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. Author manuscript; available in PMC: 2014 Oct 13.
Published in final edited form as: Mol Cell. 2013 Jun 27;50(6):818–830. doi: 10.1016/j.molcel.2013.06.004

OTULIN restricts Met1-linked ubiquitination to control innate immune signaling

Berthe Katrine Fiil 1,#, Rune Busk Damgaard 1,#, Sebastian Alexander Wagner 2, Kirstin Keusekotten 3, Melanie Fritsch 1, Simon Bekker-Jensen 1, Niels Mailand 1, Chunaram Choudhary 2, David Komander 3, Mads Gyrd-Hansen 1,§
PMCID: PMC4194427  EMSID: EMS60529  PMID: 23806334

Abstract

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after Nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling.

Introduction

An effective immunological barrier between the organism and the surrounding environment is critical for human health, particularly at the mucosal surface of the gastrointestinal tract, which constitutes the body’s largest surface (Chen et al., 2009; Maloy and Powrie, 2011). Pattern recognition receptors (PRRs) present on the cell membrane and in the cytoplasm collectively provide our cells with the capability to recognize molecular patterns on highly diverse pathogens (Takeuchi and Akira, 2010). In response, PRRs elicit a rapid and efficient immune response, in part mediated by pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukins (Baud and Karin, 2009; Takeuchi and Akira, 2010).

Stimulation of PRRs and cytokine receptors leads to assembly of signaling complexes where ubiquitin (Ub) ligases conjugate polyubiquitin (polyUb) on selected substrates to facilitate activation of mitogen-activated protein (MAP) kinases and the Inhibitor of kappa-B (IκB) kinase (IKK) complex consisting of IKKα, IKKβ and NEMO (also termed IKKγ) (Beug et al., 2012; Jiang and Chen, 2012). IKK facilitates the degradation of IκBα, leading to nuclear translocation of nuclear factor-κB (NF-κB) transcription factors. Together with transcription factors activated by MAP kinases, NF-κB promotes expression of genes orchestrating the inflammatory response (Baud and Karin, 2009).

The intracellular PRR nucleotide-oligomerization domain-containing protein 2 (NOD2) recognizes muramyl dipeptide (MDP) constituents of bacterial peptidoglycan and plays a critical role in gastro-intestinal immunity (Chen et al., 2009). Upon stimulation, NOD2 binds the proximal adaptor receptor-interacting protein kinase 2 (RIPK2), which recruits Ub ligases of the Inhibitor of Apoptosis (IAP) family (Beug et al., 2012). In turn, XIAP and cIAPs facilitate non-degradative ubiquitination of RIPK2 where polyUb formed by XIAP promotes recruitment of the linear ubiquitin chain assembly complex (LUBAC) composed of HOIL-1, HOIP and SHARPIN (Bertrand et al., 2009; Damgaard et al., 2012). LUBAC conjugates Met1-linked polyUb (Met1-Ub) to facilitate efficient NF-κB activation and transcription of inflammatory mediators. A central regulatory point for this is the activation of the IKK complex. IKK activation is dependent on phosphorylation by the K63-Ub-activated TAB/TAK1 complex as well as the conjugation of Met1-Ub, bound by the IKK subunit NEMO (Jiang and Chen, 2012; Walczak et al., 2012).

For controlled and beneficial pro-inflammatory signaling, conjugation of polyUb must be counter-balanced by deubiquitinases such as CYLD and A20 that regulate different aspects of pro-inflammatory signaling (Harhaj and Dixit, 2012). We and others recently identified the ovarian tumor (OTU) domain family deubiquitinase OTULIN (also termed FAM105B or Gumby) as a Met1-Ub-specific deubiquitinase (Keusekotten et al., 2013; Rivkin et al., 2013).

OTULIN antagonizes LUBAC-mediated Met1-Ub assembly and NF-κB activation upon TNF and poly(I:C) treatment, and regulates TNF-induced pro-inflammatory signaling and cell death (Keusekotten et al., 2013). LUBAC regulates many aspects of cellular signaling and innate immune signaling (Tokunaga and Iwai, 2012) and deregulation results in severe immune dysfunction (Boisson et al., 2012; Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011). Indeed, we recently reported that LUBAC activity is particularly important for signaling triggered by the PRRs NOD1 and NOD2 (Damgaard et al., 2012).

Here, we investigated the role of OTULIN in NOD2-mediated signaling. We find that OTULIN restricts Met1-Ub formation and that this is important for limiting pro-inflammatory signaling in response to NOD2 stimulation. SILAC-based proteomics identify RIPK2, an essential protein for NOD2 signaling, as a target for Met1-Ub conjugation after receptor stimulation, and we find that OTULIN regulates its ubiquitination. Our data suggest that OTULIN is a new regulator of NOD2 signaling and is involved in innate immune signaling.

