Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

Molecular Cell

Article

The Ubiquitin Ligase XIAP Recruits LUBACfor NOD2 Signaling in Inflammationand Innate ImmunityRune Busk Damgaard,1,2,11 Ueli Nachbur,3,4,5,11 Monica Yabal,6,11 Wendy Wei-Lynn Wong,3,4 Berthe Katrine Fiil,1

Mischa Kastirr,6 Eva Rieser,8 James Arthur Rickard,3,4 Aleksandra Bankovacki,3,4 Christian Peschel,6 Juergen Ruland,9,7

Simon Bekker-Jensen,1 Niels Mailand,1 Thomas Kaufmann,10 Andreas Strasser,4,5 Henning Walczak,8 John Silke,3,4,5

Philipp J. Jost,6,11,* and Mads Gyrd-Hansen1,2,11,*1Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research2Biotech Research and Innovation Centre

University of Copenhagen, 2200 Copenhagen, Denmark3Department of Biochemistry, La Trobe University, Victoria 3086, Australia4The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia5Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3010, Australia6III. Medizinische Klinik7Institut fur Klinische Chemie und PathobiochemieKlinikum rechts der Isar, Technische Universitat Munchen, 81675 Munich, Germany8Tumor Immunology Unit, Department of Medicine, Imperial College London, W12 0NN London, UK9Laboratory of Signaling in the Immune System, Helmholtz Zentrum Munchen - German Research Center for Environmental Health,

85764 Neuherberg, Germany10Institute of Pharmacology, University of Bern, 3010 Bern, Switzerland11These authors contributed equally to this work

*Correspondence: philipp.jost@lrz.tu-muenchen.de (P.J.J.), mads.gyrd@cpr.ku.dk (M.G.-H.)DOI 10.1016/j.molcel.2012.04.014

SUMMARY

Nucleotide-binding and oligomerization domain(NOD)-like receptors constitute a first line of defenseagainst invading bacteria. X-linked Inhibitor ofApoptosis (XIAP) is implicated in the control of bacte-rial infections, and mutations in XIAP are causallylinked to immunodeficiency in X-linked lymphoproli-ferative syndrome type-2 (XLP-2). Here, we demon-strate that the RING domain of XIAP is essential forNOD2 signaling and that XIAP contributes to exacer-bation of inflammation-induced hepatitis in experi-mental mice. We find that XIAP ubiquitylates RIPK2and recruits the linear ubiquitin chain assemblycomplex (LUBAC) to NOD2. We further show thatLUBAC activity is required for efficient NF-kB acti-vation and secretion of proinflammatory cytokinesafter NOD2 stimulation. Remarkably, XLP-2-derivedXIAP variants have impaired ubiquitin ligase activity,fail to ubiquitylate RIPK2, and cannot facilitate NOD2signaling. We conclude that XIAP and LUBAC con-stitute essential ubiquitin ligases in NOD2-mediatedinflammatory signaling andpropose that deregulationofNOD2signalingcontributes toXLP-2pathogenesis.

INTRODUCTION

Recognition of bacterial pathogens by pattern-recognition

receptors (PRRs) and appropriate activation of proinflammatory

signaling pathways is essential for immunity and host survival.

Deregulation of these processes may lead to detrimental pathol-

ogies including immunodeficiency, inflammatory diseases, and

cancer (Chen et al., 2009; Grivennikov et al., 2010). Bacterial

cell wall constituents are sensed by multiple PRRs, including

nucleotide-binding oligomerization domain (NOD)-like receptors

that respond to peptidoglycan (PGN). NOD2 (also termed

CARD15, IBD1) is activated by the PGN component muramyldi-

peptide (MDP) and plays a central role in immune regulation

and, when its function is impaired, in development of Crohn’s

disease (Chen et al., 2009; Strober et al., 2006; Van Limbergen

et al., 2009).

Stimulation of NOD2 initiates ubiquitin-dependent signaling

events that activate mitogen-activated protein (MAP) kinases

and the NF-kB-activating IkB kinase (IKK) complex composed

of IKKa, IKKb, and NEMO (also termed IKKg) (Chen et al.,

2009). Upon ligand binding, NOD2 oligomerizes leading to

recruitment of RIPK2, TRAF2, cIAP1, and cIAP2. This induces

conjugation of K63-linked ubiquitin chains on RIPK2 (Hasegawa

et al., 2008; Yang et al., 2007), reportedly mediated by cIAP1/2

(Bertrand et al., 2009). The ubiquitin chains serve as a scaffold

to recruit and activate the TAK1:TAB1:TAB2/3 and IKK kinase

complexes (Hasegawa et al., 2008; Inohara et al., 2000; Yang

et al., 2007). In turn, NF-kB transcription factors are activated

leading to transcription of target genes and production of

proinflammatory cytokines and chemokines (Bertrand et al.,

2009; Hasegawa et al., 2008; Park et al., 2007).

Conjugation of ubiquitin chains linked through the N-terminal

methionine (M1-linked; also termed linear ubiquitin chains) has

emerged as an important regulatory mechanism in activation of

kinase cascades downstream of immune receptors, including

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 1

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

tumor necrosis factor (TNF)-R1, interleukin (IL)-1b receptor,

CD40, and TLR4 (Gerlach et al., 2011; Haas et al., 2009; Ikeda

et al., 2011; Tokunaga et al., 2009, 2011). Together, K63- and

M1-linked ubiquitin chains ensure efficient activation of MAP

kinases and the IKK complex to facilitate activation of NF-kB.

The conjugation of M1-linked chains is carried out by a trimeric

protein complex named the linear ubiquitin chain assembly

complex (LUBAC). LUBAC is composed of a catalytic subunit

HOIP and the two regulatory subunits HOIL-1 and SHARPIN.

A loss-of-function mutation in the Sharpin gene causes chronic

proliferative dermatitis (cpdm) in mice, characterized by pro-

gressive inflammation in the skin and multiple organs (Seymour

et al., 2007).

