Molecular Cell Article - Laboratory of Molecular Biology · Molecular Cell Article ... SUMMARY ... NF-kB activation is dependent on its ability to disassemble

Molecular Cell

Article

OTULIN Restricts Met1-Linked Ubiquitinationto Control Innate Immune SignalingBerthe Katrine Fiil,1,4 Rune Busk Damgaard,1,4 Sebastian Alexander Wagner,2 Kirstin Keusekotten,3 Melanie Fritsch,1

Simon Bekker-Jensen,1 Niels Mailand,1 Chunaram Choudhary,2 David Komander,3 and Mads Gyrd-Hansen1,*1Department of Disease Biology2Department of ProteomicsNovo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen,

DK-2200 Copenhagen, Denmark3Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge,

CB2 0QH, UK4These authors contributed equally to this work

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.molcel.2013.06.004

SUMMARY

Conjugation of Met1-linked polyubiquitin (Met1-Ub)by the linear ubiquitin chain assembly complex(LUBAC) is an important regulatory modification ininnate immune signaling. So far, only few Met1-Ubsubstrates have been described, and the regulatorymechanisms have remained elusive. We recentlyidentified that the ovarian tumor (OTU) family deubi-quitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limitingMet1-Ub accumulation after nucleotide-oligomeriza-tion domain-containing protein 2 (NOD2) stimulation,and that OTULIN depletion augments signalingdownstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncoveredRIPK2 as the predominant NOD2-regulated sub-strate. Accordingly, Met1-Ub on RIPK2 was largelyinhibited by overexpressing OTULIN and was in-creased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1stimulation led to extensive Met1-Ub accumulationon receptor complex components. We propose thatOTULIN restricts Met1-Ub formation after immunereceptor stimulation to prevent unwarranted proin-flammatory signaling.

INTRODUCTION

An effective immunological barrier between the organism and

the surrounding environment is critical for human health, partic-

ularly 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

818 Molecular Cell 50, 818–830, June 27, 2013 ª2013 Elsevier Inc.

highly diverse pathogens (Takeuchi and Akira, 2010). In

response, PRRs elicit a rapid and efficient immune response,

in part mediated by proinflammatory 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 activa-

tion of mitogen-activated protein (MAP) kinases and the inhibitor

of kappa-B (IkB) kinase (IKK) complex consisting of IKKa, IKKb,

and NEMO (also termed IKKg) (Beug et al., 2012; Jiang and

Chen, 2012). IKK facilitates the degradation of IkBa, leading to

nuclear translocation of nuclear factor kB (NF-kB) transcription

factors. Together with transcription factors activated by MAP

kinases, NF-kB promotes expression of genes that orchestrate

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 gastrointestinal 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 nondegradative 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-kB activation and transcription of inflam-

matory 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 proinflammatory signaling,

conjugation of polyUb must be counterbalanced by deubiquiti-

nases such as CYLD and A20 that regulate different aspects of

proinflammatory 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

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Molecular Cell

OTULIN Regulates Innate Immune Signaling

Met1-Ub-specific deubiquitinase (Keusekotten et al., 2013;

Rivkin et al., 2013). OTULIN antagonizes LUBAC-mediated

Met1-Ub assembly and NF-kB activation upon TNF and poly(I:C)

treatment, and regulates TNF-induced proinflammatory signal-

ing 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 re-

ported 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 proinflammatory 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 Partof the NOD2 ComplexThe role of OTULIN in LUBAC signaling prompted us to

investigate its cellular function in context of NOD2 signaling.

