Proximity Proteomics Reveals USP44 Forms a Complex with BRCA2 in Neuroblastoma Cells and Is Required to Prevent Chromosome Breakage.

Background/objectives: The enzyme ubiquitin-specific protease 44 (USP44) is a deubiquitinating enzyme with identified physiological roles as a tumor suppressor and an oncogene. While some binding partners and substrates are known for USP44, the identification of other interactions may improve our understanding of its role in cancer. We therefore performed a proximity biotinylation study that identified products of several known cancer genes that are associated with USP44, including a novel interaction between BRCA2 and USP44.

Methods: We expressed a fusion protein that linked USP44 and mutant Escherichia coli biotin ligase BioID in SH-SY5Y neuroblastoma cells. Control experiments were performed using BioID alone. In duplicate experiments, cells were pulsed with biotin and biotinylated proteins were isolated under denaturing conditions and the proteins were identified by mass spectrometry. The resulting list of proteins were analyzed using Enrichr and cross-referenced with the COSMIC Cancer Gene Census. We validated the association with BRCA2 using immunoprecipitation. The role of USP44 in the Fanconi anemia DNA repair pathway was investigated using chromosome analysis of wild-type or Usp44-knockout cells after exposure to mitomycin C.

Results: We identified 146 proteins that were selectively retrieved by the USP44 construct and compared with cells expressing the BioID ligase alone, including 15 gene products encoded by genes on tier 1 of the COSMIC Cancer Gene Census, including BRCA2. The association between USP44 and BRCA2 was validated through immunoprecipitation. We tested the functional role of USP44 in the Fanconi anemia DNA repair pathway through chromosome breakage analysis and found that cells lacking USP44 had a significant increase in chromosome breaks and radial chromosomes. We found that high transcript was correlated with poor survival in neuroblastoma, likely due to its tight association with proliferation in these tumors.

Conclusions: Our results identified novel potential binding partners and potential substrates for USP44, including several with direct roles in cancer pathogenesis. Our results identified a novel association between BRCA2 and USP44, and a previously unknown role for USP44 in the Fanconi anemia DNA repair pathway that may contribute to its role in cancer.