Recent findings indicate that X-linked IAP (XIAP) is important

for innate immune signaling in response to intracellular

bacteria. Xiap�/� mice fail to clear certain bacterial infections,

and cells lacking XIAP fail to activate NF-kB in response to

stimulation of NOD2 (Bauler et al., 2008; Krieg et al., 2009;

Prakash et al., 2010). In humans, XIAP mutations have recently

been described in patients suffering from X-linked lymphopro-

liferative syndrome type 2 (XLP-2), a condition characterized

by deregulation of the immune system and defined by hemo-

phagocytic lymphohistiocytosis (Marsh et al., 2010; Pachlopnik

Schmid et al., 2011; Rigaud et al., 2006). XLP-2 patients

frequently develop cytopenia, splenomegaly, fever, and hemor-

rhagic colitis, suggesting that individuals with mutations in XIAP

are predisposed to the development of immunodeficiency

(Pachlopnik Schmid et al., 2011). However, the molecular

mechanism by which XIAP contributes to regulation of immune

signaling is currently unknown.

Here, we provide evidence that XIAP ubiquitylates RIPK2 and

recruits LUBAC to NOD2 for activation of NF-kB and cytokine

secretion, a process abrogated by mutations in XIAP derived

from XLP-2 patients. In addition, we demonstrate that XIAP

and LUBAC are essential ubiquitin ligases in NOD2-mediated

inflammatory signaling.

RESULTS

XIAP Is Required for NOD2-Activated Immune SignalingTreatment of bone marrow-derived macrophages (BMDMs)

with MDP revealed that XIAP, like NOD2 itself, is essential for

MDP-induced activation of the MAP kinases p38 and JNK as

well as phosphorylation and degradation of IkBa (Figure 1A;

Figure S1A available online). Accordingly, transcription and

secretion of TNF and IL-6 induced by MDP or a lipidated form

of MDP (L18-MDP) was strongly impaired in XIAP- and NOD2-

deficient BMDMs compared to wild-type (WT) BMDMs (Figures

1B and 1C; Figures S1B–S1D). Of note, Xiap�/� andWT BMDMs

showed similar differentiation and comparable survival after

MDP stimulation, indicating that the reduction in cytokine

secretion observed in Xiap�/� BMDMs was independent of the

antiapoptotic function of XIAP (Figures S1E and S1F).

To investigate the requirement for XIAP for NOD2 signaling

in vivo, we injected WT and Xiap�/� mice intraperitoneally (i.p.)

with MDP and found that the serum level of TNF and IL-6

increased in WT mice but remained unaffected in Xiap�/� mice

(Figure 1D). Accordingly, MDP treatment increased Tnf and Il6

2 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

mRNA levels in liver tissue of WT but not Xiap�/� mice

(Figure 1E).

Thus, XIAP plays an important role in NOD2-dependent acti-

vation of MAP and IKK kinase signaling, transcription of NF-kB

target genes, and secretion of proinflammatory cytokines

in vitro and in vivo.

XIAP Contributes to NOD2-Mediated Inflammation-Induced Liver Injury In VivoCoactivation of NOD2 and TLR4 leads to synergistic production

of cytokines, which constitutes an important mechanism for

mounting an effective immune response against bacteria

(Kobayashi et al., 2005; Park et al., 2007; Tsai et al., 2010)

(Figure 2A). The amplification of TNF and IL-6 secretion after

lipopolysaccharide (LPS) + MDP treatment was XIAP dependent

since cotreatment with MDP failed to increase TNF and IL-6

levels in LPS-treated Xiap�/� BMDMs when compared to WT

cells (Figure 2A). In line with a critical role for XIAP in NOD2 but

not in TLR4 signaling, LPS treatment induced comparable acti-

vation of MAP kinases p38 and JNK, and phosphorylation and

degradation of IkBa in WT and Xiap�/� BMDMs (Figure S2A).

In addition, the induction of Tnf and Il6 transcription and subse-

quent cytokine secretion after LPS treatment was similar in WT

and Xiap�/� cells, although we observed slightly diminished

levels of TNF and IL-6 in vivo after i.p. injection of LPS in Xiap�/�

mice (Figure 2A; Figures S2B–S2D).

Injection of mice with low doses of LPS plus the liver-specific

transcriptional inhibitor D(+)-galactosamine (GalN) results in

TNF-dependent fatal hepatitis (Kaufmann et al., 2009; Leist

et al., 1995). The hepatocellular destruction results from the

inflammatory response of macrophages to bacterial pathogen-

associated molecular patterns (PAMPs) and is caused by the

production of TNF and IL-6 that damage hepatocytes (Szabo

et al., 2007). To investigate the contribution of XIAP-mediated

NOD2 signaling in inflammation-induced liver injury, WT and

Xiap�/� mice were injected i.p. with MDP prior to injection of

LPS+GalN. Cotreatment withMDP plus LPS +GalN accelerated

the development of fatal hepatitis induced in WT but not Xiap�/�

mice illustrated macroscopically by blackened livers that are

indicative of hepatic destruction and intrahepatic hemorrhage

(Figure 2B). Histologically, the hepatic injury was characterized

by loss of liver architecture, severe hemorrhagic infiltration,

and increased numbers of apoptotic hepatocytes (Figures 2C

and 2D; Figure S2E). Accordingly, WT but not Xiap�/� mice pre-

sented with elevated serum levels of alanine aminotransferase

(ALT) and aspartate aminotransferase (AST) (Figure 2E). WT as

well as Xiap�/� mice treated with LPS + GalN without MDP

pretreatment sustained only minor liver damage at 5 hr of treat-

ment (Figures 2B–2E). Thus, XIAP-dependent NOD2 signaling

aggravates inflammation-induced liver injury, which is in line

with the failure of BMDMs from Xiap�/� mice to induce secretion

of TNF and IL-6 in response to NOD2 stimulation.

The Ubiquitin Ligase Activity of XIAP Is Criticalfor NOD2-Mediated Activation of NF-kBTo characterize the molecular function of XIAP in the NOD2

signaling pathway, we employed WT and XIAP-deficient HCT-

116 colon carcinoma cells that have previously been used to

Figure 1. XIAP Is Required for Signaling in Response to NOD2 Stimulation

(A) Lysates from WT and Xiap�/� BMDMs treated with MDP (10 mg/ml) were examined by immunoblotting.

(B) RelativemRNA levels weremeasured by semiquantitative RT-PCR ofWT or XIAP�/�BMDMs treated for 4 hr withMDP (10 mg/ml). Data representmean ± SEM

of six (WT) or four (Xiap�/�) independent experiments.

