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A coupled protein and probe engineering approach for selective inhibition and activity based probe labeling of the caspases

机译：一种基于蛋白和探针的工程改造方法可选择性地基于半胱氨​​酸蛋白酶抑制和活性进行探针标记

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摘要

Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8 and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.
机译：胱天蛋白酶是在细胞凋亡和炎症中起重要作用的半胱氨酸蛋白酶。不幸的是，它们高度保守的活性位点和重叠的底物特异性使得难以使用抑制剂或基于活性的探针来研究该家族单个成员的功能，激活，定位和调节。在这里，我们描述了一种使半胱天冬酶工程化以包含潜性亲核试剂的策略，该隐性亲核试剂可以被含有适当放置的亲电试剂的探针靶向，从而仅对工程化的蛋白酶进行特异性，不可逆的抑制和标记。为此，我们在所有胱天蛋白酶的小亚基上鉴定了一个非保守残基，该残基靠近底物结合口袋，并且可以突变为非催化性半胱氨酸残基。我们证明，包含不可逆结合丙烯酰胺亲电试剂的活性位点探针可以特异性地靶向该半胱氨酸残基。在这里，我们使用凋亡介质caspase-8和炎症小体效应因子caspase-1验证了该方法。我们表明，工程酶与野生型酶在功能上是相同的，并且该方法允许对细胞中工程靶进行特异性抑制和直接成像。因此，该方法可用于成像定位和激活以及单个半胱天冬酶蛋白酶对细胞死亡或炎症过程的功能贡献。

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