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com

ScienceDirect
TNFR1-activated NF-kB signal transduction: regulation
by the ubiquitin/proteasome system
Ingrid E Wertz
The Tumor Necrosis Factor Receptor-1 (TNFR1) is a central
regulator of inflammation, cell death, and cellular proliferation.
As such, alterations in TNFR1 signaling are associated with
numerous diseases ranging from autoimmune syndromes to
cancer. Understanding the regulation of TNFR1 signaling is
therefore of considerable importance. The transduction of
signaling events in the TNFR1 pathway from ligand binding
through transcriptional regulation is regulated at nearly every
step by post-translational modifications, including
ubiquitination. In this review both endogenous and
pharmacologic inhibitors of TNFR1 signaling, and how these
impact the ubiquitin system, will be discussed.
Addresses
Departments of Molecular Oncology and Early Discovery Biochemistry,
Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080,
United States
Corresponding author: Wertz, Ingrid E (wertz.ingrid@gene.com)

Current Opinion in Chemical Biology 2014, 23:7177

This review comes from a themed issue on Molecular immunology
Edited by Marcus Groettrup and Huib Ovaa
For a complete overview see the Issue and the Editorial
Available online 12th November 2014
http://dx.doi.org/10.1016/j.cbpa.2014.10.011
1367-5931/# Published by Elsevier Ltd.

Introduction
Tumor Necrosis Factor (TNF) was first described in
1975 as an endotoxin-induced factor that caused tumor
necrosis in a sarcoma mouse model [1]. It is now known that
TNF activates inflammatory responses, induces apoptosis
and necroptosis, regulates cellular proliferation, and may
even promote cancer progression. The effects of TNF are
transduced by TNF Receptor-1 (TNFR1) and TNFR2.
Studies with TNFR1-deficient and TNFR2-deficient
mice indicate that TNFR1 mediates most of the proliferation, pro-inflammatory, and cell death-activating pathways. TNFR2 signaling also impacts tissue repair and
angiogenesis and, as TNFR2 signaling is less well characterized [2,3]; this review will thus focus on TNFR1induced signaling. TNFR1 signal transduction is complex
and promotes diverse outcomes, many of which are
mediated by the transcription factor NFkB that will be
the subject of this review. Given the variable and essential
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consequences of TNFR1-mediated signaling, it is important to understand the regulation of this pathway. The
ubiquitin/proteasome system has recently emerged as a
critical regulatory mechanism of intracellular signal transduction. Avid research in the past 1520 years has identified a number of proteins in the TNFR1 transduction
cascade that are modified by ubiquitination. This review
will highlight both endogenous and pharmaceutical inhibitors of TNFR1-activated NFkB signaling, many of which
impact the ubiquitin/proteasome system.

The ubiquitin/proteasome system

Ubiquitination describes the covalent modification of
substrate proteins with the 76-amino acid protein ubiquitin and encodes signals that dictate specific outcomes for
substrate proteins. Ubiquitin modifications are achieved
by the coordinated activity of three classes of enzymes:
ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s). E1s activate ubiquitin in an ATP and Mg2+-dependent manner;
charged E1s are then competent to interact with E2s [4].
E2s subsequently interact with E3s that recruit protein
substrates for ubiquitin attachment. The E3 is important
not only because it covalently ligates ubiquitin to lysine
residues on the substrate protein but also because it
mediates substrate specificity, sometimes in collaboration
with E2s [5]. E2 and E3 enzymes coordinate to modify
substrates by addition of mono-ubiquitin or poly-ubiquitin chains by linking the C-terminal glycine of one
ubiquitin to the e-amino group of a substrate lysine, or
to one of the seven lysine (K) residues of another ubiquitin, or alternately, to a free amino-terminus of either
substrate or ubiquitin proteins [3]. E3s contain RING,
HECT, or IBR domains to achieve their ligase function
[6]. Substrate protein ubiquitination may be reversed by
de-ubiquitinating enzymes (DUBs) that are divided into
five classes based on domain structure: USP, OTU,
JAMM, UCH, and Josephin. Ligation of mono-ubiquitin
or poly-ubiquitin to recipient proteins has diverse biological consequences for the substrate. More specifically,
K48-linked and K11-linked polyubiquitination often targets substrates for degradation whereas K63 and linear
ubiquitin chains generally have scaffolding roles [7]. The
ubiquitin system regulates NFkB activity at numerous
signaling nodes via both scaffolding-promoting and
degradation-promoting functions [8].