(C) The concentration of TNF and IL-6 in supernatants from WT or Xiap�/� BMDMs stimulated with MDP (10 mg/ml). Data represent mean ± SEM of three

independent experiments each performed in triplicate. ND indicates not detectable.

(D and E) WT and Xiap�/� mice were injected i.p. with vehicle (v) or MDP (25 mg/kg body weight) 4 hr prior to collection of serum of peripheral blood and liver

tissue. Serum cytokines were measured by flow cytometry (D), and relative mRNA levels of liver tissue were measured by semiquantitative RT-PCR (E). n.s., not

significant. Replicates as indicated in the figure. The two-tailed Student’s t test was used to determine statistical significance. See Figure S1.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

investigate the function of XIAP in NOD2 signaling (Krieg et al.,

2009). In accordance with the data obtained in BMDMs, stimula-

tion with MDP (or L18-MDP) induced phosphorylation and

degradation of IkBa, Ser536 phosphorylation of the NF-kB

subunit p65/RelA, and transcription of the NF-kB target genes

IL8 and NFKBIA only in WT but not in XIAP�/y HCT-116 cells

(Figures S3A and S3B). The failure of XIAP�/y HCT-116 cells to

activate NF-kB was specific to the NOD1/2 pathways because

these cells responded normally to stimulation with TNF (Fig-

ure S3C) and to ectopic expression of TLR2 and TLR4, whereas

NF-kB activity induced by NOD1 and NOD2 expression was

impaired (Figure S3D).

The C-terminal RING domain provides XIAP with ubiquitin

ligase (E3) activity and is important for its function (Galban and

Duckett, 2010). To test if XIAP’s RING activity regulates NOD2

signaling, XIAP-deficient HCT-116 cells were reconstituted

with wild-type XIAP (XIAPWT) or a mutant variant that lacks ubiq-

uitin ligase activity due to a phenylalanine-to-alanine substitution

at position 495 (XIAPF495A) (Gyrd-Hansen et al., 2008) (Figures 3A

and 3B). Treatment withMDP showed that the E3 activity of XIAP

is required for NF-kB activation in response to NOD2-stimulation

because XIAPWT but not XIAPF495A restored MDP-induced

NF-kB activation in XIAP-deficient HCT-116 cells (Figure 3C).

Activation of the NF-kB reporter induced by ectopic expression

of NOD2 was also inhibited by overexpression of XIAPF495A in

HEK293T cells (Figure 3D). Furthermore, BMDMs isolated from

gene-targeted mice that express a truncated form of XIAP lack-

ing the RING domain (XIAPDRING; Figure 3A) (Schile et al., 2008)

failed to induce transcription of Tnf and Il6 and did not secrete

TNF when stimulated with MDP (Figures 3E and 3F). These

data demonstrate that XIAP functions as a ubiquitin ligase in

NOD2 signaling and imply that XIAP ubiquitylates one or more

proteins important for propagation of signaling downstream of

NOD2.

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 3

Figure 2. XIAP Contributes to NOD2-Mediated Inflammation-Induced Liver Injury In Vivo

(A) BMDMs from WT and Xiap�/� mice were stimulated for the indicated times with ultrapure LPS (50 ng/ml) and/or MDP (10 mg/ml) before measurement of

secreted TNF and IL-6 in supernatants. n.s., not significant. Data represent mean ± SEM of three independent experiments each performed in triplicate.

(B) WT and Xiap�/� mice were injected i.p. with PBS (vehicle) or MDP (5 mg/kg body weight) 4 hr prior to i.p. injection of ultrapure LPS (125 ng/kg body weight)

alongwith GalN (1 g/kg body weight). Macroscopic images of livers fromWT and Xiap�/�mice treated as indicated and sacrificed 5 hr after injection of LPS/GalN.

(C and D) Hematoxylin and eosin (H&E) staining (C) and quantification of TUNEL staining of liver sections (D) from mice treated as in (B). Images in (C) are

representative of three mice per genotype and time point. Scale bars: 100 mm. Quantification of TUNEL-positive cells is shown in (D) from five viewing fields per

mouse of three independent mice per time point; error bars indicate SEM.

(E) Serum levels of liver transaminases ALT and AST in mice treated as described in (B). n.s., not significant. Two-tailed Student’s t test was used for statistical

analysis; replicates as indicated in figure. See Figures S1 and S2.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

XIAPContributes to Ubiquitylation of RIPK2 in Responseto NOD2 ActivationThe presence of bacterial peptidoglycan in the cytoplasm leads

to NOD2 activation and assembly of a multiprotein signaling

complex (NOD2-SC) that includes RIPK2, cIAP1, cIAP2, and

TRAF2 (Bertrand et al., 2009; Inohara et al., 2000). XIAP has

been reported to interact with RIPK2 (Krieg et al., 2009) but it

is not clear whether XIAP is associated with the NOD2-SC. To

test this, we examined whether XIAP could be copurified with

HA-tagged NOD2 from HEK293T cells and found that endoge-

nous XIAP as well as endogenous RIPK2, cIAP1, cIAP2, and

4 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

TRAF2 copurified with NOD2 (Figure 4A). In addition, the puri-

fied NOD2-SC contained the IKK subunit NEMO, RIPK1, and

all three subunits of LUBAC (HOIP, HOIL-1, and SHARPIN)

(Figure 4A). RIPK2 interacts with several components of the

NOD2-SC and links NOD2 to downstream components of the

complex. In accordance, NOD2-SC components readily copuri-

fied with ectopically expressed HA-tagged RIPK2 (Figure S4A).

Furthermore, RNAi-mediated knockdown of RIPK2 reduced

the copurification of all tested complex components with HA-

tagged NOD2 (Figure S4B). This reiterates the central role of

RIPK2 in assembly of the NOD2-SC and shows that XIAP,

Figure 3. The Ubiquitin Ligase Activity of XIAP Is Critical for NF-kB Activation and Cytokine Secretion in Response to NOD2 Stimulation

(A) Schematic overview of XIAP variants. BIR, baculovirus IAP repeat; UBA, ubiquitin associated; RING, really interesting new gene.