Stimulation of NF-kB activity by ectopic NOD2 was inhibited

by overexpression of wild-type OTULIN (OTULINWT) but was

alleviated by mutating the catalytic cysteine (C129A), the trypto-

phan residue involved in Met1-Ub binding (W96A) (Keusekotten

et al., 2013), or both (WA/CA) (Figures 1A and 1B; see Figure S1A

online). Accordingly, OTULINWT but not OTULINWA/CA inhibited

nuclear translocation of the NF-kB subunit RelA/p65 after stimu-

lation of U2OS/FlpIn/TRex/HA-NOD2 (U2OS/NOD2) cells with

the NOD2 ligand L18-MDP (Figure 1C, quantified in Figure 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 immuno-

fluorescence staining (Figure S1B), and was detected by

western blotting only after immunoprecipitation with anti-HA

resin (Figure S1C).

RNAi-mediated depletion of OTULIN increased L18-MDP-

induced transcription of NF-kB-responsive genes (Figure 1E;

Figures S1E and S1F) and led to more pronounced degradation

of IkBa (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 deple-

tion (Figure 1F), consistent with the notion that OTULIN disas-

sembles 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 copurified RIPK2, XIAP, and the three LUBAC

components, HOIP, HOIL-1, and SHARPIN. Intriguingly, endog-

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enous OTULIN also copurified with NOD2 (Figure 2A), suggest-

ing 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 facili-

tate downstream signaling (Damgaard et al., 2012). Overex-

pressed OTULIN efficiently blocked LUBAC-induced NF-kB

activation, suggesting that OTULIN functions downstream of

LUBAC by disassembling Met1-Ub (Figure 2B; Figure S2A).

Accordingly, OTULIN impaired XIAP-mediated NF-kB activity

to an extent similar to that of 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).

To directly address if OTULIN antagonizes NF-kB activation

by disassembling Met1-Ub, we devised a system whereby a

noncleavable (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 mitochondria-derived acti-

vator of caspase (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 (Fig-

ure S2D). AVPI-Ub4GS did not increase NF-kB activity when ex-

pressed alone, but facilitated potent NF-kB activation when

coexpressed with the Ub-ligase-deficient XIAPF495A (XIAPF/A)

(Gyrd-Hansen et al., 2008), which also failed to activate NF-kB

when expressed alone (Figure 2F; Figure S2C). Substituting

Ala1 with Leu prevented NF-kB activation, confirming that

signaling by AVPI-Ub4GS depended on docking to XIAP

(Figure S2E). NF-kB activation induced by XIAPF/A and AVPI-

Ub4GS was not inhibited by overexpression of DN-LUBAC,

showing that the ectopic noncleavable Met1-Ub efficiently

bypassed the requirement for LUBAC activity (Figure 2F; Fig-

ure S2C). Importantly, under these conditions overexpressed

OTULIN also failed to inhibit NF-kB activation (Figure 2G; Fig-

ure S2B), establishing that the inhibitory effect of OTULIN on

NF-kB activation is dependent on its ability to disassemble

Met1-Ub.

RIPK2 Is a Substrate for Met1-Ub in Response to NOD2StimulationNext, 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 theUb link-

age to which it binds (vanWijk 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; Figure S3A). 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; Figure S3A).

Next, we purified Met1-Ub-modified proteins using recombinant

GST-coupled M1-SUB (for brevity henceforth referred to as

olecular Cell 50, 818–830, June 27, 2013 ª2013 Elsevier Inc. 819

Figure 1. OTULIN Regulates Signaling in Response to NOD2 Stimulation

(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-kB 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-kB subunit RelA/p65 (green) in response to L18-MDP stimulation (1 mg/ml) in U2OS/NOD2

cells transfected with empty vector (pcDNA3-3xHA; HA-vector), OTULINWT or OTULINWA/CA. Scale bar, 10 mm.

(D) Quantification of nuclear NF-kB 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 IkBa 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 themean ±SEMof at least three independent experiments, each performed in duplicate. In (D) at least 150 cells were counted per

condition in each experiment. **p < 0.01. See also Figure S1.

