Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice
Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice
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DOI：
10.1093/jb/mvab059
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发表时间：
2021-05-12
影响因子：
2.7
通讯作者：
Sugiyama, Fumihiro
中科院分区：
文献类型：
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作者：
Murata, Kazuya;Mimura, Asuka;Sugiyama, Fumihiro
邻近依赖生物素鉴定（BioID）是鉴定未知蛋白-蛋白相互作用的有效方法。很少有报道描述了生物id体内基因工程敲入小鼠模型。因此，对于生物素给药的正确方法以及适用于哪些组织，人们知之甚少。在此，我们建立了生物id敲入小鼠脑和肌肉ARNT-Like 1 （BMAL1）和BirA生物素连接酶与R118G突变（BirA*）的模型。采用BMALl-BioID小鼠模型，研究生物素日粮饲喂对几种组织中蛋白质生物素化的影响。BMAL1- bira *融合蛋白在HEK293T细胞中保留了BMAL1的胞内定位和与CLOCK蛋白的结合。小鼠胚胎成纤维细胞的生物素标记实验显示，敲入小鼠细胞中表达的BMAL1-BirA*蛋白生物素化活性依赖于生物素的补充。最后，饲喂0.5%生物素饲料7 d可诱导BMAL1-BioID小鼠脑、心脏、睾丸和肝脏的蛋白质生物素化，对精子发生无不利影响。在肾脏中，生物素饮食增加了BMAL1-BioID和对照小鼠的生物素化蛋白水平，表明存在内源性生物素化活性。这些结果为优化体内生物id程序提供了有价值的信息。
Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMALl-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.