A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell.
A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell.
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DOI：
10.1038/s41467-018-04440-0
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发表时间：
2018-05-16
影响因子：
16.6
通讯作者：
Ward TR
中科院分区：
文献类型：
--
作者：
Okamoto Y;Kojima R;Schwizer F;Bartolami E;Heinisch T;Matile S;Fussenegger M;Ward TR
由于细胞内存在大量的功能，用均相或异相催化剂补充天然环境中的酶具有挑战性。为了补充这些努力，人造金属酶正在引起人们的关注，因为它们结合了均相催化剂和酶的诱人特征。在此，我们展示了这种由金属辅因子、细胞穿透模块和蛋白质支架组成的混合催化剂被吸收到 HEK-293T 细胞中，在那里它们催化激素的解封。这种生物正交反应导致基因回路上调，进而导致纳米荧光素酶的表达。依靠生物素-链霉亲和素技术，生物素化钌复合物的变异：生物素化细胞穿透性聚二硫键比例可以与链霉亲和素上的点突变相结合，以优化烯丙基氨基甲酸酯保护的甲状腺激素三碘甲状腺原氨酸的催化解笼。这些结果表明，人工金属酶提供了高度模块化的工具来在活 HEK 细胞中进行生物正交催化。人工酶可用于引发细胞内的反应。在这里，作者开发了一种与基因开关相结合的人工催化剂，并表明它很容易被人体细胞吸收，并能够启动级联反应，从而导致所需产物的生物合成。
Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells. Artificial enzymes can be used to elicit reactions in cells. Here, the authors developed such an artificial catalyst combined with a genetic switch, and showed that it was readily taken up by human cells and able to kick off a reaction cascade resulting in the biosynthesis of the desired product.
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DOI：
10.1073/pnas.1514383113
发表时间：
2016-02-02
影响因子：
11.1
作者：
Saxena，Pratik;Charpin-El Hamri，Ghislaine;Fussenegger，Martin
通讯作者：
Fussenegger，Martin
影响因子：
15
作者：
Derivery E;Bartolami E;Matile S;Gonzalez-Gaitan M
通讯作者：
Gonzalez-Gaitan M
影响因子：
15
作者：
Li, Nan;Lim, Reyna K. V.;Edwardraja, Selvakumar;Lin, Qing
通讯作者：
Lin, Qing
影响因子：
15
作者：
Izumi, Saki;Urano, Yasuteru;Nagano, Tetsuo
通讯作者：
Nagano, Tetsuo
影响因子：
16.6
作者：
Qian, Linghui;Fu, Jiaqi;Yao, Shao Q.
通讯作者：
Yao, Shao Q.