Results

OTULIN regulates NOD2 signaling and is part of the NOD2 complex

The role of OTULIN in LUBAC signaling prompted us to investigate its cellular function in context of NOD2 signaling. Stimulation of NF-κB activity by ectopic NOD2 was inhibited by overexpression of wild-type OTULIN (OTULINWT), but was alleviated by mutating the catalytic cysteine (C129A), the tryptophan residue involved in Met1-Ub binding (W96A) (Keusekotten et al., 2013) or both (WA/CA) (Figures 1A and 1B; Figure S1A). Accordingly, OTULINWT but not OTULINWA/CA inhibited nuclear translocation of the NF-κB subunit RelA/p65 after stimulation of U2OS/FlpIn/TRex/HA-NOD2 (U2OS/NOD2) cells with the NOD2 ligand L18-MDP (Figure 1C, quantified in 1D). Of note, the U2OS/NOD2 cells responded to L18-MDP without addition of doxycycline (DOX). Under these conditions HA-NOD2 was expressed at low levels and was not detectable by immunofluorescence staining (Figure S1B), and was detected by Western blotting only after immunoprecipitation with anti-HA resin (Figures S1C).

Figure 1. OTULIN regulates signaling in response to NOD2 stimulation.

Figure 1

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(A) Schematic depiction of human OTULIN. Circles illustrate residues required for ubiquitin binding and stars denote residues required for cleavage of Met1-Ub. (B) NF-κB activity in HEK293T cell lysates transfected with HA-NOD2 alone or with OTULINWT or the indicated point mutants. (C) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (green) in response to L18-MDP stimulation (1 μg/mL) in U2OS/NOD2 cells transfected with empty vector (pcDNA3-3xHA; HA-vector), OTULINWT or OTULINWA/CA. Scalebar, 10 μm (D) Quantification of nuclear NF-κB translocation after L18-MDP stimulation of cells treated as in (C). (E) Relative levels of TNF, IL8 and IL6 transcripts measured by qRT-PCR on cDNA from U2OS/NOD2 control and OTULIN-depleted cells treated with L18-MDP (200 ng/mL). Immunoblot of OTULIN levels in control (MM) and siOTULIN-treated cells. (F) Immunoblotting of IκBα degradation and phosphorylation of signaling components in response to stimulation with L18-MDP (200 ng/mL) in control and OTULIN-depleted cells. Data in (B, D and E) represent the mean ± SEM of at least three independent experiments, each performed in duplicate. In (D) at least 150 cells were counted per condition in each experiment. Asterisks (**) indicates p < 0.01. See also Figure S1.

RNAi-mediated depletion of OTULIN increased L18-MDP-induced transcription of NF-κB responsive genes (Figure 1E; Figures S1E and S1F) and led to more pronounced degradation of IκBα (Figure 1F) compared with cells transfected with mismatch control siRNAs (siMM). Activation of the MAP kinases p38 and JNK1/2 was only slightly increased by OTULIN depletion (Figure 1F) consistent with the notion that OTULIN disassembles Met1-Ub primarily involved in IKK activation. Of note, OTULIN migrated as two distinct bands in the Tris-Glycine-SDS buffer system (Figure 1E) but only as a single species in MOPS (Figure S1D). The nature of the second band is currently unknown, but might represent an alternative variant or a modified form of OTULIN.

Immunoprecipitation of HA-tagged NOD2 from DOX-treated U2OS/NOD2 cells co-purified RIPK2, XIAP and the three LUBAC components HOIP, HOIL-1 and SHARPIN. Intriguingly, endogenous OTULIN also co-purified with NOD2 (Figure 2A), suggesting that OTULIN could regulate signaling at the NOD2 signaling complex. XIAP enhances recruitment of LUBAC to the NOD2 signaling complex, where LUBAC conjugates Met1-Ub to facilitate downstream signaling (Damgaard et al., 2012). Overexpressed OTULIN efficiently blocked LUBAC-induced NF-κB activation, suggesting that OTULIN functions downstream of LUBAC by disassembling Met1-Ub (Figure 2B; Figure S2A). Accordingly, OTULIN impaired XIAP-mediated NF-κB activity to a similar extent as overexpression of RING-mutated HOIP (Haas et al., 2009) and HOIL-1 (termed dominant-negative (DN)-LUBAC) (Figures 2C and 2D; Figures S2B and S2C).

Figure 2. OTULIN is part of the NOD2 receptor complex and antagonizes Met1-Ub-dependent signaling.

Figure 2

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(A) Immunoprecipitation with anti-HA in U2OS/NOD2 cells. HA-NOD2 expression was induced for 24 hours with doxycycline (DOX). Immunoprecipitates were examined for co-purification of OTULIN and known members of the NOD2 receptor complex. (B-D) NF-κB activity in HEK293T cell lysates transfected with luciferase reporters and OTULIN, LUBAC (HOIP, HOIL-1), DN-LUBAC (RING-mutated HOIP, HOIL-1) or XIAP as indicated. (E) Schematic depiction of the engineered AVPI-Ub4GS protein, which binds to XIAP BIR2 and BIR3 domains via an N-terminal IBM to activate NF-κB. DUB, deubiquitinase. (F, G) NF-κB activity in HEK293T cell lysates transfected luciferase reporters and XIAPF/A, AVPI-Ub4GS, DN-LUBAC or OTULIN as indicated. Data in (B-D and F-G) represent the mean ± SEM of at least three independent experiments (except ‘OTULIN’ in 2B where n = 2), each performed in duplicate. Asterisks (**) indicates p < 0.01. n.s., not significant. See also Figure S2.