(B) Ubiquitin conjugates were purified from cells expressing Strep-ubiquitin and HA-tagged XIAPWT or XIAPF495A.

(C and D) NF-kB activity in lysates of cells transfected as indicated and stimulated with MDP (10 mg/ml) for 24 hr (C) or transfected with HA-NOD2 (D). Data

represent mean ± SEM of three independent experiments, each performed in triplicate. n.s., not significant.

(E) Relative mRNA levels were measured by semiquantitative RT-PCR of WT, XIAP�/�, and XIAPDRING BMDMs treated for 4 hr with MDP (10 mg/ml). Data

represent mean ± SEM of 3–6 independent experiments.

(F) TNF in supernatants from WT, XIAP�/�, and XIAPDRING BMDMs that had been stimulated with MDP (10 mg/ml) for the indicated time points. A line connects

data points from BMDM cultures isolated from individual mice. Each data point represents the average of triplicate measurements. The two-tailed Student’s

t test was used to determine statistical significance. See Figure S3.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

LUBAC (indicated here by HOIP) and RIPK1 specifically copur-

ify with HA-tagged NOD2 in a RIPK2-dependent manner. We

were surprised that RIPK1 was present in the NOD2-SC and

therefore investigated whether RIPK1 has an unappreciated

role in NOD2-dependent signaling akin to its role in TNF-R1

signaling. However, RIPK1-depletion in HEK293T did not affect

NOD2-induced NF-kB activation whereas depletion of RIPK2

essentially blocked the activation of NF-kB (Figure S4C).

Furthermore, secretion of TNF and IL-6 by MDP was strongly

impaired in fetal liver derived macrophages from gene-targeted

mice deficient for RIPK2 but not RIPK1 compared to WT or

Ripk1+/� macrophages (Figure S4D). We conclude that RIPK1

is not rate limiting for NOD2-dependent NF-kB activation and

cytokine secretion.

To investigate potential targets for ubiquitylation by XIAP

within the NOD2-SC, tandem ubiquitin-binding entities (TUBEs)

were employed to affinity purify endogenous ubiquitin conju-

gates after NOD2 stimulation. Stimulation of THP-1 human

monocytic cells with L18-MDP induced ubiquitylation of RIPK2

(Figures 4B and 4C). The timing of RIPK2 ubiquitylation coin-

cided with phosphorylation and degradation of IkBa and

preceded S536 phosphorylation of the p65 subunit of NF-kB

(Figure 4B; Figure S5A). This is consistent with the notion that

ubiquitylation of RIPK2 facilitates activation of IKK (Hasegawa

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 5

Figure 4. XIAP Ubiquitylates RIPK2 in Response to Stimulation of NOD2

(A) HA-NOD2 was immunoprecipitated from lysates of HEK293T cells transfected with empty vector or HA-NOD2 vector. Immunoprecipitates were examined for

copurified proteins by immunoblotting.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

6 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

et al., 2008). Intriguingly, RIPK2 appears to be the major target

for ubiquitylation in the activated NOD2-SC as none of the

other proteins in the complex were ubiquitylated in response to

stimulation with L18-MDP (Figure 4B; Figure S5B). Conversely,

treatment of monocytic THP-1 cells with TNF induced rapid

ubiquitylation of RIPK1 and degradation of IkBa, whereas

RIPK2 remained unmodified (Figure 4B). These observations

indicate that ubiquitylation within the NOD2-SC is tightly regu-

lated and specifically directed toward RIPK2.

We next investigated the role of XIAP in RIPK2 ubiquitylation.

XIAP was depleted by RNAi-mediated knockdown in THP-1

cells, which resulted in a reduced average size of ubiquitin-

RIPK2 conjugates and a decrease in overall RIPK2 ubiquitylation

in response to NOD2 stimulation (Figure 4C). MDP-stimulation of

XIAP-deficient HCT-116 and DLD-1 cells also led to formation of

RIPK2-ubiquitin conjugates of lower molecular weight than the

RIPK2-ubiquitin conjugates detected in the corresponding WT

cells (Figure 4D, compare lanes 1–3 with lanes 7–9; Figure S5C).

This indicates that XIAP contributes to ubiquitylation of RIPK2

in response to NOD2 activation. In support of this, ectopic

expression of XIAPWT in HEK293T cells stimulated ubiquitylation

of endogenous RIPK2, whereas the ubiquitin ligase-defective

XIAPF495A failed to induce RIPK2 ubiquitylation (Figure 4F).

cIAP1 and cIAP2 are reported to conjugate K63- and K48-linked

ubiquitin chains onto RIPK2 (Bertrand et al., 2009). To test if XIAP

modifies RIPK2 with K63- and K48-linked ubiquitin chains, we

coexpressed XIAP with ubiquitin mutants in which all lysine (K)

residues except K63 (K63 only) or K48 (K48 only) had been

mutated to arginine (R) in HEK293T cells. Intriguingly, XIAP-

mediated ubiquitylation of RIPK2 was lost by coexpression of

K63-only ubiquitin and was strongly reduced by coexpression

of K48-only ubiquitin (Figure 4G, compare lanes 5 and 6 with

lane 4). Consistently, coexpression of ubiquitin mutants in which

only K63 or K48 had been mutated (K63R and K48R, respec-

tively) did not alter XIAP-mediated RIPK2 ubiquitylation (Fig-

ure 4H, compare lanes 5 and 6 with lane 4). This suggests that

XIAP primarily conjugates ubiquitin chains on RIPK2 that are

linked through lysine residues other than K63 and K48.

To determine the relative contributions of cIAP1/2 and XIAP

in MDP-induced RIPK2 ubiquitylation, HCT-116 cells were

treated with the IAP antagonist LBW-242. LBW-242 causes

rapid degradation of cIAP1/2 without affecting XIAP stability

(Gaither et al., 2007) (Figure 4D, compare lanes 1–3 with lanes

4–6). As reported, pharmacological depletion of cIAP1/2

impaired TNF-induced ubiquitylation of RIPK1 (Bertrand et al.,

2008; Haas et al., 2009) (Figure 4E, compare lanes 1–3 with lanes

4–6). Surprisingly, the depletion of cIAP1/2 did not detectably

impair MDP-induced ubiquitylation of RIPK2 in WT cells despite

(B) Purification of endogenous ubiquitin conjugates from THP-1 lysates after st

examined for ubiquitylated RIPK1 and RIPK2 proteins by immunoblotting.

