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A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN
Cell Chemical Biology ( IF 8.6 ) Pub Date : 2017-09-14 00:00:00 , DOI: 10.1016/j.chembiol.2017.08.006
Aurelia Weber 1 , Paul R Elliott 2 , Adan Pinto-Fernandez 3 , Sarah Bonham 3 , Benedikt M Kessler 3 , David Komander 2 , Farid El Oualid 4 , Daniel Krappmann 1
Affiliation  

The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe UbG76Dha-Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1-cleaving DUBs, including CYLD. The only detectable cross-reactivities were the labeling of USP5 (Isopeptidase T) and an ATP-dependent assembly of polyOTULIN ABP chains via Ub-activating E1 enzymes. Both cross-reactivities were abolished by the removal of the C-terminal Gly in the ABP's proximal Ub, yielding the specific OTULIN probe UbG76Dha-UbΔG76(OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective ABP for OTULIN that will facilitate studying the cellular function of this essential DUB.

中文翻译:

一种基于双泛素的线性探针,用于高效和选择性检测去泛素化酶 OTULIN

甲硫氨酸 1 (M1) 特异性去泛素化酶 (DUB) OTULIN 作为核因子 κB 信号传导和免疫稳态的负调节剂。通过用脱氢丙氨酸替代远端泛素 (Ub) 中的 Gly76,我们设计了基于双泛素 (diUb) 活性的探针 UbG76Dha-Ub(基于 OTULIN 活性的探针 [ABP]),它与 OTULIN 的催化位点偶联,从而在其活性中捕获 OTULIN构象。OTULIN ABP 对 OTULIN 显示出高选择性,并且不会标记其他 M1 裂解 DUB,包括 CYLD。唯一可检测到的交叉反应是 USP5(异肽酶 T)的标记和通过 Ub 激活 E1 酶的 polyOTULIN ABP 链的 ATP 依赖性组装。通过去除 ABP 近端 Ub 中的 C 端 Gly,两种交叉反应都被消除了,产生特定的 OTULIN 探针 UbG76Dha-UbΔG76(OTULIN ABPΔG76)。下拉表明底物结合的 OTULIN 与线性泛素链组装复合物 (LUBAC) 相关联。因此,我们为 OTULIN 提供了一种高度选择性的 ABP,这将有助于研究这种基本 DUB 的细胞功能。
更新日期:2017-09-15
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