To directly address if OTULIN antagonizes NF-κB activation by disassembling Met1-Ub, we devised a system where a non-cleavable (Gly76->Ser76; GS) Met1-Ub was docked to XIAP via an IAP-binding motif (IBM). The engineered protein comprised Ub fused to the N-terminal Ala-Val-Pro-Ile (AVPI) IAP-binding motif (IBM) of Second mitochondrial-derived activator of caspases (Smac), followed by a FLAG epitope and four Ub(GS) moieties in tandem (Figure 2E). The N-terminal Ub is rapidly removed by deubiquitinases to expose the Smac IBM (Hunter et al., 2003), which enables binding of AVPI-Ub4GS to XIAP in a manner dependent on an N-terminal alanine (Figure S2D). AVPI-Ub4GS did not increase NF-κB activity when expressed alone, but facilitated potent NF-κB activation when co-expressed with the Ub-ligase deficient XIAPF495A (XIAPF/A) (Gyrd-Hansen et al., 2008), which also failed to activate NF-κB when expressed alone (Figure 2F; Figure S2C). Substituting Ala1 with Leu prevented NF-κB activation, confirming that signaling by AVPI-Ub4GS depended on docking to XIAP (Figure S2E). NF-κB activation induced by XIAPF/A and AVPI-Ub4GS was not inhibited by overexpression of DN-LUBAC showing that the ectopic non-cleavable Met1-Ub efficiently bypassed the requirement for LUBAC activity (Figure 2F; Figure S2C). Importantly, under these conditions overexpressed OTULIN also failed to inhibit NF-κB activation (Figure 2G; Figure S2B), establishing that the inhibitory effect of OTULIN on NF-κB activation is dependent on its ability to disassemble Met1-Ub.

RIPK2 is a substrate for Met1-Ub in response to NOD2 stimulation

Next, we sought to uncover potential OTULIN substrates by identifying proteins modified by Met1-Ub in response to NOD2 stimulation. For this, we employed a Met1-linkage specific Ub-binder (M1-SUB) based on NEMO’s UBAN region (Rahighi et al., 2009). Ectopic expression of Ub-linkage-selective binders can inhibit cellular signaling processes dependent on the Ub-linkage it binds to (van Wijk et al., 2012). Indeed, expression of the GFP-coupled M1-SUB (GFP-M1-SUB) inhibited nuclear localization of RelA/p65 after NOD2 stimulation (Figures 3A and 3B). Mutation of residues in the M1-SUB required for Ub binding (GFP-M1-SUBmut) (Rahighi et al., 2009; Wu et al., 2006) reversed the inhibitory effect on RelA/p65 translocation, showing that the M1-SUB inhibited NOD2 signaling by binding to Ub (Figures 3A and 3B). Next, we purified Met1-Ub-modified proteins using recombinant GST-coupled M1-SUB (for brevity henceforth referred to as M1-SUB) (Keusekotten et al., 2013). For comparison, we used a Tandem Ubiquitin Binding Entity (TUBE) (Hjerpe et al., 2009) to purify all types of polyubiquitinated proteins. The specificity of the M1-SUB towards Met1-Ub-modified proteins was determined in extracts of cells overexpressing LUBAC (HOIP and HOIL-1) and OTULIN. The M1-SUB almost exclusively purified Ub-conjugates from lysates of OTULINC129A-overexpressing cells, consistent with the notion that OTULINC129A stabilizes Met1-Ub formed by LUBAC (Figure 3C) (Keusekotten et al., 2013). In comparison, the non-selective TUBE pulled down comparable amounts of Ub from all extracts (Figure 3C).

Figure 3. Proteome-wide quantification of NOD2-regulated Met1-Ub.

Figure 3

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(A) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (red) in response to L18-MDP stimulation (1 μg/mL) in U2OS/NOD2 cells transfected with GFP vector, GFP-M1-SUB, or a Ub-binding mutant GFP-M1-SUBmut. Scalebar, 10 μm (B) Quantification of nuclear NF-κB translocation after L18-MDP stimulation of cells treated as in (A). (C) Purification of endogenous Ub conjugates using GST-coupled M1-SUB (Met1-Ub), or TUBE (all Ub-linkages). Pulldown with glutathione sepharose beads on HEK293T cell lysates transfected with LUBAC (HOIP and HOIL) and the indicated OTULIN mutants. Purified material and lysate was examined by immunoblotting. (D) Outline of the SILAC-based proteomics approach for quantification of protein ubiquitination in response to NOD2 stimulation. (E) MS data from TUBE1-purification of ubiquitinated proteins from THP-1 cells treated or not with L18-MDP (200 ng/mL) for 60 min. The circle representing RIPK2 is marked in red. The mass spectrum shows the relative abundance of the RIPK2 peptide TQNILLDNEFHVK in L18-MDP treated (SILAC heavy) cells compared to untreated (SILAC light). (F) Same as in (E) but after purification of ubiquitinated proteins with M1-SUB. RIPK2 is marked in red. (G) SILAC H/L ratios of proteins identified in (E) and (F) with reported function in the NOD2 signaling complex. NA, not available. (H) Time course analysis of RIPK2 ubiquitination with TUBE and M1-SUB in lysates of U2OS/NOD2 cells treated with L18-MDP (200 ng/mL). Purified material and lysate was examined by immunoblotting. Data in (B) represent the mean ± SEM of three independent experiments. At least 140 cells were counted per condition in each experiment. Asterisks (*) indicates p < 0.05. See also Figure S3 and Table S1.