(C) THP-1 cells were transfected with siRNA targeting XIAP and were stimulated

(D and E) Cells were left untreated or treated with the IAP antagonist LBW-242

(10 ng/ml) (E). Ubiquitin conjugates were isolated from cell lysates and analyzed

(F) Ubiquitin conjugates were purified with StrepTactin-Agarose resin from lysates

was examined for ubiquitylated proteins by immunoblotting.

(G and H) Ubiquitin conjugates were purified with anti-HA-Agarose resin from lysa

Purified material was examined for ubiquitylated proteins by immunoblotting. Se

the rapid and almost complete disappearance of cIAP1 and

cIAP2 in the treated cells (Figure 4D, compare lanes 1–3 with

lanes 4–6). However, treatment of XIAP-deficient cells with

LBW-242, which essentially rendered the cells deficient for all

three IAP proteins, reduced MDP-induced RIPK2 ubiquitylation

considerably (Figure 4D, compare lanes 7–9 with lanes 10–12).

Similar results were obtained in THP-1 cells where RNAi-medi-

ated depletion of XIAP resulted in a stronger reduction in

RIPK2 ubiquitylation in LBW-242-treated cells compared to cells

not treated with LBW-242 (Figure S5D, compare lanes 3 and 4

with lanes 7 and 8). The residual ubiquitylation of RIPK2 after

L18-MDP treatment in the XIAP- and cIAP1/2-depleted cells is

likely due to the incomplete depletion of XIAP and cIAP1/2 in

these cells (Figure S5D, lane 8). Of note, LBW-242 treatment

slightly increased RIPK2 ubiquitylation in response to L18-

MDP in THP-1 cells (Figure S5D, compare lane 3 with lane 7)

and in WT HCT-116 cells (Figure 4D, compare lane 3 with lane

6). These results indicate that XIAP, together with cIAP1/2,

constitutes the major ubiquitin ligase activity that ubiquitylates

RIPK2 and that cIAP1/2 appear only to be rate limiting when

XIAP is not present.

XLP-2-DerivedMutations in XIAPAffect Its RINGActivityand Impair NOD2 SignalingXLP-2 patients often develop hemorrhagic colitis, which may

imply that defects in NOD2 signaling contribute to the pathogen-

esis of the disease. To investigate this, we functionally character-

ized two XLP-2-derived XIAP alleles carrying single base-pair

mutations that were identified in patients with detectable XIAP

expression (Marsh et al., 2010; Pachlopnik Schmid et al.,

2011). Both of the mutations located to the region encoding the

RING domain of XIAP: One is a non-sense mutation that intro-

duces a premature stop codon at position 466 (G466stop),

whereas the other mutation results in a proline to arginine sub-

stitution at position 482 (P482R) (Figure 5A). The position and

evolutionary conservation of these residues in the XIAP RING

suggested that they perturb the core RING structure and ligase

activity (Figures 5A and 5B). Consistent with this notion,

XIAPG466stop and XIAPP482R behaved like the RING mutant

XIAPF495A and were unable to auto-ubiquitylate, ubiquitylate

endogenous RIPK2 or activate NF-kB when ectopically overex-

pressed (Figures 5C–5E). Significantly, reconstitution of XIAP-

deficient cells with the patient-derived XIAP variants XIAPG466stop

or XIAPP482R failed to efficiently restore MDP-induced NOD2

signaling (Figure 5F). Akin to the XIAPF495A mutant, the XLP-2-

associated XIAP mutants also inhibited NOD2-induced NF-kB

activation when coexpressed with NOD2 in HEK293T cells

(Figure 5G). Together, this implicates the ubiquitin ligase activity

imulation with L18-MDP (200 ng/ml) or TNF (10 ng/ml). Purified material was

with L18-MDP. Cell lysates were analyzed as in (B).

(20 mM) for 15 min before stimulation with L18-MDP (200 ng/ml) (D) or TNF

as in (B).

of cells expressing Strep-Ubiquitin and XIAPWT or XIAPF495A. Purified material

tes of cells expressing HA-Ubiquitin variants and XIAP as indicated in the figure.

e Figures S4 and S5.

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 7

Figure 5. XLP-2-Derived Mutations in XIAP Affect Its RING Activity and Impair NOD2 Signaling

(A) Alignment of RING domain and C-terminal region of XIAP-like proteins. Blue andmagenta colors indicate Zn-coordinating Cys and His residues, respectively.

The green and red labels indicate residues altered in XIAP variants identified in XLP-2 patients. The XLP-2-associatedmutations studied here are indicated above

the alignment. Species abbreviations are as follows: Hs (Homo sapiens), Mm (Mus musculus), Clf (Canis lupus familiaris), Ss (Sus scrofa), Xl (Xenopus laevis),

Dr (Danio rerio), Dm (Drosophila melanogaster).

(B) Cartoon depiction of NMR structure of the RING domain of human XIAP (Protein Data Bank [PDB] ID: 2ECG). The red and green labels indicate the residues

mutated in XLP-2 patients.

(C and D) Ubiquitin conjugates were purified with StrepTactin-Agarose resin from lysates of cells expressing Strep-Ubiquitin and XIAP variants as indicated.

Purified material was examined by immunoblotting as indicated. Note that lanes 1–3 in (C) are also shown in Figure 3B.

(E–G) NF-kBactivity in lysates of cells transfected as indicated and treatedwith L18-MDP (200 ng/ml) for 24 hr (F). Data representmean ± SEMof 3–6 independent

experiments, each performed in triplicate. The two-tailed Student’s t test assuming unequal variance was used to determine statistical significance.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

8 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

Figure 6. LUBAC Is Recruited to NOD2 by XIAP

(A) Cells expressing HA-NOD2 were untreated or treated with LBW-242 (20 mg/ml) 1 hr prior to lysis and immunoprecipitation of HA-NOD2. Immunoprecipitates

were examined by immunoblotting for copurification of components of the NOD2 signaling complex.