TNFR1-activated NFkB signaling

TNFR1 may be activated by TNF, LTa3, or LTa2b1
ligands [9]. Figure 1 summarizes TNF-activated TNFR1
Current Opinion in Chemical Biology 2014, 23:7177

72 Molecular immunology

Figure 1

TNF
antagonists

TNF
TNFR1

A20 ZnF4 Ud Ud Ud Ud RIPK1 Ub TRADD

Ub
TRAF2/5
Ub
A20 OTU
OTULIN

Ub Ub

Ub
Ub Ub Ub

Ub

cIAP1/2

Ub

TAB1 CYLD Ub
Ub
TAB2/3
TAK1
NEMO

Ub
Ub
Ub

IAP
antagonists

Ub

IKK IKK
P

LUBAC
proteasome
inhibitors

MKK3
MKK4
Ub Ub Ub

Ub

IB P

p50 p65

JNK
p38

proteasome
IB

NFB

c-jun
ATF2
Ubiquitin chain key
Ub

K48-linked

Ub

K63-linked

Ub

linear

transcription

cell survival
proliferation
inflammation

Current Opinion in Chemical Biology

TNFR1 signal transduction to NFkB activation. The liganded receptor is shown with the proximal signaling complex assembled. Ubiquitin
modifications are also shown, as are phosphorylation events. Ubiquitin-mediated assembly of signaling complexes via K63-linked and linear
chains promotes sequential kinase activation that ultimately results in transcription factor activation. Endogenous inhibitors are indicated in red
whereas pharmaceutical inhibitors are indicated in blue. Not shown: the LUBAC ubiquitin ligase complex promotes linear polyubiquitination of
TNFR1 signaling components including NEMO and RIPK1.

NFkB signaling. Upon ligand binding, TNFR1 forms a

trimer, thereby promoting recruitment of the adaptor
protein TRADD and the RIP1 kinase (RIPK1) [10,
11]. TRADD recruits TRAF2, TRAF5, and the ubiquitin ligases cIAP1 and cIAP2. The cIAP proteins
promote K11-linked and K63-linked ubiquitination of
signaling proteins including RIPK1, cIAP1/2 (autoubiquitination), and possibly other proteins within the TNFR1
signaling complex [12]. The K63 ubiquitination of cIAP1/
Current Opinion in Chemical Biology 2014, 23:7177

2 subsequently recruits the LUBAC complex that

promotes linear polyubiquitination of signaling proteins
including RIPK1 and NEMO [13]. K63 ubiquitin chains
on RIPK1 promote recruitment of the TAK1/TAB2/3
complex [1416], whereas linear ubiquitin chains on
RIPK1 promote IkK kinase complex recruitment via
NEMO [17,18]. Kinase complex recruitment promotes
their subsequent activation and propagation of downstream JNK, p38, and NFkB signaling pathways. Free
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Inhibiting TNFR1-activated NF-kB signaling Wertz 73

Table 1
A summary of endogenous inhibitors of TNFR1-activated NFkB signaling
Cellular targets

Endogenous
inhibitor

Mechanisms of action

Knockout phenotypea
of protein inhibitor

Human diseases attributed

to aberrant expression
or mutations

CYLD

RIPK1, TAK1, TRAF2,

TRAF6, free K63-linked
polyubiquitin chains

Disassembles all types of

polyubiquitin chains and
unachored polyubiquitin chains

Enhanced inflammation
and tumor formation

Skin tumors (cylindromatosis),

other tumors including colon
and liver

A20

RIPK1, RIPK2, NEMO,

TRAF6, UBCH5, UBCH13

OTU domain: disassembles

K48-linked chains in vitro,
K63-linked chains in cellular
assays
ZnF4 motif: binds K63-linked
chains and facilitates
polyubiquitination
ZnF7 motif: binds linear chains

Perinatal lethality due

to uncontrolled
inflammation in multiple
organ systems

 Autoimmune and inflammatory

syndromes including rheumatoid
arthritis, psoriasis, celiac disease,
Crohns disease, systemic sclerosis,
type 1 diabetes
 Lymphomas and other hematological
malignancies

OTULIN

HOIP, Disheveled-2

Not yet described

Linear polyubiquitin-specific DUB Embryonic lethal with
defects in angiogenesis,
neural development, Wnt,
and NFkB signaling

IkB proteins Bind to NFkB heterodimeric Sequester NFkB heterodimers

transcription factors
in the cytosol to inhibit
transcription
a

Enhanced inflammation
Hypermorphic mutants have
due to constitutive NFkB impaired responses to infectious
activity
diseases

Knockout studies are performed in mice unless otherwise noted.

