A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.
A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.
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DOI：
10.1074/mcp.m110.002188
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发表时间：
2011-05
期刊：
影响因子：
--
通讯作者：
Mayor U
中科院分区：
文献类型：
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作者：
Franco M;Seyfried NT;Brand AH;Peng J;Mayor U
泛素化在神经元的发育和功能中具有重要作用。酵母和HeLa细胞的泛素蛋白质组学研究已被证明是非常有益的，但仍然有一个关于神经元组织特异性泛素化的差距。在生物体中，神经元蛋白泛素化的直接证据甚至更少。在这里，我们报告了一种新的蛋白质组学策略的基础上，在体内的生物素化的泛素分离泛素共轭物的果蝇胚胎的神经元。我们确定了48种神经元泛素底物，其中没有一种已知是泛素化的。早期在非神经元细胞类型中的蛋白质组学和生物化学研究已经确定了其中一些的直系同源物，但没有确定其他的直系同源物。新的泛素底物，没有已知的泛素化的直系同源物，在这里的鉴定表明，蛋白质组学研究必须进行神经元细胞，以确定不共享的其他细胞类型的泛素化途径。重要的是，一些新发现的神经元泛素底物是突触发生的关键参与者。质谱分析的结果进行了验证，Western印迹法，以确认这些蛋白质确实是在果蝇胚胎神经系统中的泛素化，并阐明他们是否是单或多泛素化。除了泛素底物，我们还确定了在突触发生过程中活跃的泛素载体。在特定的细胞类型中，在特定的发育阶段，并在一个活的有机体的背景下，识别内源性泛素化的蛋白质将允许理解这些蛋白质的组织特异性功能是如何由泛素系统调节。
Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.