A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN

The methionine1 (M1)-specific deubiquitinase OTULIN acts as a negative regulator of NF-ΚB signaling and immune homeostasis. By replacing G76 in distal ubiquitin by dehydroalanine we designed the diubiquitin (diUb) probe UbG76Dha-Ub (OTULIN ABP) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. OTULIN ABP displays high selectivity for OTULIN and is not labeling to other M1-specific DUB including CYLD. In fact, labeling of USP5/IsopeptidaseT was the only detectable cross-reactivity. Further, ATP-dependent association of UbG76Dha-Ub probe with E1 activating enzymes induces assembly of polyOTULIN ABP chains, but USP5 labelling and E1 binding was abolished by deleting the C-terminal glycine in proximal ubiquitin (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 DUB for OTULIN that will facilitate studying the cellular function of this critical deubiquitinase.