K63-linked polyubiquitin chains may also directly activate

TAK1 and the IkK kinases [19]. IkKb phosphorylates
Inhibitor of kB (IkB) proteins that creates a recognition
motif for the SCFbTrCP ubiquitin ligase complex [20].
Subsequent IkB ubiquitination and proteasomal degradation releases the NFkB heterodimeric transcription factor from inhibition, thereby promoting nuclear translocation
and transcription of NFkB target genes that promote cell
survival, proliferation, and inflammation [21]. Non-canonical NFkB signaling is regulated by other receptors and
signaling proteins and will not be discussed in this review.

Inhibitors of TNFR1-activated NFkB signaling

Given the importance of TNFR1 signaling in maintaining normal homeostasis, it is no surprise that Nature has
engineered multiple checks and balances at numerous
steps in the TNFR1 pathway in order to fine-tune signaling output. The pharmaceutical industry has also
recognized the ample therapeutic opportunities that
abound in designing chemical modulators of this pathway. Both naturally occurring inhibitors and pharmaceutical modulators of TNFR1 signaling will be highlighted
in this review, and are summarized in Tables 1 and 2.

Table 2
A summary of pharmaceutical inhibitors of TNFR1-activated NFkB signaling
Chemical inhibitor

Knockout phenotypea
of inhibitor target

Cellular targets

Mechanisms of action

TNF antagonists

TNF and, in the case

of Remicade,
lymphotoxin family
members

Ligand binding precludes

activation of cognate
receptors

Depressed recovery
from infectious challenges,
decreased granuloma
formation, enhanced LPS
toxicity of D-gal-treated mice

Autoimmune syndromes
including rheumatoid arthritis,
inflammatory bowel disease,
psoriasis

IAP antagonists

IAP proteins

 Promote cIAP
autoubiquitination and
degradation
 Block XIAP caspase
binding to enhance
cell death

 Defective in NF-kB and TNF

cell survival responses
 Embryonic lethality of double
IAP null animals

Numerous malignancies,
chemotherapy resistance,
X-linked lymphoproliferative
syndrome type-2

Proteasome
inhibitors

Primarily PSMB5
(chymotrypsin-like
activity)

Reversible and
irreversible enzyme
inhibition

Yeast PSMB5 homolog Pre2

knockout is lethal

Multiple myeloma and other

hematological malignancies

Common diseases targeted

by inhibitors

Knockout studies are performed in mice unless otherwise noted.

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Current Opinion in Chemical Biology 2014, 23:7177

74 Molecular immunology

Naturally occurring inhibitors

Numerous proteins that attenuate TNFR1 NFkB activity

by a variety of mechanisms have been reported. This
review will focus on those endogenous inhibitors that
have been validated by genetic ablation studies.
CYLD

CYLD was first identified as a mutated gene in familial

cylindromatosis [22], a condition that predisposes
patients to the development of skin appendage tumors.
CYLD also has a high mutation incidence in multiple
myeloma and in other types of cancers. These data,
combined with the findings that reduced CYLD expression contribute to tumorigenesis and cause increased
sensitivity to chemically induced skin tumors in mice,
implicate CYLD as a tumor suppressor gene [22,23].
The carboxy-terminal USP catalytic domain of CYLD is
critical for its tumor suppressor function and is reported to
cleave both K63 and linear polyubiquitin chains, though it
is less effective in cleaving K48-linked ubiquitin chains
[24]. CYLD substrates regulate NFkB signaling pathways
and others have roles in cell cycle regulation and in
calcium signaling [25,26]. For example, the CYLD substrate Bcl3 is an IkBa homolog that functions as a transcriptional coactivator for NFkB DNA-binding subunits
p50 and 52 [27]. CYLD removes K63-linked ubiquitin
chains from Bcl3 and thereby limits Bcl3 proliferative
activity [25]. RIPK1 is another CYLD substrate [28]. In
addition to promoting NFkB signaling RIPK1 also prevents the formation of a TNFR1-activated apoptotic
signaling complex that includes caspase-8 and FADD.
Thus, by deubiquitinating RIPK1, CYLD is reported to
block NFkB activation and also stimulates caspase-8
dependent apoptosis [29]. Nonubiquitinated RIPK1 is
also reported to associate with RIPK3 to promote necroptosis when caspase activation is blocked [30,31]. Thus,
by deubiquitinating RIPK1, CYLD can shift TNFR1
signaling from NFkB activation to promoting caspasedependent and caspase-independent cell death pathways.
A20

