Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion.
Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion.
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DOI：
10.1002/cpch.37
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发表时间：
2018-03
影响因子：
--
通讯作者：
DeLisa MP
中科院分区：
文献类型：
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作者：
Baltz MR;Stephens EA;DeLisa MP
现在存在许多用于减少细胞蛋白质的表达而不需要基因组修饰的技术。一种这样的技术涉及将E3泛素连接酶的泛素化活性与设计结合蛋白（DBP）的结合亲和力和底物特异性结合的工程蛋白嵌合体，所述工程蛋白嵌合体联合收割机。这些嵌合体，称为“泛素”，能够选择性地和可控地操纵几乎任何蛋白质到泛素蛋白酶体途径（UPP）进行降解，使泛素成为蛋白质敲除工具箱的有力补充。泛体的一个显著特征是它们的模块性-简单地交换DBP可以产生对不同底物蛋白具有特异性的新泛体。此外，通过采用识别特定蛋白质状态（例如，活性构象与非活性构象、突变体与野生型、翻译后修饰），因此有可能耗尽某些蛋白质亚群而保留其它亚群。该方案概述了设计和功能评估用于可定制的细胞蛋白质沉默的泛素所需的步骤。
A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called “ubiquibodies”, are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity – simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active vs. inactive conformation, mutant vs. wildtype, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins.