IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF INTERACTION PARTNERS OF THE TUMOR SUPPRESSOR PALB2

INTRODUCTION AND OBJECTIVES: The DNA damage response (DDR) proper function maintenance is fundamental for tumor suppression activity. DNA double-strand breaks (DSB) pose the greatest threat to DNA stability and the homologous recombination repair (HR) pathway is the most accurate DSB repair mechanism. Following DSB, RAD51 recombinase is recruited to DNA damage sites in HR repair, promoted by the BRCA1-PALB2-BRCA2 complex. PALB2 (PArtner and Localizer of BRCA2) interacts with BRCA1 at DNA damage sites and functions as a linker between BRCA1 and BRCA2. BRCA1-PALB2-BRCA2 complex formation is crucial for the appropriate HR function. Mutations in the genes encoding for these proteins are associated with a high risk of developing breast cancer and other tumor types. Besides being associated with hereditary breast cancer, the PALB2 gene, also known as FANCN, is also associated with pancreatic cancer and Fanconi Anemia syndrome. Little is known about PALB2 protein complexes formation and, since its identification, few studies have explored its protein interaction partners. In view of PALB2 relevance for HR repair and the need for better understanding its functions, this work aims to characterize PALB2 WD40 domain new interaction partners by conducting a yeast two-hybrid (Y2H) screening. MATERIAL AND METHODS: We have conducted a Y2H screening using PALB2 C-terminal (WD40 domain) segment as a bait and a universal human cDNA library (Clontech, USA) as prey. Isolated clones were sequenced and the results analyzed by sequence alignment using BLAST. Interactions identified will be validated by GST-pull-down assays using human cell lines. Functional characterization of the validated PALB2 heterodimers will be performed by co-immunoprecipitation and co-immunofluorescence microscopy assays. Other assays to better assess the identified targets' role in the BRCA1-PALB2-BRCA2 complex formation and function will be conducted. RESULTS AND CONCLUSION: We have identified 7 new PALB2 WD40 interactions. We highlight the newly identified partners COPS5 and SNAPIN. We identified two independent clones coding for COPS5. Both COPS5 and SNAPIN were also detected in an unpublished Y2H screening conducted by our group using PALB2 full-length as a bait, strongly suggesting they could be bonafide PALB2 functional partners. COPS5 is part of the COP9 signalosome (CSN) deneddylase complex. Neddylation/deneddylation, similar to ubiquitination and sumoylation, is involved in different cell signaling processes, including DDR. SNAPIN is associated with organelles protein docking and has a potential role in cell cycle regulation. We expect that the resulting data from this project will validate and characterize new interacting partners of PALB2, contributing to the better understanding of the cellular functions of this important tumor suppressor.