A20 was initially characterized as a primary response gene of

inflammatory cytokines, including TNF [32] and is a key
negative regulator of inflammatory responses [33]. A20 null
mice succumb perinatally to systemic inflammation and
multi-organ failure as a result of unchecked NFkB activity
[34]. A20 is an OTU domain-containing DUB and also
harbors seven C-terminal zinc finger (ZnF) motifs. The A20
protein attenuates TNFR1-mediated NFkB signaling via
multiple mechanisms related to ubiquitin signaling. One
mechanism, known as ubiquitin editing, can be broadly
conceptualized as the removal of modifications that
promote signaling complex assembly and activation, such
as K63-linked polyubiquitination, followed by the addition
of modifications that promote substrate degradation, such as
K11 or K48 polyubiquitination [35]. The net result of
ubiquitin editing by A20 is attenuation of signaling. More
Current Opinion in Chemical Biology 2014, 23:7177

specifically, A20 is reported to attenuate TNF-activated

NFkB signaling by removing K63 chains from RIPK1 via
the OTU DUB domain and promotes the addition of
degradative polyubiquitin chains on RIPK1 via the ZnF4
motif [3638]. A20 is also reported to attenuate NFkB
activity by facilitating the degradation of E2 enzymes that
promote signaling downstream of TNFR1 and other proinflammatory receptors [39]. However, conflicting reports
regarding the contributions of the enzymatic functions of
the OTU domain and the ZnF motifs in regulating NFkB
signaling indicate that more in-depth studies are warranted
[40,41]. Finally, A20 has also been shown to antagonize
NFkB activity via non-catalytic mechanisms whereby
ZnF7 mediates recruitment to linear poly-ubiquitin chains
in the activated TNFR1 signaling complex. A20 recruitment in turn inhibits interactions between key signaling
proteins such as LUBAC and NEMO [42,43] and obstructs
signal propagation, including IKK activation by TAK1 [44].
OTULIN

OTULIN is an OTU domain-containing DUB that attenuates TNF-activated NFkB signaling, similar to A20 and
CYLD, yet is unique in its selectivity for linear ubiquitin
chains [45,46]. OTULIN functionally reverses
LUBAC-mediated linear ubiquitination of TNFR1 signaling complex proteins to attenuate NFkB activity [46]
and has also been implicated in angiogenesis, WNT signaling and in Toll-like receptor and NOD2-mediated
immunity [45,47]. OTULIN is recruited to TNFR1
via HOIP within the LUBAC complex, and phosphorylation of OTULIN or mutation of critical HOIP-interacting
residues blocks association with HOIP, thereby resulting
in enhanced TNF-activated NFkB activity [48,49].
IkB proteins

In resting cells, NFkB heterodimers are retained in the

cytosol in an inactive state by IkB proteins that include
IkBa, IkBb, IkBe, and Bcl3. TNF-mediated activation of
the NFkB signaling cascade activates the IkK complex
that subsequently phosphorylates IkB. Only phosphoIkB is recruited to the SCFbTrCP E3 ligase, thereby
promoting IkB K48-linked polyubiquitination and subsequent degradation [20]. The NFkB heterodimer is then
released to enter the nucleus and promote transcription of
pro-inflammatory, anti-apoptotic, and proliferation-inducing genes [50]. In this way, the ubiquitin system couples
cytokine stimulation with transcriptional activation. IkB
protein knockout studies have confirmed their roles in
attenuating NFkB signaling [20]. Furthermore, an IkB
super-repressor, that cannot be phosphorylated by the
IkK complex, blocks constitutively active NFkB activity
in lymphoma cells and promotes cell death [51].
Chemical compounds

Chemical modulators that target TNFR1 signaling and

that are FDA-approved, or are in clinical trials, will be
highlighted in this review.
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Inhibiting TNFR1-activated NF-kB signaling Wertz 75