(B) HA-NOD2 was immunoprecipitated from lysates of cells expressing HA-NOD2 and FLAG-XIAP (WT or F495A). Immunoprecipitates were examined for

copurification of components of the LUBAC complex. Asterisk indicates unspecific band detected by the anti-FLAG antibody.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

of XIAP in regulation of NOD2-dependent immune regulation

and suggests that XIAP RING domain mutations may contribute

to the pathogenesis of colitis in XLP-2 patients.

XIAP Recruits the LUBAC to NOD2LUBAC contributes to immune signaling through conjugation of

M1-linked ubiquitin chains and is recruited to the TNF- and CD40

receptor complexes by cIAP1/2. Having observed that all three

LUBAC subunits are constituents of the NOD2-SC, we investi-

gated the role of IAPs in recruiting LUBAC to the NOD2-SC.

Immunoprecipitation of HA-tagged NOD2 revealed that the

amounts of HOIP, SHARPIN, and HOIL-1 copurified were

reduced in XIAP-deficient HCT-116 cells, whereas the amounts

of RIPK2, TRAF2, and NEMO were unaffected (Figure 6A,

compare lanes 9 and 10). Importantly, reconstitution of XIAP-

deficient HCT-116 cells with XIAPWT increased the recruitment

of all three LUBAC subunits to the NOD2-SC to similar levels

as observed in WT cells, whereas expression of the RING-

mutated form XIAPF495A did not (Figure 6B). In contrast, pharma-

cological depletion of cIAP1/2 did not affect the copurification

of LUBAC components with HA-NOD2 (Figure 6A, compare

lanes 9 and 11, and lanes 10 and 12). This indicates that XIAP-

mediated ubiquitylation of RIPK2 specifically contributes to the

association of LUBAC with the NOD2-SC.

LUBAC Is Required for Efficient NF-kB Activationin NOD2 SignalingRNAi-mediated depletion of HOIP or HOIL-1 plus SHARPIN in

HCT-116 cells reduced activation of NF-kB in response to

MDP treatment, and NF-kB activation was further reduced by

depletion of all three LUBAC subunits (Figure 7A). Consistently,

stable knockdown of HOIL-1 in HeLa cells decreased activation

of NF-kB induced by ectopic expression of NOD2 (Figure 7B).

Although the catalytic activity of LUBAC is provided by HOIP,

SHARPIN has recently been demonstrated to be critical for

LUBAC function, which has been attributed to destabilization

of the LUBAC complex in mice lacking SHARPIN (Gerlach

et al., 2011). To further investigate the contribution of LUBAC

function, we prepared BMDMs from cpdm mice that harbor

a spontaneous mutation in the Sharpin gene, which causes

absence of the protein in all tested cell types, including macro-

phages (Gerlach et al., 2011; Seymour et al., 2007). In accor-

dance with the results obtained with RNAi-mediated depletion

of LUBAC subunits, we found no induction of Tnf and Il6

mRNA or secretion of these cytokines in cpdm BMDMs after

MDP treatment (Figures 7C and 7D).

To address the role for LUBAC ubiquitin ligase activity in NOD2

signaling, NOD2 was coexpressed with a HOIP mutant with

mutations in key cysteine residues in the two RING domains

(termed HOIPmutR2), which lacks ubiquitin ligase activity and

cannot activate NF-kB (Haas et al., 2009; Tokunaga et al.,

2009) (Figure S6A). Expression of HOIPmutR2 alone or together

with HOIL-1 inhibited NOD2-induced activation of NF-kB,

whereas coexpression of HOIPWT and HOIL-1 resulted in

a modest increase in activation of NF-kB (Figure 7E, compare

lane 2 with lanes 5–7). Expression of HOIPWT or HOIL-1 alone

did not change NOD2-induced activation of NF-kB (Figure 7E,

compare lane 2 with lanes 3 and 4). Depletion of HOIL-1 or

coexpression of HOIPmutR2 and HOIL-1 also inhibited NF-kB

activation induced by ectopic expression of XIAP (Figures 7B

and 7F, compare lane 2 with lane 5 in Figure 7F). Conversely,

RNAi-mediated depletion of XIAP only led to a minor decrease

in the activation of NF-kB induced by coexpression of HOIPWT

and HOIL-1, although the depletion of XIAP did inhibit NOD2-

induced NF-kB activity (Figure S6A). This is consistent with our

observation that XIAP recruits LUBAC components to the

NOD2-SC and indicates that LUBAC functions as a ubiquitin

ligase downstream of XIAP.

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 9

Figure 7. LUBAC Is Required for Efficient NF-kB Activation and Cytokine Secretion in Response to NOD2 Stimulation

(A) NF-kB activity in lysates of cells depleted for LUBAC subunits as indicated and treated with MDP (10 mg/ml) for 24 hr.

(B) NF-kB activity in lysates of cells with or without stable knockdown of HOIL-1 after transfection with HA-NOD2 or FLAG-XIAPWT vectors.

(C) Relative mRNA levels of Tnf and Il6 in WT or cpdm BMDMs treated with L18-MDP (200 ng/ml). Data represent mean ± SEM of four (WT) or three (cpdm)

independent experiments). n.s., not significant.

(D) TNF and IL-6 in supernatants from WT and cpdm BMDMs stimulated with L18-MDP (200 ng/ml). A line connects data points from BMDM cultures isolated

from individual mice. Each data point represents the average of triplicate measurements.

(E and F) NF-kB activity in lysates of cells transfected with the expression vectors indicated. Data in (A), (B), (C), (E), and (F) represent mean ± SEM of at least three

independent experiments, each performed in triplicate. The two-tailed Student’s t test was used to determine statistical significance. See Figure S6.

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

10 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

DISCUSSION

IAP proteins have emerged as key ubiquitin ligases in the

regulation of innate immunity (Gyrd-Hansen and Meier, 2010).

Whereas cIAP1 and cIAP2 have been shown to regulate

signaling cascades downstream of several immune receptors,

including NOD1 and NOD2, the role for XIAP in these processes

has remained poorly defined. O’Riordan and colleagues demon-

strated that Xiap�/� mice have a defect in clearance of intra-

cellular bacteria and suggested a role for XIAP in NOD2 signal

transduction (Bauler et al., 2008). In line with this, we show that

Xiap�/� mice are unable to induce Tnf and Il6 transcription and

cytokine secretion after MDP treatment and that XIAP contrib-

utes to organ damage triggered by bacterial PAMPs in a mouse

model of inflammation-induced hepatitis.