We then employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics (Ong et al., 2002) together with purification of Ub-modified proteins by M1-SUB or TUBE. THP1 cells labeled with heavy- or light isotope containing amino acids were exposed to L18-MDP for 60 min or left untreated, and TUBE or M1-SUB were used to purify all polyubiquitinated proteins or Met1-Ub-modified proteins, respectively. Purified proteins were digested with trypsin, and peptides were quantified by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) (Figure 3D). Strikingly, we found that RIPK2 was by far the most highly enriched protein (SILAC H/L ratio >20) after L18-MDP stimulation in the TUBE purification (Figures 3E and 3G; Table S1). This is consistent with previous reports showing that RIPK2 is the predominant target for ubiquitination in the NOD2 signaling complex (Damgaard et al., 2012; Hasegawa et al., 2008). Remarkably, in the M1-SUB purification RIPK2 was enriched >12 fold after NOD2 stimulation and was the only detected protein with a SILAC H/L ratio >2.5 (Figures 3F and 3G; Table S1). NEMO, XIAP, LUBAC subunits and other NOD2 signaling complex components were not enriched after L18-MDP stimulation, suggesting that RIPK2 is a major target of Met1-linked ubiquitination in response to NOD2 stimulation (Figure 3G; Table S1). Time course analysis confirmed the mass spectrometry data and showed that L18-MDP-induced ubiquitination of RIPK2 occurred at 60 min, was detectable both by TUBE and M1-SUB, and was decreased after 120 and 240 min. Notably, the ubiquitination of RIPK2 coincided with IκBα phosphorylation and degradation and phosphorylation of RelA/p65 and MAP kinases (Figure 3H).

OTULIN limits Met1-Ub accumulation on RIPK2 to control NOD2 signaling

To test if OTULIN regulated Met1-linked ubiquitination of RIPK2, Ub-conjugates were isolated from L18-MDP-treated U2OS/NOD2 cells depleted of OTULIN. Indeed, RNAi-mediated OTULIN knockdown led to extensive accumulation of Met1-ubiquitinated RIPK2 that correlated with enhanced degradation of IκBα in response to NOD2 stimulation with 200 ng/mL L18-MDP (Figure 4A, lanes 1-6). Conversely, OTULIN overexpression suppressed Met1-Ub of RIPK2 in a manner dependent on its catalytic activity (Figure 4B).

Figure 4. OTULIN limits Met1-linked ubiquitination of RIPK2 after NOD2 stimulation.

Figure 4

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(A, B) Purification of endogenous Ub conjugates with M1-SUB from (A) OTULIN-depleted U2OS/NOD2 cells treated with either 200 ng/mL L18-MDP for 1 hour or 5 ng/mL L18-MDP for 2 hours (B) OTULIN overexpressing U2OS/NOD2 cells treated with L18-MDP (200 ng/mL) for the indicated times. Purified material and lysate from (A, B) was examined by immunoblotting. (C) Ub-chain restriction analysis of ubiquitinated RIPK2 isolated with M1-SUB from L18-MDP treated and siMM or siOTULIN transfected cells. Purified Ub-conjugates were incubated with the indicated deubiquitinases (DUBs) for 1h, and samples were examined by immunoblotting. (D) Coomassie staining of the recombinant DUBs used in (C). (E) Immunoprecipitation of endogenous NEMO from OTULIN-depleted cells treated with 200 ng/mL L18-MDP for 1 hour. Purified material and lysate was examined by immunoblotting (F) Immunoprecipitation of endogenous NEMO from OTULIN overexpressing cells treated with 200 ng/mL L18-MDP for 1 hour or 5 ng/mL L18-MDP for 2 hours. Purified material and lysate was examined by immunoblotting.