Molecular Cell



Figure 2. OTULIN Is Part of the NOD2 Receptor Complex and Antagonizes Met1-Ub-Dependent Signaling(A) Immunoprecipitation with anti-HA in U2OS/NOD2 cells. HA-NOD2 expression was induced for 24 hr with doxycycline (DOX). Immunoprecipitates were

examined for copurification of OTULIN and known members of the NOD2 receptor complex.

(B–D) NF-kB 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-kB. DUB,

deubiquitinase.

(F and G) NF-kB activity in HEK293T cell lysates transfected luciferase reporters and XIAPF/A, AVPI-Ub4GS, DN-LUBAC, or OTULIN as indicated.

Data in (B)–(D), (F), and (G) represent themean ±SEMof at least three independent experiments (except ‘‘OTULIN’’ in B, where n = 2), each performed in duplicate.

**p < 0.01. n.s., not significant. See also Figure S2.

Molecular Cell


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 toward Met1-Ub-modified proteins was deter-

mined 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 nonselective TUBE pulled down com-

parable amounts of Ub from all extracts (Figure 3C).

M

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. THP-1 cells labeled with heavy or light

isotope-containing amino acids were exposed to L18-MDP for

60min or left untreated, and TUBE orM1-SUBwas 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-tandem

mass spectrometry (LC-MS/MS) (Figure 3D). Strikingly, we

found that RIPK2 was by far the most highly enriched protein


Figure 3. Proteome-wide Quantification of NOD2-Regulated Met1-Ub

(A) Immunofluorescence analysis of nuclear translocation of the NF-kB subunit RelA/p65 (red) in response to L18-MDP stimulation (1 mg/ml) in U2OS/NOD2 cells

transfected with GFP vector, GFP-M1-SUB, or a Ub-binding mutant GFP-M1-SUBmut. Scale bar, 10 mm.

(B) Quantification of nuclear NF-kB translocation after L18-MDP stimulation of cells treated as in (A).

(C) Purification of endogenous Ub conjugates using GST-coupledM1-SUB (Met1-Ub) or TUBE (all Ub-linkages). Pull-down with glutathione Sepharose beads on

HEK293Tcell lysates transfectedwith LUBAC (HOIP andHOIL) and the indicatedOTULINmutants. Purifiedmaterial and lysatewere examinedby immunoblotting.

(D) Outline of the SILAC-based proteomics approach for quantification of protein ubiquitination in response to NOD2 stimulation.

(E) MS data from TUBE purification of ubiquitinated proteins from THP-1 cells treated or not with L18-MDP (200 ng/ml) for 60min. The circle representing RIPK2 is

marked in red. Themass spectrum shows the relative abundance of the RIPK2 peptide TQNILLDNEFHVK in L18-MDP-treated (SILAC heavy) cells comparedwith

untreated (SILAC light).

(legend continued on next page)

Molecular Cell



Molecular Cell


(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 (Dam-

gaard 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 andM1-SUB, andwas decreased after

120 and 240 min. Notably, the ubiquitination of RIPK2 coincided

with IkBa phosphorylation and degradation and phosphorylation

of RelA/p65 and MAP kinases (Figure 3H).

OTULIN Limits Met1-Ub Accumulation on RIPK2to Control NOD2 SignalingTo 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 IkBa 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).

These data strongly suggest that RIPK2 is a physiological sub-

strate 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., 2012, 2013). 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 (Fig-

ure 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

(F) Same as in (E) but after purification of ubiquitinated proteins with M1-SUB. R

(G) SILAC H/L ratios of proteins identified in (E) and (F) with reported function in

(H) Time course analysis of RIPK2 ubiquitination with TUBE and M1-SUB in lysate

lysate were examined by immunoblotting.

Data in (B) represent the mean ±SEM of three independent experiments. At least

Figure S3 and Table S1.

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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 pre-existing 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

(Figures 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 feed-

back 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 (Fig-

ure 5B). Also, RelA/p65 phosphorylation and IkBa 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; Fig-

ure S4A). Interestingly, treatment of U2OS/NOD2 cells with

L18-MDP diluted 40-fold (5 ng/ml) induced only marginal degra-

dation of IkBa 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 (Figures 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).

