023), pp. 1-11.
Viola Previtali, Samuel H. Myers, Laura Poppi, Kieran Wynne, Irene Casamassima Stefania Girotto, Giuseppina Di Stefano, Fulvia Farabegoli, Marinella Roberti, Giorgio Oliviero, Andrea Cavalli
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/969069
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