Accepted Manuscript Title: IAPs, regulators of innate immunity and inflammation Author: Yann Estornes Mathieu J.M. Bertrand PII: DOI: Reference:

S1084-9521(14)00073-1 http://dx.doi.org/doi:10.1016/j.semcdb.2014.03.035 YSCDB 1555

To appear in:

Seminars in Cell & Developmental Biology

Received date: Revised date: Accepted date:

14-11-2013 6-2-2014 28-3-2014

Please cite this article as: Estornes Y, Bertrand MJM, IAPs, regulators of innate immunity and inflammation, Seminars in Cell and Developmental Biology (2014), http://dx.doi.org/10.1016/j.semcdb.2014.03.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The role of IAPs extent apoptosis inhibition XIAP, cIAP1 and cIAP2 regulate innate immune responses downstream of various PRRs and TNFRs IAPs regulate inflammatory responses through their E3 ubiquitin ligase activities IAPs opposite roles in regulating the canonical and the non-canonical NFkB pathways IAPs are inhibitors of necroptosis, or programmed necrosis

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IAPs, regulators of innate immunity and inflammation

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Yann Estornes1,2 and Mathieu JM Bertrand1,2,#

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Inflammation Research Center, VIB, Technologiepark 927, Zwijnaarde-Ghent, 9052, Belgium. Department of Biomedical Molecular Biology,

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Ghent University, Technologiepark 927, Zwijnaarde-Ghent, 9052, Belgium.

To whom correspondence should be addressed: Inflammation Research Center, VIB-Ghent University, Technologiepark 927, Zwijnaarde-Ghent, 9052, Belgium. Tel: +32 09 33 13720 Fax: +32 09 33 13609. Email: [email protected]

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Abstract

As indicated by their name, members of the Inhibitor of APoptosis (IAP) family were first believed to be functionally restricted to

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apoptosis inhibition. It is now clear that IAPs have a much wider spectrum of action, and recent studies even suggest that some of its members

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primarily regulate inflammatory responses. Inflammation, the first response of the immune system to infection or tissue injury, is highly regulated by ubiquitylation -a posttranslational modification of proteins with various consequences. In this review, we focus on the recently

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reported functions of XIAP, cIAP1 and cIAP2 as ubiquitin ligases regulating innate immunity and inflammation.

Keywords: TNF, RIG-I, TLR, NOD, IAP

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1. From direct caspase inhibitors to key regulators of signal transduction

The first member of the Inhibitor of APoptosis (IAP) family was identified 20 years ago as a baculovirus protein contributing to

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efficient viral replication by sustaining survival of the infected insect host cell [1]. Since then, IAP orthologs have been identified in various

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organisms, and the human genome was shown to encode eight of them (BIRC1/NAIP, BIRC2/cIAP1, BIRC3/cIAP2, BIRC4/XIAP, BIRC5/Survivin, BIRC6/BRUCE, BIRC7/ML-IAP and BIRC8/ILP2) [2, 3]. The defining feature of IAPs is the presence of at least one

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baculovirus IAP repeat (BIR) domain, a protein-protein interaction motif that is required for the anti-apoptotic potential of some IAP family

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members [4-6]. Another important characteristic of certain IAPs, such as mammalian XIAP, cIAP1 and cIAP2 (cIAP1/2), is the presence of a C-terminal RING domain conferring upon them ubiquitin (Ub)-ligase activity. Protein ubiquitylation, the covalent attachment of Ub -a 76-amino acid polypeptide -to a target protein, has crucial roles in the

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regulation of many physiological processes, such as protein degradation, signal transduction, or even protein trafficking. It is a dynamic process that is catalyzed by the concerted action of a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2) and a Ub-ligase (E3), and which is negatively regulated by de-ubiquitylases (DUBs) [7] (Figure 1). The wide range of consequences of protein ubiquitylation originates from the fact that this process can result in the conjugation of either one Ub molecule (mono-ubiquitylation) or various Ub chains (poly-ubiquitylation) to the substrate. Ubiquitin polymers are generated out of eight possible Ub-Ub linkages. Importantly, each Ub linkage has a different topology, which allows specificity in their recognition by the different Ub binding domain (UBD)-containing proteins that function as Ub receptors [8].