XIAP serves well-described antiapoptotic functions (Sriniva-

sula and Ashwell, 2008) including its role in preventing FAS-

induced apoptosis in hepatocytes (Jost et al., 2009). It has

been suggested that XIAP contributes to bacterial clearance

by protecting macrophages from apoptosis triggered by infec-

tion with Chlamydophila pneumonia (Prakash et al., 2010).

However, this antiapoptotic role is unlikely to contribute to the

observed signaling defect described here since MDP alone or

combined with LPS failed to induce apoptosis in WT, Xiap�/�

or XiapDRING macrophages (Figure S1E).

The ubiquitylation of RIPK2 is a critical step for signal trans-

duction in response to NOD2 stimulation. Employing XiapDRING

macrophages and RING-mutated XIAP variants, we demon-

strate that XIAP is an essential ubiquitin ligase in the NOD2

signaling pathway and functions in the formation of the

receptor-signaling complex. This finding, together with previous

reports, indicates that cIAP1/2 and XIAP collectively constitute

the major (possibly sole) ubiquitin ligases for RIPK2 in response

to NOD2 stimulation. Analyzing the ubiquitylation of components

in the NOD2-SC, we uncovered a remarkable specificity in ubiq-

uitylation of RIPK2. This indicates that the ubiquitin ligase activ-

ities of IAP proteins in the NOD2-SC are tightly regulated and

specifically directed to RIPK2. This notion is supported by the

observation that the absence of XIAP results in a defined reduc-

tion in the apparent molecular weight of the ubiquitin-RIPK2

species induced after NOD2 activation, which may reflect that

XIAP conjugates a distinct ubiquitin chain onto RIPK2 that is

required to propagate the signal and enable IKK activation.

This is supported by our finding that XIAP modifies RIPK2 with

ubiquitin chain types that differ from the K63- and K48-linked

ubiquitin chains conjugated on RIPK2 by cIAP1/2. In addition,

our data are consistent with the idea that cIAP1 and cIAP2

contribute to RIPK2 ubiquitylation in response to NOD2 activa-

tion, but suggest that they are not rate limiting when XIAP is

present. This implies that cIAP1/2 may have functions in addition

to ubiquitylation of RIPK2 that are important for NOD2 signaling.

cIAP1/2 are reported to facilitate the association of LUBAC

with the TNF-R1 complex by ubiquitylating RIPK1 (Gerlach

et al., 2011; Haas et al., 2009), whereas XIAP is dispensable

for TNF-R1 signaling (Harlin et al., 2001; Vince et al., 2007).

However, the RING of XIAP proved to play the pivotal role in

the recruitment of LUBAC to the NOD2-SC, whereas cIAP1

and cIAP2 were not needed. The underlying molecular explana-

tion for these differential functions of XIAP and cIAP1/2 in the

respective NOD2- and TNF-R1-signaling complexes are not

yet completely understood. Nonetheless, our data provide

evidence for a unifying concept where IAPs regulate immune

receptor signaling by generating ubiquitin platforms that

promote the recruitment of LUBAC and facilitate the activation

of downstream kinase cascades.

LUBAC-dependent conjugation of M1-linked ubiquitin chains

may be a general regulatory mechanism to modulate the extent

of activation of NF-kB transcription factors in response to stim-

ulation of immune receptors. Our finding that LUBAC is recruited

to the NOD2-SC and is functionally relevant within the complex

extends the repertoire of known signaling pathways regulated

by LUBAC to also include intracellular pattern-recognition

receptor pathways. Our data indicate that XIAP and LUBAC

regulate NOD2 signaling in a hierarchical manner in which XIAP

functions upstream of LUBAC. The function of M1-linked ubiqui-

tin chains has not been fully resolved but it has been proposed

that NEMO, upon binding to M1-linked ubiquitin chains,

undergoes a conformational change to facilitate more efficient

activation of the catalytic subunits of the kinase complex (Bloor

et al., 2008; Rahighi et al., 2009). Thus, LUBAC may contribute

to NOD2 signaling by providing the type of ubiquitin chain

directly needed to fully activate IKK, whereas XIAP provides

the chain needed to efficiently recruit and/or retain LUBAC at

the NOD2-SC.

The X-linked lymphoproliferative syndrome (XLP-1/-2) was

first described in families carrying null mutations in SH2D1A

(SAP; XLP-1); however, disease progression varies between

patients with XLP-1 (SAP) and XLP-2 (XIAP) (Marsh et al.,

2010; Pachlopnik Schmid et al., 2011). Some XLP-2 patients

develop chronic hemorrhagic colitis leading to inflammatory

bowel disease (IBD), whereas this is not observed in XLP-1

patients. NOD2 mutations similarly predispose to development

of Crohn’s disease which points to a specific role for XIAP in

NOD2-dependent immune regulation. In support of this notion,

we find that mutations in the XIAP RING, identified in XLP-2

patients, interfere with XIAP’s ubiquitin ligase activity, its ability

to ubiquitylate RIPK2, and to facilitate NF-kB activation in

response to NOD2 stimulation. XLP syndromes are defined by

hemophagocytic lymphohistiocytosis and patients frequently

develop splenomegaly and cytopenia. This may indicate that

XIAP also contributes to signaling downstream of immune

receptors other than NOD2 and that deregulation of these

signaling pathways contributes to the pathogenesis of XLP-2.

The key function of XIAP as a ubiquitin ligase in immune

signaling indicates that pharmacological inhibition may be

a feasible strategy for the treatment of patients suffering from

overactivation of the immune system. Targeting XIAP and

cIAP1/2 function with IAP antagonists might provide therapeutic

benefit by lowering the response of macrophages to certain

proinflammatory stimuli. Several IAP antagonists (e.g., Smac

mimetics) have recently been developed and are currently

used in clinical trials for treatment of cancer (Gyrd-Hansen and

Meier, 2010; Kaufmann et al., 2011); however, their relevance

in immunology has only recently emerged (Damgaard and

Gyrd-Hansen, 2011). Most IAP antagonists do not display selec-

tivity against individual members of the IAP family, but the ability

Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc. 11

Molecular Cell

XIAP Recruits LUBAC for NOD2 Signaling

Please cite this article in press as: Damgaard et al., The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immu-nity, Molecular Cell (2012), doi:10.1016/j.molcel.2012.04.014

to specifically target the more restricted role of XIAP in immune

signaling with specific inhibitors may be desirable to avoid

unwanted side effects caused by inactivation of cIAP1/2.

