Daniel James A, Cooper Benjamin H, Palvimo Jorma J, Zhang Fu-Ping, Brose Nils, Tirard Marilyn
Max Planck Institute of Experimental Medicine, Molecular Neurobiology, Göttingen, Germany.
Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
Elife. 2017 Jun 9;6:e26338. doi: 10.7554/eLife.26338.
SUMO1-conjugation of proteins at neuronal synapses is considered to be a major post-translational regulatory process in nerve cell and synapse function, but the published evidence for SUMO1-conjugation at synapses is contradictory. We employed multiple genetic mouse models for stringently controlled biochemical and immunostaining analyses of synaptic SUMO1-conjugation. By using a knock-in reporter mouse line expressing tagged SUMO1, we could not detect SUMO1-conjugation of seven previously proposed synaptic SUMO1-targets in the brain. Further, immunostaining of cultured neurons from wild-type and SUMO1 knock-out mice showed that anti-SUMO1 immunolabelling at synapses is non-specific. Our findings indicate that SUMO1-conjugation of synaptic proteins does not occur or is extremely rare and hence not detectable using current methodology. Based on our data, we discuss a set of experimental strategies and minimal consensus criteria for the validation of SUMOylation that can be applied to any SUMOylation substrate and SUMO isoform.
蛋白质在神经元突触处的SUMO1缀合被认为是神经细胞和突触功能中的主要翻译后调控过程，但已发表的关于突触处SUMO1缀合的证据相互矛盾。我们采用了多种基因工程小鼠模型，对突触SUMO1缀合进行严格控制的生化和免疫染色分析。通过使用表达带标签SUMO1的敲入报告小鼠品系，我们在大脑中未能检测到七个先前提出的突触SUMO1靶标的SUMO1缀合。此外，对野生型和SUMO1基因敲除小鼠培养神经元的免疫染色表明，突触处的抗SUMO1免疫标记是非特异性的。我们的研究结果表明，突触蛋白的SUMO1缀合不会发生或极其罕见，因此使用当前方法无法检测到。基于我们的数据，我们讨论了一套用于验证SUMO化的实验策略和最低共识标准，这些策略和标准可应用于任何SUMO化底物和SUMO异构体。
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