Jiangsu Key Laboratory for Translational Research and Therapeutics of Neuro-Psycho- Diseases, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu, People's Republic of China.
Program in Molecular Structure and Function, The Hospital for Sick Children, Department of Molecular Genetics, University of Toronto, Toronto, M5G 0A4, Canada.
J Hematol Oncol. 2017 Jul 3;10(1):132. doi: 10.1186/s13045-017-0499-7.
UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.
Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively.
UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway.
UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.
UBE2O 被认为是一种泛素连接酶，但它的功能在很大程度上是未知的。
质谱法用于鉴定 c-Maf 泛素化相关蛋白。免疫沉淀法用于 c-Maf 和 UBE2O 相互作用。免疫印迹法用于 Maf 蛋白稳定性。荧光素酶 assay 用于 c-Maf 转录活性。慢病毒感染用于多发性骨髓瘤（MM）细胞中的 UBE2O 功能。流式细胞术和裸鼠异种移植分别用于 MM 细胞凋亡和肿瘤生长测定。
通过亲和纯化/串联质谱分析和 co-immunoprecipitation 分析发现，UBE2O 与 c-Maf 相互作用，c-Maf 是 MM 中的关键转录因子。随后的研究表明，UBE2O 介导 c-Maf 多泛素化和降解。此外，UBE2O 下调了 c-Maf 的转录活性和 cyclin D2 的表达，cyclin D2 是一种典型的由 c-Maf 调节的基因。DNA 微阵列显示，UBE2O 在正常骨髓细胞中表达，但在 MGUS、冒烟型 MM 和 MM 细胞中下调，在原代 MM 细胞中通过 RT-PCR 得到证实，这表明其在骨髓瘤发病机制中具有潜在作用。当 UBE2O 被恢复时，MM 细胞中的 c-Maf 蛋白显著减少，MM 细胞发生凋亡。此外，裸鼠的人 MM 异种移植显示，UBE2O 的重新表达与 c-Maf 下调和凋亡途径的激活相关，可延迟裸鼠骨髓瘤异种移植的生长。
UBE2O 介导 c-Maf 多泛素化和降解，诱导 MM 细胞凋亡，并抑制骨髓瘤肿瘤生长，为理解骨髓瘤发生机制和 UBE2O 生物学提供了新的视角。