TNF antagonists

Remicade (Infliximab) is a chimeric fusion protein that

binds TNF and LT family members whereas Humeria
(Adalimumab) Etanercept (Enbrel), Simponi (Golimumab), and Cimzia (Certolizumab pegol) are monoclonal
antibodies that bind TNF. These agents thereby
antagonize TNFR1-mediated, TNFR2-mediated and,
in the case of Remicade, LTbR-mediated signaling [9].
As such, TNF antagonists antagonize ligand binding and
activation of TNFR1 downstream signaling, including
NFkB activation. TNF antagonists have shown benefit
in autoimmune diseases and inflammatory syndromes
such as rheumatoid arthritis, inflammatory bowel disease,
and psoriasis. Given the potential benefits that may be
achieved with selective TNFR1 antagonism, strategies to
engineer such agents are also being explored [9,52].
IAP antagonists

The IAP proteins are key regulators of cell viability that

regulate multiple signaling pathways, including TNFR1
[12]. IAPs antagonize pro-apoptotic proteins including
caspases and second mitochondrial-derived activator of
caspases (SMAC). The IAP1 and cIAP2 proteins are
RING domain E3s. Expression of IAP proteins is elevated in numerous tumor types and promotes inappropriate cell survival. Thus, a number of pharmaceutical
programs are aimed at developing IAP antagonists [53].
Cellular studies confirmed that IAP antagonists prevent
association of IAPs with caspases and SMAC to promote
cell death and structural studies have revealed the mechanism by which IAP antagonists promote cIAP1 degradation [54,55]. The antagonists promote a conformational
change that results in RING dimerization, autoubiquitination, and rapid degradation [56,57]. Thus IAP antagonists
transiently activate cIAP ligase activity and a burst of
NFkB signaling, but the end result is cIAP protein degradation. Given their remarkable potency and selectivity,
IAP antagonists have been used to explore the roles of
cIAP1 and cIAP2 regulating NFkB signaling. Such studies
reveal that the cIAP proteins are key ubiquitin ligases for
RIPK1 and TRAF2 in the TNFR1 signaling pathway [12].
Importantly, the therapeutic efficacy of IAP antagonists is
being evaluated in clinical trials [53].

yeast Pre2, the PSMB5 ortholog [59]. Given the ubiquitous function of the proteasome, it is no surprise that
proteasome inhibitors are reported to promote cell death
via a multitude of molecular mechanisms. These include
stabilization of pro-apoptotic Bcl-2 family members, inhibition of canonical and noncanonical NFkB pathways,
disruption of interactions between tumor and stromal
cells, accumulation of misfolded proteins and proteasome
substrates such as b-catenin, and induction of cell cycle
arrest due to accumulation of cyclin B1 and p21 cell
cycle regulators [6062]. More specifically, Bortezomib
inhibits NFkB pro-survival function by blocking TNFinduced IkBa degradation and thereby promotes cell
death [63]. On the other hand, in chronic lymphocytic
leukemia cells, Carfilzomib has been shown to promote
atypical NFkB activation as indicated by phosphorylation
and loss of IkBa and increased DNA binding of the
NFkB heterodimer, yet without subsequent increases
in canonical NFkB target gene transcription [64].

Future directions
Here we have reviewed some of the endogenous proteins
and pharmaceutical compounds that regulate TNFR1activated NFkB signaling, with a focus on ubiquitinmediated regulation. While the complexity of this signaling pathway poses challenges to the facile and complete understanding of the molecular regulatory
mechanisms, it also presents numerous opportunities
for pharmacological intervention. The pharmaceutical
industry is avidly exploring such opportunities and, given
the clinical success in these endeavors combined with the
multi-faceted roles of the targeted proteins in various
pathological conditions, it makes sense to not only
identify novel targets for pharmacological intervention
but also to repurpose the utility of selective compounds in
alternate disease indications [65].

Acknowledgement
Thank you to Allison Bruce for graphical assistance.

References and recommended reading

Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest

Proteasome inhibitors

Bortezomib (Velcade) was the first FDA-approved proteasome inhibitor and has shown efficacy for the treatment of multiple myeloma and mantle cell lymphoma
[58]. Bortezomib is a slowly reversible peptide boronate
inhibitor that primarily targets proteasomal chymotrypsin-like activity, that is afforded by the PSMB5 subunit.
Carfilzomib (Kyprolis) is a second-generation epoxyketone proteasome inhibitor that is a highly selective,
irreversible inhibitor of chymotrypsin-like activity. The
importance of proteasomal chymotrypsin-like activity is
confirmed by the lethal phenotype of knockout strains of
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