Division for Therapies Against Intractable Diseases, Center for Medical Science, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi, 470-1192, Japan.
Open Facility Center, Research Promotion Headquarters, Fujita Health University, Toyoake, Aichi, 470-1192, Japan.
Mol Brain. 2024 Aug 15;17(1):57. doi: 10.1186/s13041-024-01131-4.
Discovery of novel post-translational modifications provides new insights into changes in protein function, localization, and stability. They are also key elements in understanding disease mechanisms and developing therapeutic strategies. We have previously reported that ubiquitin-like 3 (UBL3) serves as a novel post-translational modifier that is highly expressed in the cerebral cortex and hippocampus, in addition to various other organs, and that 60% of proteins contained in small extracellular vesicles (sEVs), including exosomes, are influenced by UBL3. In this study, we generated transgenic mice expressing biotinylated UBL3 in the forebrain under control of the alpha-CaMKII promoter (Ubl3). Western blot analysis revealed that the expression of UBL3 in the cerebral cortex and hippocampus was 6- to 7-fold higher than that in the cerebellum. Therefore, we performed immunoprecipitation of protein extracts from the cerebral cortex of Ubl3 and Ubl3 mice using avidin beads to comprehensively discover UBL3 interacting proteins, identifying 35 new UBL3 interacting proteins. Nine proteins were annotated as extracellular exosomes. Gene Ontology (GO) analysis suggested a new relationship between sEVs and RNA metabolism in neurodegenerative diseases. We confirmed the association of endogenous UBL3 with the RNA-binding proteins FUS and HPRT1-both listed in the Neurodegenerative Diseases Variation Database (NDDVD)-and with LYPLA1, which is involved in Huntington's disease, using immunoprecipitation (IP)-western blotting analysis. These UBL3 interacting proteins will accelerate the continued elucidation of sEV research about proteins regulated by novel post-translational modifications by UBL3 in the brain.
发现新的翻译后修饰为蛋白质功能、定位和稳定性的变化提供了新的见解。它们也是理解疾病机制和开发治疗策略的关键要素。我们之前报道过，泛素样蛋白 3 (UBL3) 作为一种新型翻译后修饰物，除了各种其他器官外，在大脑皮层和海马体中高度表达，并且小细胞外囊泡 (sEV) 中包含的 60%的蛋白质，包括外泌体，都受到 UBL3 的影响。在这项研究中，我们生成了在α-CaMKII 启动子控制下在前脑中表达生物素化 UBL3 的转基因小鼠 (Ubl3)。Western blot 分析显示，UBL3 在大脑皮层和海马体中的表达比小脑高出 6-7 倍。因此，我们使用亲和素珠对 Ubl3 和 Ubl3 小鼠大脑皮质的蛋白质提取物进行免疫沉淀，以全面发现 UBL3 相互作用蛋白，鉴定出 35 种新的 UBL3 相互作用蛋白。其中 9 种蛋白被注释为细胞外外泌体。基因本体论 (GO) 分析表明，sEV 与神经退行性疾病中的 RNA 代谢之间存在新的关系。我们通过免疫沉淀 (IP)-western blot 分析证实了内源性 UBL3 与 RNA 结合蛋白 FUS 和 HPRT1（均列于神经退行性疾病变异数据库 (NDDVD) 中）的关联，以及与 LYPLA1 的关联，后者与亨廷顿病有关。这些 UBL3 相互作用蛋白将加速对由 UBL3 在大脑中调节的 sEV 研究中受新型翻译后修饰调控的蛋白质的进一步阐明。
