May, Danielle G

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 BRCA2 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.

Abstract The mechanistic target of rapamycin (mTOR) is a central regulator of cellular proliferation and metabolism. Depending on its binding partners, mTOR is at the core of 2 complexes that either promote protein biosynthesis (mTOR complex 1; mTORC1) or provide survival and proliferation signals (mTORC2). Protein biosynthesis downstream of mTORC1 plays an important role in MYC-driven oncogenesis with translation inhibitors garnering increasing therapeutic attention. The germinal center B-cell oncogene UCHL1 encodes a deubiquitinating enzyme that regulates the balance between mTOR complexes by disrupting mTORC1 and promoting mTORC2 assembly. While supporting mTORC2-dependent growth and survival signals may contribute to its role in cancer, the suppression of mTORC1 activity is enigmatic, as its phosphorylation of its substrate 4EBP1 promotes protein biosynthesis. To address this, we used proximity-based proteomics to identify molecular complexes with which UCH-L1 associates in malignant B cells. We identified a novel association of UCH-L1 with the translation initiation complex eIF4F, the target of 4EBP1. UCH-L1 associates with and promotes the assembly of eIF4F and stimulates protein synthesis through a mechanism that requires its catalytic activity. Because of the importance of mTOR in MYC-driven oncogenesis, we used novel mutant Uchl1 transgenic mice and found that catalytic activity is required for its acceleration of lymphoma in the Eμ-myc model. Further, we demonstrate that mice lacking UCH-L1 are resistant to MYC-induced lymphomas. We conclude that UCH-L1 bypasses the need for mTORC1-dependent protein synthesis by directly promoting translation initiation, and that this mechanism may be essential for MYC in B-cell malignancy.