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：

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基于线性泛素化探针的去泛素化酶OTULIN的高效和选择性检测

甲硫氨酸1（M1）特异性去泛素酶（DUB）OTULIN充当核因子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遍在蛋白（Ub）中的Gly76，我们设计了基于双遍在蛋白（diUb）活性的探针UbG76Dha-Ub（基于OTULIN活性的探针[ABP]），该探针与OTULIN的催化位点偶联，从而在其活性中捕获OTULIN构象。OTULIN ABP显示出对OTULIN的高选择性，并且不标记其他M1裂解DUB，包括CYLD。唯一可检测到的交叉反应性是USP5的标记（异肽酶T）和通过Ub激活E1酶的ATP依赖的polyOTULIN ABP链组装。通过去除ABP近端Ub中的C端Gly消除了两种交叉反应性，产生特异性OTULIN探针UbG76Dha-UbΔG76（OTULINABPΔG76）。下拉列表显示与底物结合的OTULIN与线性泛素链组装复合体（LUBAC）相关联。因此，我们提出了OTULIN的高度选择性ABP，它将有助于研究该基本DUB的细胞功能。