These data strongly suggest that RIPK2 is a physiological substrate for Met1-linked polyubiquitination, and is regulated by OTULIN. RIPK2 is also modified by Ub-linkages other than Met1, and genetic deletion of XIAP or pharmacological inhibition of XIAP dramatically reduces RIPK2 ubiquitination (Damgaard et al., 2013; Damgaard et al., 2012). To directly address the contribution of Met1-linkages to Ub-modified RIPK2, we subjected M1-SUB-purified Ub-RIPK2 to in vitro Ub chain restriction analysis by OTULIN and other linkage-selective or promiscuous deubiquitinases (Hospenthal et al., 2013; Mevissen et al., 2013) (Figures 4C and 4D). The promiscuous deubiquitinase USP21 removed virtually all Ub moieties from RIPK2, leading to a collapse of the RIPK2 ‘smear’ into a single band representing unmodified RIPK2 (Figure 4C, see lanes 1-3 and lanes 8-10). In contrast, incubation with the K48-linkage-selective OTUB1 did not alter the migration pattern of Ub-RIPK2 on SDS-PAGE (Figure 4C, compare lanes 2 and 7, and lanes 9 and 14). Importantly, incubation with OTULINWT but not catalytic inactive OTULINC129A resulted in a downshift in the migration pattern of Ub-RIPK2, which was particularly evident for Ub-RIPK2 isolated from OTULIN-depleted cells (Figure 4C, compare lanes 2, 4 and 5, and lanes 9, 11 and 12). Interestingly, OTULIN did not give rise to an increased level of unmodified RIPK2 suggesting that the Met1-linkages are formed on preexisting polyubiquitin on RIPK2. Accordingly, the viral deubiquitinase vOTU that disassembles all Ub-linkages except Met1 (Mevissen et al., 2013) removed most Ub from RIPK2, and increased the amount of unmodified and monoubiquitinated RIPK2 (Figure 4C, see lanes 6 and 13).

NEMO facilitates IKK activation through interaction with Met1-Ub-modified proteins (Rahighi et al., 2009). Accordingly, depletion of OTULIN enhanced the recruitment of NEMO to Met1-ubiquitinated RIPK2 in response to L18-MDP whereas NEMO recruitment was inhibited by OTULIN overexpression (Figure 4E and 4F). Notably, ubiquitinated RIPK2 was readily detected in the lysates from OTULIN-depleted cells treated with L18-MDP, illustrating extensive ubiquitination of RIPK2 when OTULIN is depleted (Figure 4E).

This finding, together with the observation that OTULIN was not transcriptionally induced by L18-MDP (Figure 5A), led us to speculate that OTULIN might regulate the initial response to the NOD2 ligand rather than function as part of a negative feedback mechanism. This notion was supported by an extended time course analysis of NOD2 signaling, which showed that Met1-linked ubiquitination of RIPK2 in OTULIN-depleted cells decreased with kinetics similar to that of the control cells (Figure 5B). Also, RelA/p65 phosphorylation and IκBα degradation in OTULIN-depleted cells was temporally comparable to the L18-MDP response observed in cells transfected with control siRNA (Figure 5B and Figure 1F). To test if OTULIN depletion sensitized cells to NOD2 stimulation, we treated U2OS/NOD2 cells with lower concentrations of L18-MDP (Figure 5C; Figure S4A). Interestingly, treatment of U2OS/NOD2 cells with L18-MDP diluted 40-fold (5 ng/mL) induced only marginal degradation of IκBα and RelA/p65 phosphorylation in control cells whereas signaling was readily detected OTULIN-depleted cells, albeit with slower kinetics than after treatment with higher ligand concentrations (Figure 5C and 1F). In line with this, Met1-Ub on RIPK2 was barely detectable in control cells subjected to the low concentration of L18-MDP, but accumulated in the OTULIN-depleted cells to levels comparable with those in control cells treated with 200 ng/mL of L18-MDP (Figure 4A, compare lane 4 with lanes 7-9). Accordingly, the transcriptional response to 5 ng/mL L18-MDP was up to 20-fold higher in OTULIN-depleted cells than in control siRNA-transfected cells (Figure 5D).

Figure 5. OTULIN regulates early NOD2 signaling and sensitivity to L18-MDP.

Figure 5

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(A) Relative levels of OTULIN transcripts measured by qRT-PCR with two different primer sets on cDNA from U2OS/NOD2 control cells treated with 200 ng/mL L18-MDP for the times indicated. (B) Purification of endogenous Ub conjugates with M1-SUB from OTULIN-depleted cells treated with L18-MDP for up to 4 h. Purified material and lysate was examined by immunoblotting. (C) Immunoblotting of IκBα degradation and phosphorylation of signaling components in response to stimulation with 5 ng/mL of L18-MDP in control and OTULIN-depleted U2OS/NOD2 cells. (D) Relative levels of TNF, IL8 and IL6 transcripts measured by qRT-PCR on cDNA from U2OS/NOD2 control and OTULIN-depleted cells treated with 5 ng/mL of L18-MDP. Data in (A, D) represent the mean ± SEM of at least three independent experiments. See also Figure S4.

Decreased OTULIN function leads to promiscuous Met1-Ub accumulation

In non-stimulated cells, Met1-Ub assembled by LUBAC does not accumulate to detectable levels suggesting that it is rapidly turned over. Met1-Ub can however be stabilized by exogenous OTULINC129A (Figure 3A) (Keusekotten et al., 2013). Moreover, Met1-Ub accumulates on overexpressed HOIP when OTULIN is depleted (Keusekotten et al., 2013) or when OTULINC129A is overexpressed (Figure 6A). This suggested that OTULIN globally restricts Met1-Ub formation under basal conditions and led us to investigate if OTULIN regulates Met1-Ub of LUBAC components at the endogenous level. Indeed, depletion of OTULIN or expression of OTULINC129A resulted in accumulation of Met1-linked ubiquitination of endogenous LUBAC components in non-stimulated cells (Figure 6B and 6C). This was particularly evident for HOIP and HOIL-1 whereas SHARPIN was not appreciably modified (Figure 6B). L18-MDP stimulation further increased Met1-Ub on HOIP and HOIL-1, and under these conditions Met1-linked ubiquitination of SHARPIN was also detected (Figure 6B). Together with the observation that OTULIN interacts with the LUBAC complex at the endogenous level (Keusekotten et al., 2013), this suggested that OTULIN prevents auto-ubiquitination of LUBAC under basal conditions and restricts accumulation of Met1-Ub on LUBAC substrates after NOD2 stimulation.