EXPERIMENTAL PROCEDURES

Generation of BMDMs and Fetal Liver-Derived Macrophages

BMDMs were prepared from bone marrow cells derived from femora and

tibiae of WT, Xiap�/�, XiapDRING, and SHARPIN-deficient cpdm mice as

previously described (Hammer et al., 2005). Fetal liver-derived macrophages

were generated from E14.5 livers of Ripk1+/� and Ripk2�/� parental matings.

Cells were differentiated for 5 days in 20% L929 conditioned medium

before treatment. Identity of macrophages was confirmed by F4/80 and

Gr-1 stainings and analysis by flow cytometry. Both BMDM and fetal liver-

derived macrophages were primed with IFN-g (10 ng/ml; R&D Systems,

Minneapolis, MN), unless stated otherwise, for 2 hr prior to PRR stimulation

for the indicated times. Cell viability was measured 24 hr after PRR stimulation

with CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) or

MTT assay (Invitrogen, Paisley, UK). Animal experiments were performed

under ethics approval 09-57B of La Trobe University, Bundoora, Australia

and ethics approval AEC 2007.005 of the Walter and Eliza Hall Institute of

Medical Research, Melbourne, Australia.

PRR and TNF-R1 Stimulation

Cells were stimulated for the indicated times with ultrapure LPS from

Escherichia coli K12 (50 ng/ml; InvivoGen, San Diego, CA), MDP (10 mg/ml;

InvivoGen), L18-MDP (200 ng/ml; InvivoGen), or TNF (10 ng/ml; R&D Systems)

as indicated. LPS, L18-MDP, and TNF were added directly to the culture

medium, whereas MDP was added together with FuGENE 6 (Roche Ltd.,

Basel, Switzerland). MDP (10 mg) mixed with 40 ml serum-free medium and

1 ml FuGENE 6 per ml of cell culture medium was incubated at room temper-

ature for 20–30 min before the mixture was added drop-wise to the cells.

For BMDM stimulations, MDP was added directly to the culture medium.

Induction of Cytokine Secretion or Hepatitis

For cytokine secretion, female 6- to 8-week-old C57BL/6 mice were injected

i.p. with MDP (25 mg/kg body weight; InvivoGen) or ultrapure LPS (10 mg/kg

body weight, InvivoGen), and serum or liver was harvested. Cytokines were

measured with Cytokine Bead Array (BD Biosciences, San Jose, CA), and total

RNAwas isolated with TRIzol reagent (Invitrogen) according to manufacturer’s

instructions. For induction of fatal hepatitis, mice were primed for 4 hr by i.p.

injection of MDP (5 mg/kg body weight; InvivoGen). All animals were injected

i.p. with ultrapure LPS (125 ng/kg body weight, InvivoGen) plus GalN (1 g/kg

body weight, Sigma, Gillingham, UK). For survival, animals were monitored

every 15 min and sacrificed when sick. ALT and AST were measured in 50–

100 ml of serum from peripheral blood and analyzed by modified International

Federation of Clinical Chemistry and Laboratory Medicine method run on

a Beckman/Coulter AU2700.

Purification of Endogenous Ubiquitin Conjugates

Tandem Ubiquitin Binding Entities (TUBEs) were used to purify endogenous

Ub conjugates from cell lysates according to the manufacturer’s recommen-

dations with minor modifications. TUBEs consist of four UBA domains in

tandem and bind polyubiquitin chains with high affinity (Hjerpe et al., 2009).

Briefly, lysis buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 1% NP-40, 2 mM

EDTA) was supplemented with 1 mM DTT, 13 protease inhibitor mix (Sigma),

and 50 mg/ml of GST-TUBE1 (Lifesensors, Malvern, PA). THP-1 cells were

lysed in ice-cold 100 ml lysis buffer per 5 3 106 cells. For HCT-116 and

DLD-1 cells, one 70%–80% confluent 10 cm dish per condition was lysed in

300 ml lysis buffer.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and Supplemental Experimental

Procedures and can be found with this article online at doi:10.1016/j.molcel.

2012.04.014.

12 Molecular Cell 46, 1–13, June 29, 2012 ª2012 Elsevier Inc.

ACKNOWLEDGMENTS

We thank Drs. H. Steller, R.A. Flavell, M. Kelliher, C. Borner, C.H. Emmerich,

P. Meier, O. Gross, C. Duckett, M. Jaattela, P. Schneider, P. Eitz-Ferrer,

G. Nunez, B. Vogelstein, and L.A. O’Reilly for gifts of mice, antibodies, and

reagents; Dr. M. Frodin for laboratory space (M.G.-H. and R.B.D.); Dr. R.

Czajko from the Department of Biochemistry at the Royal Melbourne Hospital

for measurements of ALT and AST; and Dr. D. Neuberg from the Department of

Biostatistics, School of Public Health and Biostatistics and Computational

Biology, Dana-Farber Cancer Institute, Harvard for statistical help. We thank

Jan Christian for experimental help and Leigh Zawel and Novartis for LBW-

242. This work was supported by a Max-Eder Program grant from the Mildred

Scheel-Stiftung/Deutsche Krebshilfe (P.J.J.), a Steno Fellowship from the

Danish Council for Independent Research - Natural Sciences (M.G.-H.), the

Danish Cancer Society (M.G.-H.), a fellowship from the Swiss National Science

foundation (# PA00P3_126249 to U.N.), the NHMRC (Canberra, program

#461221 and fellowship #461299 to A.S.), and the Leukemia and Lymphoma

Society (SCOR grant #7413 to A.S.). This work was made possible through

Victorian State Government Operational Infrastructure Support and Australian

Government NHMRC IRIISS.

Received: November 17, 2011

Revised: March 19, 2012

Accepted: April 12, 2012

Published online: May 17, 2012

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