Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.
Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.
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DOI：
10.1186/s12896-018-0452-z
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发表时间：
2018-06-11
影响因子：
3.5
通讯作者：
Nakashima K
中科院分区：
文献类型：
--
作者：
Watanabe Y;Matsuba T;Nakanishi M;Une M;Hanajima R;Nakashima K
破伤风神经毒素(帐篷)在神经末梢被摄取，并经历逆行迁移。帐篷的毒性性质存在于毒素轻链(L)，但与完整的帐篷一样，帐篷重链(TTH)和C末端结构域(TTC)可以单独结合并进入神经元。在这里，我们探索了无毒的帐篷碎片是否可以作为神经元中的药物输送工具。在这项研究中，我们使用了一种已知在体内和体外都具有抗凋亡特性的蛋白-2作为包裹，将其偶联到帐篷碎片上。我们单独表达了Bcl2和TTC片段，并尝试在哺乳动物(Cos7细胞)和大肠杆菌系统中表达与TTH N端偶联的Bcl2(Bcl2-TTH)和TTC的N端和C端偶联的融合蛋白(TTC-Bcl2和Bcl2-TTC)。TTC和Bcl2分别在大肠杆菌和Cos7细胞中高效表达，但Bcl2和融合蛋白在大肠杆菌中不能很好地表达。融合蛋白在COS7细胞中也不表达。为了提高融合蛋白的产量和纯度，我们从Bcl2-TTC融合中删除了TTC的N端半部分，从而产生了Bcl2-hTTC。纯化的bcl2-hTTC能与神经元结合，并能抑制线粒体细胞色素C的释放，从而阻止血清和NGF剥夺诱导的神经元PC12细胞死亡。在体内试验中，Bcl2-hTTC被注射到小鼠的舌头中，并被发现通过逆行轴突运输选择性地迁移到小鼠舌下核的脑干。这些结果表明，bcl2-hTTC既保留了Bcl2-2的功能，又保留了TTC的功能，因此可能是一种有效的治疗神经疾病的药物。
Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.
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影响因子：
4.7
作者：
Chaïb-Oukadour, I;Gil, C;Aguilera, J
通讯作者：
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