Decreased OTULIN Function Leads to PromiscuousMet1-Ub AccumulationIn nonstimulated 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

IPK2 is marked in red.

the NOD2 signaling complex. NA, not available.

s of U2OS/NOD2 cells treated with L18-MDP (200 ng/ml). Purified material and

140 cells were counted per condition in each experiment. *p < 0.05. See also


Figure 4. OTULIN Limits Met1-Linked Ubiquitination of RIPK2 after NOD2 Stimulation

(A and 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 hr or

5 ng/ml L18-MDP for 2 hr. (B) OTULIN-overexpressing U2OS/NOD2 cells treated with L18-MDP (200 ng/ml) for the indicated times. Purified material and lysate

from (A) and (B) were 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 1 hr, and samples were examined by immunoblotting.

(legend continued on next page)

Molecular Cell



Figure 5. OTULIN Regulates Early NOD2 Signaling and Sensitivity to L18-MDP

(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 hr. Purified material and lysate were

examined by immunoblotting.

(C) Immunoblotting of IkBa degradation and phosphorylation of signaling components in response to stimulation with 5 ng/ml of L18-MDP in control andOTULIN-

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) and (D) represent the mean ±SEM of at least three independent experiments. See also Figure S4.

Molecular Cell


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 whether OTULIN regulates Met1-Ub of LUBAC com-

ponents at the endogenous level. Indeed, depletion of OTULIN or

expression of OTULINC129A resulted in accumulation of Met1-

(D) Coomassie staining of the recombinant DUBs used in (C).

(E) Immunoprecipitation of endogenous NEMO fromOTULIN-depleted cells treate

by immunoblotting.

(F) Immunoprecipitation of endogenous NEMO from OTULIN-overexpressing cell

material and lysate were examined by immunoblotting.

M

linked ubiquitination of endogenous LUBAC components in

non-stimulated cells (Figures 6B and 6C). This was particularly

evident for HOIP and HOIL-1, whereas SHARPIN was not appre-

ciably 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

d with 200 ng/ml L18-MDP for 1 hr. Purifiedmaterial and lysate were examined

s treated with 200 ng/ml L18-MDP for 1 hr or 5 ng/ml L18-MDP for 2 hr. Purified


Figure 6. Decreased OTULIN Function Leads to Promiscuous Met1-Ub Accumulation

(A–C) Samples used in Figure 3C (A), Figure 4A (B), and Figure 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 were 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.

Molecular Cell


detected (Figure 6B). Together with the observation that OTULIN

interacts with the LUBAC complex at the endogenous level (Keu-

sekotten et al., 2013), this suggested that OTULIN prevents au-

toubiquitination of LUBAC under basal conditions and restricts

accumulation of Met1-Ub on LUBAC substrates after NOD2

stimulation.

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 nonstimulated 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 ubiquitina-

tion of TNFR1 at 5 min after TNF stimulation (Figure 6E).

Conversely, overexpression of OTULIN decreased the level of

Molecular Cell


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 re-

stricting the accumulation of Met1-Ub in innate immune receptor

signaling complexes, and that this is essential for preventing

excessive proinflammatory signaling in response to NOD2

stimulation.

DISCUSSION

Met1-Ub assembled by LUBAC has emerged as a versatile pro-

tein modification that regulates several cellular functions, in

particular proinflammatory signaling (Tokunaga and Iwai,

2012). We and others recently reported that NOD2-mediated

transcription of NF-kB-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 tran-

scription of inflammatory mediators after NOD2 stimulation. In

comparison, OTULIN and LUBAC seem to have more subdued

functions in regulating NF-kB activation in response to TNFR1

stimulation. Instead, deregulation of Met1-Ub formation after

TNF treatment leads to excessive programmed cell death (Bois-

son et al., 2012; Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga

et al., 2009). The underlying mechanisms dictating these differ-

ences 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 protein

substrates (NEMO and RIPK1) have been reported so far (Bois-

son 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 remark-

able 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

nonselective 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 stimula-

tion 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,

M

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 regu-

lated 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 stimu-

lated 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 sub-

strates 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 overexpress-

ing 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 pre-

sent at very low levels in cells, and why its abundance is not

significantly increased by overexpression of LUBAC, despite

the fact that 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, sug-

gesting 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-kB transcription factors. Consequently, A20 is essential for

termination of proinflammatory 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 that