Figure 6. Decreased OTULIN function leads to promiscuous Met1-Ub accumulation.

Figure 6

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(A-C) Samples used in Figure 3A (C), 4A (B), and 4B (C) were immunoblotted for LUBAC components as indicated. (D) Purification of endogenous Ub conjugates with M1-SUB in U2OS/NOD2 control and OTULIN-depleted cells treated with TNF (10 ng/mL). Purified material and lysate was examined by immunoblotting. (E) TNF-RSC purification by FLAG-TNF (100 ng/mL) immunoprecipitation from TREx293 control or OTULIN-depleted cells at different times after FLAG-TNF stimulation. Purified material was analyzed by immunoblotting. See also Figure S5.

OTULIN also regulates Met1-linked ubiquitination of RIPK1 and recruitment of NEMO to ubiquitinated RIPK1 after TNFR1 activation (Keusekotten et al., 2013). Moreover, TNF stimulation strongly increased Met1-Ub of HOIP and HOIL-1 in OTULIN-depleted U2OS cells compared with non-stimulated cells (Figure 6D). Purification of the TNF-receptor signaling complex (TNF-RSC) from TRex-293 cells using FLAG-tagged TNF revealed significant ubiquitination of the TNFR1 itself, consistent with previous reports (Haas et al., 2009). Analysis of the TNF-RSC in TRex-293 cells expressing an OTULIN-targeting miRNA showed that OTULIN depletion led to both accelerated and increased accumulation of Met1-Ub, and increased ubiquitination of TNFR1 at 5 min after TNF stimulation (Figure 6E). Conversely, overexpression of OTULIN decreased the level of Met1-Ub in the TNF-RSC and reduced the apparent molecular weight of ubiquitinated TNFR1 (Figure S5).

Together, our data suggest that OTULIN is important for restricting the accumulation of Met1-Ub in innate immune receptor signaling complexes, and that this is essential for preventing excessive pro-inflammatory signaling in response to NOD2 stimulation.

Discussion

Met1-Ub assembled by LUBAC has emerged as a versatile protein modification that regulates several cellular functions, in particular pro-inflammatory signaling (Tokunaga and Iwai, 2012). We and others recently reported that NOD2-mediated transcription of NF-κB-responsive genes is largely dependent on LUBAC function (Damgaard et al., 2012; Warner et al., 2013). In agreement with this, we show here that OTULIN has an important role in regulating NOD2-mediated signaling and that reducing OTULIN levels leads to a striking increase in transcription of inflammatory mediators after NOD2 stimulation. In comparison, OTULIN and LUBAC seem to have more subdued functions in regulating NF-κB activation in response to TNFR1 stimulation. Instead, deregulation of Met1-Ub formation after TNF treatment leads to excessive programmed cell death (Boisson et al., 2012; Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2009). The underlying mechanisms dictating these differences between NOD2- and TNFR1-dependent signaling are not well understood, but will be important to dissect in future studies.

Identifying physiological substrates for ubiquitination by LUBAC has proven exceptionally difficult and only few proteins substrates (NEMO and RIPK1) have been reported so far (Boisson et al., 2012; Gerlach et al., 2011; Keusekotten et al., 2013; Tokunaga et al., 2009). One reason for this is that Met1-Ub assembly is incompatible with N-terminally modified Ub such as epitope-tagged exogenous ubiquitin (Gerlach et al., 2011; Kirisako et al., 2006), a strategy widely used to study Lys-linked ubiquitination. We therefore used a proteomics approach that combined affinity purification of endogenous Met1-Ub-modified proteins using a Met1-linkage-selective affinity reagent with SILAC-based quantitative proteomics. This revealed a remarkable specificity in ubiquitination after NOD2 stimulation and identified RIPK2 as the predominant Met1-Ub target. Mass spectrometry analysis of Ub-modified proteins purified by the non-selective TUBE and our previous analysis of L18-MDP-induced ubiquitination (Damgaard et al., 2012) further supported that RIPK2 is the major substrate for ubiquitination of the NOD2-signaling complex. We believe that this methodology might be applicable for analysis of a wide range of ubiquitin-regulated processes, and for the detection of other Ub-linkages in innate immune signaling.

