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: [email protected] (P.J.J.), [email protected] (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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