Ali Asma, Hussain Sajjad, Bedekovics Tibor, Jeon Raymond H, May Danielle G, Roux Kyle J, Galardy Paul J
Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Department of Family Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Biomedicines. 2024 Dec 20;12(12):2901. doi: 10.3390/biomedicines12122901.
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.
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.
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.
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.
背景/目的：泛素特异性蛋白酶44（USP44）是一种去泛素化酶，已确定其作为肿瘤抑制因子和癌基因的生理作用。虽然已知USP44的一些结合伴侣和底物，但鉴定其他相互作用可能会增进我们对其在癌症中作用的理解。因此，我们进行了一项邻近生物素化研究，鉴定了与USP44相关的几种已知癌症基因的产物，包括BRCA2与USP44之间的一种新相互作用。
我们在SH-SY5Y神经母细胞瘤细胞中表达了一种将USP44与突变型大肠杆菌生物素连接酶BioID连接的融合蛋白。单独使用BioID进行对照实验。在重复实验中，用生物素脉冲处理细胞，在变性条件下分离生物素化蛋白，并通过质谱鉴定这些蛋白。使用Enrichr分析所得的蛋白质列表，并与COSMIC癌症基因普查进行交叉参考。我们使用免疫沉淀法验证了与BRCA2的关联。在暴露于丝裂霉素C后，通过对野生型或Usp44基因敲除细胞进行染色体分析，研究了USP44在范可尼贫血DNA修复途径中的作用。
我们鉴定了146种通过USP44构建体选择性回收的蛋白质，并与单独表达BioID连接酶的细胞进行了比较，其中包括COSMIC癌症基因普查第1层基因编码的15种基因产物，包括BRCA2。通过免疫沉淀验证了USP44与BRCA2之间的关联。我们通过染色体断裂分析测试了USP44在范可尼贫血DNA修复途径中的功能作用，并发现缺乏USP44的细胞中染色体断裂和放射状染色体显著增加。我们发现高转录水平与神经母细胞瘤患者的不良生存相关，这可能是由于其与这些肿瘤增殖的紧密关联。
我们的结果鉴定了USP44的新潜在结合伴侣和潜在底物，包括几种在癌症发病机制中具有直接作用的物质。我们的结果鉴定了BRCA2与USP44之间的新关联，以及USP44在范可尼贫血DNA修复途径中以前未知的作用，这可能有助于其在癌症中的作用。
