KB-project – University of Copenhagen

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Department of Cellular and Molecular Medicine > icmm-staff > lopez-contreras, andres > KB-project

Project

Identification of novel targets for the BRCA1 ubiquitin ligase activity

BRCA1 is an essential tumor suppressor, being the most frequently mutated gene in familial breast cancer. BRCA1 has several roles in cell cycle regulation, DNA repair, centrosome amplification, chromatin regulation and transcriptional regulation, processes that prevent genomic instability (Yarden and Papa, Mol Cancer Ther, 2006). It is generally accepted that BRCA1 tumor suppressor function is due to its role in DNA repair. However, the precise molecular mechanisms by which BRCA1 exerts its function remain unclear.

The only known activity on BRCA1 is an E3 UQ ligase. Importantly, the generation of a mouse model with a mutation in a catalytic residue of the RING domain (C61G) demonstrated that BRCA1 E3 UQ ligase activity is essential for its tumor suppressor function (Drost et al., Cancer Cell, 2011). Therefore, identifying which proteins are ubiquitinated by BRCA1 will be crucial for understanding the molecular function of BRCA1. To date, a limited number of BRCA1 UQ ligase activity substrates have been identified. Importantly, large-scale analysis of UQ modifications in a proteome is now feasible (Kim et al., Mol Cell, 2011). One limitation of this published model is, however, its constitutive nature, which does not allow for the isolation of primary cells deficient in BRCA1 UQ ligase activity. We have now resolved this problem by generating a conditional knock-in mutant allele, so that we can inactivate BRCA1 UQ ligase activity in any primary cell of choice.

Fig 3. Conditional KI C61G BRCA1 mouse model based on pFLEX technology: “UQ ligase dead”. A) BRCA1 gene structure. Exons encoding the RING (UQ ligase activity) and the BRCT domains (phospho-proteins interaction) are underlined. B) Cre-mediated recombination diagram. WT exons 3, 4 and 5 are colored in blue, and exon 4 containing the C61G mutation is colored in orange. White and black triangles represent LoxP and Lox511 that recombine among them. In basal conditions, mutated exon 4 is inverted and not transcribed. When Cre recombinase is active, WT exon 4 is excised and mutant exon 4 is inverted giving raise to the expression of mutant BRCA1.

In this project, we aim to perform a comprehensive proteomic study to identify novel BRCA1 UQ ligase substrates in vivo and to determine their potential clinical impact in breast and ovarian cancer. The use of a conditional knock-in mouse model, will allow us to induce mutated BRCA1 expression in adult tissues and to investigate BRCA1 UQ ligase targets in vivo in clinically relevant cells and tissues such as mammary gland, ovary and tumors. The novel BRCA1 UQ targets identified will be validated and functionally characterized by different biochemical and cellular approaches.

This project is funded by a KBVU Project from the Danish Cancer Society.

Participants:
Andres J Lopez-Contreras (Group Leader)
Alexandra Avram (Lab technician
Mauro Sbroggió (postdoc) 
Eliene Albers (PhD student)

Collaborators:
Dr. Oscar Fernandez-Capetillo, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Prof. Ian D. Hickson, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark
Prof. Rafael Peñafiel-Garcia, University of Murcia, Spain
Dr. Javier Muñoz, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Dr. Andre Nussenzweig, National Cancer Institute, National Institutes of Health, Bethesda, USA
Dr. Jeremy A. Daniel, Center for Protein Research, University of Copenhagen, Denmark.