Using recombinant deubiquitinases for Ub chain restriction analysis of Ub-modified RIPK2, we provide compelling evidence that the polyubiquitin conjugated on RIPK2 after NOD2 stimulation comprises at least two distinct Ub-linkages. OTULIN trimmed away the slowest migrating Ub-RIPK2 species without increasing the amount of unmodified RIPK2 suggesting that Met1-Ub is conjugated predominantly on existing polyubiquitin, conjugated on RIPK2 by other Ub ligase(s). Ectopically expressed XIAP conjugates polyubiquitin on RIPK2 linked through lysines other than K48 and K63 (Damgaard et al., 2012), implying that atypical polyubiquitin might play a role in NOD2 signaling. Detailed Ub chain restriction analysis using a panel of linkage-selective deubiquitinases (Mevissen et al., 2013) might thus uncover unappreciated roles for atypical polyubiquitin in regulation of NOD2 signaling.

Our analysis of Met1-Ub in NOD2 signaling and how it is regulated by OTULIN led to several interesting observations. First, OTULIN was rate limiting for the accumulation of Met1-Ub on RIPK2, which was particularly evident when NOD2 was stimulated with low amounts of ligand. Second, OTULIN continuously disassembled Met1-Ub that, in the absence of OTULIN activity, accumulated on LUBAC components. Third, stimulation of NOD2 and TNFR1 resulted in promiscuous Met1-Ub of receptor components unless counterbalanced by endogenous OTULIN. These data imply that LUBAC is active under basal conditions and continuously ubiquitinates itself and possibly other substrates in a not strictly selective manner, and that the Met1-Ub chains are rapidly disassembled by OTULIN. This is supported by the observation that OTULIN binds to the LUBAC complex (Keusekotten et al., 2013), by the strong increase in steady-state levels of Met1-Ub and HOIP ubiquitination in cells overexpressing LUBAC together with catalytically inactive OTULIN, as well as by the accumulation of Met1-Ub on endogenous HOIP and HOIL-1 in cells with decreased OTULIN activity. The balanced activity of LUBAC and OTULIN may explain why Met1-Ub is present at very low levels in cells, and why its abundance is not significantly increased by overexpression of LUBAC, despite LUBAC readily assembles Ub chains in vitro (Kirisako et al., 2006; Stieglitz et al., 2012).

While LUBAC appears to be active under basal conditions, Met1-Ub formation on LUBAC components was increased by NOD2 and TNFR1 stimulation in OTULIN-depleted cells, suggesting that the propensity of LUBAC for assembling Met1-Ub is regulated by environmental cues such as microbial products and cytokines. This might occur through direct activation of the ligase complex or as a consequence of induced proximity of LUBAC within the formed receptor complexes. LUBAC activity was recently reported to be regulated by the Ub ligase Parkin in response to cellular stress signals, exemplifying that LUBAC activity may indeed be regulated (Muller-Rischart et al., 2013). Further investigation of LUBAC function in innate immune receptor signaling may thus reveal additional mechanisms for modulating formation of productive Met1-Ub.

Other deubiquitinases such as A20 and CYLD function as negative regulators of immune signaling (Harhaj and Dixit, 2012). A20 contains a K48-Ub-specific OTU domain (Mevissen et al., 2013) but is thought to disassemble K63-Ub chains in cells (Wertz et al., 2004), and A20/TNFAIP3 is a transcriptional target of NF-κB transcription factors. Consequently A20 is essential for termination of pro-inflammatory signaling (Boone et al., 2004; Lee et al., 2000). In contrast, our data suggest that OTULIN functions to restrict Met1-Ub accumulation and regulate early signaling processes. This was supported by the observation that OTULIN was not transcriptionally activated by NOD2 stimulation and OTULIN levels were not elevated, consistent with data obtained using TNF (Keusekotten et al., 2013). Also, the temporal profile of RIPK2 ubiquitination, degradation of IκBα, or transcription of NF-κB responsive genes after NOD2 stimulation was not markedly prolonged by OTULIN depletion. Instead, OTULIN knockdown rendered cells hyper-responsive to NOD2 stimulation, which was particularly evident when ligand concentration was decreased. We speculate that OTULIN might be involved in determining the threshold for pro-inflammatory signaling in response to NOD2 ligands.

In conclusion, we have shown that OTULIN is essential for restricting accumulation of Met1-Ub in cells and for limiting NOD2-dependent proinflammatory signaling. Our data thus provide evidence that OTULIN is a physiological regulator of innate immune responses and inflammation. The analysis of OTULIN/FAM105B-ablated mice will be important for revealing the in vivo function of OTULIN in innate immunity.

Experimental procedures

Plasmids and cloning, Cell lines, RNA interference and Antibodies and affinity resin

(see Supplemental Experimental Procedures).

Receptor stimulation

THP-1 and U2OS/NOD2 cells were treated with the NOD2 ligand L18-MDP (InvivoGen, San Diego, CA) for the indicated times with 5-1000 ng/mL or TNF 1-100 ng/mL (R&D systems, Minneapolis, MN), both were added directly to the culture medium (see Supplemental Experimental Procedures).

Luciferase reporter assays

HEK293T cells were co-transfected with the NF-κB luciferase reporter construct pBIIX-luc and a thymidine kinase-renilla luciferase construct for normalization of transfection efficiency. Cells were either co-transfected with additional plasmids or treated with compounds as indicated elsewhere and luciferase assays were performed as previously described (Damgaard et al., 2012). Individual experiments were performed in duplicate.

