A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases.
A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases.
复制标题
DOI：
10.1021/ja403521u
复制
发表时间：
2013-06-19
影响因子：
15
通讯作者：
Bogyo, Matthew
中科院分区：
文献类型：
--
作者：
Xiao, Junpeng;Broz, Petr;Puri, Aaron W.;Deu, Edgar;Morell, Montse;Monack, Denise M.;Bogyo, Matthew
半胱氨酸蛋白酶是一种半胱氨酸蛋白酶，在细胞凋亡和炎症中发挥重要作用。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、定位和调节变得困难。在这里，我们描述了一种策略，使半胱氨酸天冬氨酸氨基转移酶包含潜在的亲核体，该亲核体可以被含有适当位置的电泳体的探针靶向，从而允许仅对工程酶进行特定的、不可逆的抑制和标记。为了实现这一点，我们在所有caspase的小亚基上发现了一个非保守残基，该残基位于底物结合口袋附近，可以突变为非催化半胱氨酸残基。我们证明了含有不可逆结合的丙烯酰胺电泳体的活性位点探针可以特异性地靶向这个半胱氨酸残基。在这里，我们使用凋亡调节因子caspase-8和炎症体效应因子caspase-1来验证该方法。我们表明，工程酶在功能上与野生型酶相同，并且该方法允许对细胞中的工程靶标进行特定的抑制和直接成像。因此，这种方法可以用来定位和激活以及单个caspase蛋白酶在细胞死亡或炎症过程中的功能贡献。
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.
登录
查看更多内容
影响因子：
15
作者：
Morell, Montse;Thinh Nguyen Duc;Willis, Amanda L.;Syed, Salahuddin;Lee, Jiyoun;Deu, Edgar;Deng, Yang;Xiao, Junpeng;Turk, Benjamin E.;Jessen, Jason R.;Weiss, Stephen J.;Bogyo, Matthew
通讯作者：
Bogyo, Matthew
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
--
作者：
Edgington, Laura E.;van Raam, Bram J.;Bogyo, Matthew
通讯作者：
Bogyo, Matthew
影响因子：
14.8
作者：
Blair, Jimmy A.;Rauh, Daniel;Shokat, Kevan M.
通讯作者：
Shokat, Kevan M.
DOI：
10.1007/978-1-61779-228-1_27
发表时间：
2011-01-01
期刊：
MOLECULAR METHODS FOR EVOLUTIONARY GENETICS
影响因子：
--
作者：
Nelson, Matthew D.;Fitch, David H. A.
通讯作者：
Fitch, David H. A.