OTULIN levels were not elevated, consistent with data obtained


Molecular Cell


using TNF (Keusekotten et al., 2013). Also, the temporal profile of

RIPK2 ubiquitination, degradation of IkBa, or transcription of

NF-kB-responsive genes after NOD2 stimulation was not

markedly prolonged by OTULIN depletion. Instead, OTULIN

knockdown rendered cells hyperresponsive to NOD2 stimula-

tion, which was particularly evident when ligand concentration

was decreased. We speculate that OTULIN might be involved

in determining the threshold for proinflammatory 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 pro-

vide 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, Antibodies, and

Affinity Resin

See the 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 5–1,000 ng/ml or

TNF 1–100 ng/ml (R&D Systems, Minneapolis, MN); both were added directly

to the culture medium (see the Supplemental Experimental Procedures).

Luciferase Reporter Assays

HEK293T cells were cotransfected with the NF-kB luciferase reporter

construct pBIIX-luc and a thymidine kinase-renilla luciferase construct for

normalization of transfection efficiency. Cells were either cotransfected 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 the 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 the Supplemental Experi-

mental 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 (full sequence will be made available upon

request). Purified material was analyzed by immunoblotting or mass spec-

trometry analysis (see the Supplemental Experimental Procedures).

Deubiquitinase Assays

Ubiquitin conjugates from L18-MDP-treated siMM or siOTULIN#2 were iso-

lated byM1-SUB onGST-beads, washed in PBS Tween-20 (0.1%), and resus-

pended in deubiquitinase-buffer (50 mMHEPES [pH 7.5], 100 mMNaCl, 2 mM


DTT, 1 mM MnCl2, 0.01% Brij-35) without or with deubiquitinases (USP21

[0.5 mM], OTULIN [1 mM], OTULIN CA [1 mM], vOTU [0.4 mM], OTUB1

[15 mM]). Samples incubated for 1 hr 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 (Keuse-

kotten et al., 2013) from U2OS/NOD2 cells transfected and treated as indi-

cated. HA-NOD2 from U2OS/NOD2 cells treated with doxycycline (2 mg/ml)

for 24 hr 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 the Sup-

plemental 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)- or GFP (M1-SUB)-positive

cells per condition were counted in each experiment (see the 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 60min 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 Scienti-

fic, Rockford, IL) as described (Michalski et al., 2011). Raw data files were

analyzed using MaxQuant (Cox and Mann, 2008) (see the Supplemental

Experimental Procedures).

Statistical Analysis

The two-tailed Student’s t test was used to determine statistical significance.

Error bars represent SEM.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures, one table, Supplemental

Experimental Procedures, and Supplemental References and can be found

with this article at http://dx.doi.org/10.1016/j.molcel.2013.06.004.

ACKNOWLEDGMENTS

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 sup-

ported by 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, D.K.), the European Research

Council (D.K.), the EMBO Young Investigator Programme (D.K.), and the Lister

Institute for Preventive Medicine (to D.K.). The Center for Protein Research is

supported by a grant from the Novo Nordisk Foundation.

Received: May 4, 2013

Revised: June 5, 2013

Accepted: June 6, 2013

Published: June 27, 2013


Molecular Cell


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Molecular Cell Article - Laboratory of Molecular Biology · Molecular Cell Article ... SUMMARY ... NF-kB activation is dependent on its ability to disassemble