Quantitative RT-PCR

Total RNA was isolated from U2OS/NOD2 using RNeasy Mini Kit (Qiagen, Hilden, Germany) and DNase digestion was performed on-column with the RNase-Free DNase Set (Qiagen) according to manufacturer’s protocol. Total RNA was reverse transcribed with SuperScript® III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo(d)T primers, in the presence of RNasin® (Promega, Madison, WI). QPCR was performed using Brilliant III Ultra-Fast SYBR® Green QPCR Master (Agilent Technologies, Santa Clara, CA). Gene-specific primers were used to amplify cDNA (see Supplemental Experimental Procedures).

Purification of endogenous ubiquitin conjugates

Ubiquitin conjugates from cell lysates were pulled down in THP-1 and U2OS/NOD2 cells using affinity reagents. For isolation of Met1-Ub, recombinant protein containing one copy of the UBAN region from human NEMO (residues 257-346) fused to Glutatione-S-transferase (GST) was used (M1-SUB; full sequence will be made available upon request). TUBE (TUBE1; Lifesensors, Malvern, PA, was used for the mass spectrometry analysis) consists of four UBA domains in tandem fused to GST and was used to purify all polyubiquitin linkages. Purified material was analyzed by immunoblotting or mass spectrometry analysis (see Supplemental Experimental Procedures).

Deubiquitinase assays

Ubiquitin conjugates from L18-MDP treated siMM or siOTULIN#2 were isolated by M1-SUB on GST-beads, washed in PBS Tween-20 (0.1%), and resuspended in deubiquitinase-buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 1 mM MnCl2, 0.01% Brij-35) without or with deubiquitinases (USP21 (0.5 μM), OTULIN (1 μM), OTULIN CA (1 μM), vOTU (0.4 μM), OTUB1 (15 μM)). Samples incubated for 1h at 30 °C, and LSB buffer was added to end the reaction. Cloning, expression and purification of the deubiquitinases used here is described elsewhere (Mevissen et al., 2013).

Immunoprecipitation

Endogenous NEMO was immunopurified as previously described (Keusekotten et al., 2013) from U2OS/NOD2 cells transfected and treated as indicated. HA-NOD2 from U2OS/NOD2 cells treated with doxycycline (2 μg/mL) for 24 h was immunopurified as described for NEMO, except that anti-HA-agarose resin (Sigma-Aldrich, Gillingham, UK) was used as affinity reagent. TNF-RSC purification was performed as previously described but using 100 ng/mL FLAG-TNF (Haas et al., 2009; Keusekotten et al., 2013) (see Supplemental Experimental Procedures).

Immunofluorescence staining and microscopy

U2OS/NOD2 cells were fixed in 4% formaldehyde, permeabilized with PBS containing 0.2% Triton X-100 for 5 min and incubated with antibodies. Cover slips were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing the DNA stain DAPI. Images were acquired with an LSM 780 confocal microscope (Carl Zeiss Microimaging, Jena, Germany). For data quantification, at least 100 HA (OTULIN)-positive cells per condition were counted in each experiment (see Supplemental Experimental Procedures).

Mass spectrometry based analysis of L18-MDP induced ubiquitination

For SILAC labeling, THP-1 cells were cultured in media containing either L-arginine and L-lysine or L-arginine-U-13C6-15N4 and L-lysine-U-13C6-15N2 (Cambridge Isotope Laboratories, Andover, MA) as described previously (Ong et al., 2002). Ubiquitinated proteins from cells treated for 60 min with L18-MDP were purified using TUBE1 or M1-SUB. The enriched proteins were resolved by SDS-PAGE and digested in-gel with trypsin. Peptide fractions were analyzed on a quadrupole Orbitrap (Q-Exactive, Thermo Scientific) mass spectrometer equipped with a nanoflow HPLC system (Thermo Scientific, Rockford, IL) as described (Michalski et al., 2011). Raw data files were analyzed using MaxQuant (Cox and Mann, 2008) (see Supplementary Experimental Procedures).

Statistical Analysis

The two-tailed Student’s t-test was used to determine statistical significance. Error bars represent standard error of the mean (SEM).

Supplementary Material

SI
Supplementary Table S1

Acknowledgements

We thank Dr. Henning Walczak for reagents and members of the Ubiquitin Signaling group for helpful suggestions and reading the manuscript. We thank the Protein Production Facility at the Novo Nordisk Foundation Center for Protein Research for production of TUBE and M1-SUB. This work was supported by the Novo Nordisk Foundation, a Steno Fellowship from the Danish Council for Independent Research – Natural Sciences (M.G.-H.), The Lundbeck Foundation (M.G.-H.), the Medical Research Council (U105192732, DK), the European Research Council (DK) and the EMBO Young Investigator Programme (DK). The Center for Protein Research is supported by a grant from the Novo Nordisk Foundation.

Footnotes

The authors declare no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SI
NIHMS60529-supplement-SI.pdf (340.9KB, pdf)
Supplementary Table S1
NIHMS60529-supplement-Supp_Table_S1.xlsx (356.6KB, xlsx)

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