Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.
Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.
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DOI：
10.1021/acschembio.5b00971
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发表时间：
2016-06-17
影响因子：
4
通讯作者：
Hardy JA
中科院分区：
文献类型：
--
作者：
Hill ME;MacPherson DJ;Wu P;Julien O;Wells JA;Hardy JA
常规工程蛋白酶特异性的能力可以使我们更好地了解和调节它们的生物学，以扩大治疗和工业应用。在这里，我们报告了一种新的方法的基础上笼绿色荧光蛋白（CA-GFP）的报告，允许流式细胞术为基础的选择在细菌或其他细胞类型，使选择细胞内蛋白酶的特异性，无论组成的蛋白酶的复杂性。在这里，我们应用这种方法将caspase-6的特异性引入到caspase-7中，caspase-7是一种在细胞重塑和细胞死亡中重要的细胞内半胱氨酸蛋白酶。我们发现，从caspase-6到caspase-7的底物接触残基的取代是无效的，产生一个无活性的酶，而在这些位置的饱和诱变和选择定向进化产生活性caspase。该过程产生了许多不明显的突变，使caspase-7特异性转化为匹配caspase-6。进化特异性胱天蛋白酶-7（esCasp-7）的结构揭示了底物的交替结合模式，包括活性位点环的重组。通过N-末端组学分析esCasp-7的整个人类蛋白质组表明，对天然蛋白质底物的全局特异性与caspase-6的特异性非常相似。由于esCasp-7保持了caspase-7的核心，我们能够鉴定出caspase-6底物，核纤层蛋白C，我们预测其依赖于外部位点进行底物识别。这些重编程的蛋白酶可能是第一个工具，建立明确的意图，区分外部位点依赖性或独立的基板。这种特异性重编程的方法也应该在广泛的蛋白酶中推广。
The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of non-obvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.