Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.
Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.
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DOI：
10.1021/acssynbio.0c00397
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发表时间：
2020-12-18
影响因子：
4.7
通讯作者：
Gu L
中科院分区：
文献类型：
--
作者：
Huang Z;Li Z;Zhang X;Kang S;Dong R;Sun L;Fu X;Vaisar D;Watanabe K;Gu L
由红光控制的蛋白质二聚系统随着组织穿透深度的增加是临床应用(如细胞和基因治疗)的迫切需要的工具。然而，现有的红光诱导二聚系统在哺乳动物中的应用受到其两个组分的限制：通常需要哺乳动物外源发色团的光敏蛋白(或光感受器)和具有复杂结构和非理想结合特性的自然产生的光感受器结合蛋白。在这里，我们介绍了一种有效的、可推广的方法(联合LID)，用于创建高度特异的可逆光诱导异二聚化系统，而不依赖于任何现有的光感受器结合剂。它涉及组合纳米体库的两步结合筛选(噬菌体展示和酵母双杂交)，以获得选择性地与光激活形式的光开关蛋白或结构域结合的结合物，而不是暗形式。通过设计基于纳米体的红光诱导二聚(NanReD)系统提供了原理证明，该系统包括使用哺乳动物内源发色团、胆绿素和光形式的特定纳米体的截断的细菌光敏色素传感模块。选择的纳米ReD系统进行了生化表征，显示出低的暗活性和高的诱导特异性，进一步证明了对小鼠蛋白质转位和基因表达激活的可逆控制。总体而言，联合盖子为创造用于生物过程的光学操作的遗传编码的致动器开辟了新的机会。
Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and non-ideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
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影响因子：
64.8
作者：
Chevalier A;Silva DA;Rocklin GJ;Hicks DR;Vergara R;Murapa P;Bernard SM;Zhang L;Lam KH;Yao G;Bahl CD;Miyashita SI;Goreshnik I;Fuller JT;Koday MT;Jenkins CM;Colvin T;Carter L;Bohn A;Bryan CM;Fernández-Velasco DA;Stewart L;Dong M;Huang X;Jin R;Wilson IA;Fuller DH;Baker D
通讯作者：
Baker D
影响因子：
48
作者：
Kennedy, Matthew J.;Hughes, Robert M.;Peteya, Leslie A.;Schwartz, Joel W.;Ehlers, Michael D.;Tucker, Chandra L.
通讯作者：
Tucker, Chandra L.
影响因子：
64.8
作者：
Bhoo, SH;Davis, SJ;Vierstra, RD
通讯作者：
Vierstra, RD
影响因子：
3.4
作者：
Niu, Jacqueline;Ben Johny, Manu;Inoue, Takanari
通讯作者：
Inoue, Takanari
影响因子：
48
作者：
Kaberniuk AA;Shemetov AA;Verkhusha VV
通讯作者：
Verkhusha VV