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Consequently, the type of Ub-linkage will determine the consequence of ubiquitylation. It is for example well established that K48-linkages are

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implicated in proteasomal degradation while K63-and linear-linkages serve as docking sites promoting signal transduction. As indicated by their name, IAPs were first believed to be functionally restricted to inhibition of apoptosis, mostly by direct

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interference, via their BIR domains, with the proteolytic activity of caspases [9]. Several later studies however demonstrated that not all IAPs

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protect cells from apoptotic stimuli, and that amongst the mammalian IAPs, XIAP is probably the only family member capable of direct caspase inhibition [10, 11]. Other pro-survival IAPs, such as cIAP1 and cIAP2, bind to the effector caspase-7 and -3, but are inefficient in

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physically interfering with their proteolytic activities. Instead, these IAPs were suggested to neutralize caspase-7 and 3 by conjugating them

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with K48-Ub chains that promote their proteasomal degradation [12]. Of note, the pro-survival function of cIAP1/2 is not limited to caspase regulation but also involves their ability to activate, in an E3-dependent manner, the canonical Nuclear Factor-κB (NF-κB) pathway, which drives expression of various pro-survival molecules [13-16]. In addition, cIAP1/2 protect cells from death by regulating Receptor Interacting

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Protein Kinase (RIPK)-1 and -3 activities [17, 18]

It is now clear that IAPs have a much broader spectrum of action than promoting cell survival by caspase regulation [4, 19]. Recent findings may even suggest that the primary function of some IAPs consist in the regulation of inflammatory and innate immune signaling pathways, a function attributed to their E3 Ub-ligase activities [4, 20-28]. In this review, we focus on the recently reported roles of XIAP, cIAP1 and cIAP2 as Ub-ligases regulating innate immunity and inflammation.

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2. IAPs are major regulators of inflammatory responses

Inflammation, the first response of the immune system to infection or tissue injury, is initiated by the sensing of “stress” signals by

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Pathogen-Associated

Molecular

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(PAMPs),

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intracellular

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components,

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members of the innate immune Pattern-Recognition Receptors (PRRs) superfamily. These receptors detect the presence of conserved microbial

Danger/Damage-Associated Molecular Patterns (DAMPs), that are released by dying cells [29]. Upon binding of their respective agonists,

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many PRRs activate the Mitogen-Activated Protein Kinase (MAPK) and NF-κB signaling pathways, which collectively lead to the

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transcriptional upregulation of genes encoding various mediators of inflammation (such as pro-inflammatory cytokines, chemokines, adhesion molecules, acute phase proteins and antimicrobial peptides). Some of these PRRs can also activate transcription factors of the Interferon Regulatory Factor (IRF) family, which are responsible for the type I InterFeroN (IFN)-dependent antiviral responses. Other PRRs do not

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activate transcription factors but instead lead to the assembly of macromolecular platforms, called inflammasomes, which regulate the proteolytic maturation of the pro-inflammatory cytokines InterLeukin-1 (IL-1) and IL-18 [30]. At the site of infection, pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF) regulate the inflammatory response by inducing further production of cytokines and chemokines, and by regulating the eventual death of the inflammatory cells [31].

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Ubiquitylation has emerged as a crucial molecular mechanism regulating various levels of the inflammatory responses [32], and recent

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cascades activated by pro-inflammatory cytokines, such as TNF.

