A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN

摘要

class="head no_bottom_margin" id="sec1title">IntroductionBy assembling covalent chains with distinct linkages, the post-translational modifier ubiquitin (Ub) controls nearly all aspects of cell biology and cellular homeostasis (). Conjugation of methionine 1 (M1)-linked polyubiquitin (polyUb) is catalyzed by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIP/RNF31, HOIL-1, and SHARPIN (, ). Upon stimulation, LUBAC is recruited to immune receptors, e.g., tumor necrosis factor receptor (TNFR), interleukin-1 receptor, and Toll-like receptors, where it decorates signaling mediators such as RIPKs, MYD88, IRAKs, and NEMO with M1-linked Ub chains to facilitate nuclear factor κB (NF-κB)-dependent immune and inflammatory responses. In turn, LUBAC activity needs to be tightly controlled and the deubiquitinases (DUBs) CYLD and OTULIN cleave M1-linked polyUb with high specificity and efficiency (, , ). Indeed, OTULIN and CYLD bind to LUBAC via a PUB (peptide:N-glycanase/UBA- or UBX-containing proteins) domain in HOIP. While OTULIN binds HOIP directly through its PUB-interacting motif (PIM) (, ), CYLD is recruited via a PIM in the bridging factor SPATA2 (, , , ). PUB-PIM interactions with HOIP are mutually exclusive, and while OTULIN-LUBAC and CYLD/SPATA2-LUBAC complexes are readily detectable, only CYLD/SPATA2-LUBAC is efficiently recruited to TNFR complexes after TNF stimulation (, , , href="#bib55" rid="bib55" class=" bibr popnode">Wagner et al., 2016). Nevertheless, OTULIN is also a negative regulator of TNF-α signaling (href="#bib6" rid="bib6" class=" bibr popnode">Damgaard et al., 2016, href="#bib22" rid="bib22" class=" bibr popnode">Hrdinka et al., 2016, href="#bib26" rid="bib26" class=" bibr popnode">Keusekotten et al., 2013). Furthermore, it serves to prevent auto-ubiquitination of LUBAC components at steady state, and accumulation of M1-linked chains in general (href="#bib14" rid="bib14" class=" bibr popnode">Elliott et al., 2014, href="#bib18" rid="bib18" class=" bibr popnode">Fiil et al., 2013, href="#bib26" rid="bib26" class=" bibr popnode">Keusekotten et al., 2013, href="#bib47" rid="bib47" class=" bibr popnode">Schaeffer et al., 2014). Genetically, an OTULIN loss-of-function mouse model displayed developmental defects in angiogenesis, leading to embryonic lethality (href="#bib44" rid="bib44" class=" bibr popnode">Rivkin et al., 2013). Conditional knockout (KO) mice with OTULIN deletion in immune cells demonstrated that OTULIN is critical for preventing spontaneous inflammation and maintaining immune homeostasis (href="#bib6" rid="bib6" class=" bibr popnode">Damgaard et al., 2016). This correlated with hypomorphic mutations in human patients and led to the description of an OTULIN-related auto-inflammatory syndrome (href="#bib6" rid="bib6" class=" bibr popnode">Damgaard et al., 2016, href="#bib59" rid="bib59" class=" bibr popnode">Zhou et al., 2016). Despite its pathophysiological importance, questions remain regarding whether and how the DUB activity and LUBAC interaction of OTULIN are regulated in cells.Activity-based probes (ABPs) are powerful tools to study enzyme activities in vitro and in vivo and have been helpful for studying the activity of DUBs (href="#bib10" rid="bib10" class=" bibr popnode">Ekkebus et al., 2014). Whereas many DUBs react with ABPs containing an electrophilic group at the C terminus of a monoUb (href="#bib7" rid="bib7" class=" bibr popnode">de Jong et al., 2012), the substrate-assisted Ub chain hydrolysis of OTULIN relies on a native M1-linked diubiquitin (diUb) as a substrate (href="#bib26" rid="bib26" class=" bibr popnode">Keusekotten et al., 2013, href="#bib36" rid="bib36" class=" bibr popnode">Mevissen et al., 2013). The design of an electrophile that mimics the Gly-Met environment of the linear diUb linkage has remained a challenge to date. href="#bib35" rid="bib35" class=" bibr popnode">McGouran et al. (2013) designed an ABP based on an M1-linked diUb which more promiscuously labeled ubiquitin-specific protease (USP) DUBs that normally show little reactivity for linear Ub chains. OTULIN, however, was not labeled with this probe. The outcome was attributed to the greater flexibility of the used electrophile and the loss of key residues such as the M1 side chain and the amide bond between M1 and Gln2 of the proximal Ub (href="#bib26" rid="bib26" class=" bibr popnode">Keusekotten et al., 2013, href="#bib35" rid="bib35" class=" bibr popnode">McGouran et al., 2013). href="#bib38" rid="bib38" class=" bibr popnode">Mulder et al. (2014) reported the use of a chemical ligation handle that allowed the generation of linkage-specific diUb ABPs based on all seven lysine- and thus isopeptide-linked diUb chains (i.e., K6, K11, K27, K29, K33, K48, and K63). Whereas the design of the electrophile was well-suited to mimic the isopeptide bond between two Ub proteins, the differences in chemistry imposed by the “linear” peptide linkage made this strategy impractical for the M1-linked chain type. In another attempt to create an ABP based on linear diUb, M1 of proximal Ub was replaced by the electrophilic dehydroalanine (Dha) residue (href="#bib2" rid="bib2" class=" bibr popnode">Bernardes et al., 2008, href="#bib20" rid="bib20" class=" bibr popnode">Haj-Yahya et al., 2014). However, the probe was cleaved by OTULIN and USP2 rather than reacting covalently with the active site cysteine residues.We report here on the total chemical synthesis of biotinylated linear diUb in which Gly76 of the distal Ub is replaced by Dha (bio-UbG76Dha-Ub: OTULIN ABP) to yield an ABP that covalently labels active OTULIN. By crystallizing the OTULIN-(UbG76Dha-Ub) complex, we show that the probe captures OTULIN in its active state. Proteome-wide mass spectrometry (MS) experiments reveal that the OTULIN ABP cross-reacts only with USP5 (Isopeptidase T) and the Ub-activating E1 enzymes UBA1 and UBA6. Deletion of the C-terminal glycine in the proximal Ub moiety in UbG76Dha-UbΔG76 abolishes USP5 labeling and E1 association, yielding a specific probe for OTULIN (OTULIN ABPΔG76). We show that the probes can be used to monitor cellular OTULIN activity, pull-down (PD) active OTULIN, and co-precipitate the OTULIN-associated E3 ligase LUBAC from cells.