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studies identified cIAP1, cIAP2 and XIAP as E3s promoting ubiquitylation both downstream of various PRRs but also in the signaling

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2.1 Regulation of PRRs signaling by IAP proteins

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2.1.1. NOD1/2 signaling

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NOD1 and NOD2 are mammalian cytosolic PRRs of the Nod-Like Receptor (NLR) family that provide immune defenses against intracellular bacterial infection by reacting to the bacterial cell wall PeptidoGlycaN (PGN) [33, 34] (Figure 2A). NOD1 senses the PGN constituent DiAminoPimelic acid (DAP), while NOD2 detects the PGN derivative MuramylDiPeptide (MDP). Upon activation, both receptors

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oligomerize and recruit the adaptor and effector protein RIPK2 via homotypic Caspase Activating and Recruiting Domain (CARD) interactions [33, 34]. The conjugation of Ub chains to RIPK2 then allows downstream activation of the MAPKs and of the canonical NF-κB pathways that collectively transduce signals leading to the expression of pro-inflammatory cytokines, chemokines and antimicrobial peptides [35-37]. The UBDs of TAB2/3 and of NEMO are believed to permit recruitment and close proximity between the TAB2/3-TAK1 complex and of the IκB kinase (IKK) complex (composed of IKK, IKK and NEMO) on RIPK2 Ub chains [38-40]. This allows TAK1 to activate downstream MAPKs (such as JNK, p38 and ERK) and IKK by phosphorylation. Once activated, IKKκphosphorylates IκB, which is a signal for its K48-ubiquitylation and proteasomal degradation. At steady state level, IκB sequesters the p50/RelA NF-κB dimers in the cytosol. Its 7 oftarget 39 signal-dependent degradation therefore enables the nuclear translocation of the NF-κB dimers and the subsequent expression ofPage NF-κB

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genes (canonical NF-κB pathway) [41].

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Recent studies have demonstrated non-redundant functions of XIAP, cIAP1 and cIAP2 as positive regulators of the NOD1/2-dependent

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immune responses [2023]. Primary cultures of XIAP-, cIAP1- or cIAP2-deficient macrophages, or human colonocytes depleted of each IAP by siRNA, were shown to be defective for MAPKs and NF-κB activation, as well as subsequent cytokine secretion, following NOD1 or NOD2

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stimulation [21-23]. This defect in NOD signaling was also observed in vivo when cIAP1-, cIAP2-, or XIAP-null mice were challenged with

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NOD1/2 agonists [21, 22], or during infection of XIAP-null mice with Listeria monocytogenes [20]. At the molecular level, all these IAPs were shown to positively regulate NOD1/2 signaling by promoting RIPK2 ubiquitylation [21, 22]

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(Figure 2A). Upon receptor activation, XIAP and cIAP1/2 are recruited to the receptor-signaling complex [21-23]. These IAPs directly bind to

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RIPK2 via a region that overlaps with the Second Mitochondria-derived Activator of Caspase (SMAC)-binding portion of their BIR2/3 domains [4, 23, 42, 43]. Importantly, while cIAP1 and cIAP2 conjugate RIPK2 with K48-and K63-linked Ubs [21], XIAP promotes RIPK2 ubiquitylation via attachment of Ub moieties linked through lysine residues other than K63 and K48 [22, 43]. In addition, the enzymatic

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activity of XIAP, but not that of cIAP1/2, is required for the recruitment of LUBAC, a tri-partite E3 complex generating linear Ub chains, to the NOD signaling complex [22]. LUBAC promotes linear ubiquitylation of NEMO, an important step in NF-κB activation [44-47]. Consistently, both K63-and linear-Ub linkages were shown to be important for the recruitment and/or activation of the TAB2/3-TAK1 and IKK complexes [22, 35, 38-40, 45, 48]. Together, these results provide potential molecular explanations for the non-redundant functions of XIAP, cIAP1 and cIAP2 in the regulation of NOD1/2-dependent immune responses.

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In humans, the BIR2 domain of XIAP has been identified as a hotspot for missense mutations causing X-linked LymphoProliferative

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syndrome type 2 (XLP2), an immune disease with potential lethal consequences. Importantly, these mutations were shown to affect

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NOD2-dependent signaling by impairing the XIAP-RIPK2 interaction [43].

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2.1.2 TLR signaling

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The Toll-like receptors (TLRs) were the first identified PRRs and represent the most largely described family of PRRs. Ten members

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have been identified in humans, which together play a central role in the initiation of innate immune responses by detecting a wide range of PAMPs and DAMPs [49]. TLRs are type I trans-membrane proteins that can be divided in two sub-groups depending on their cellular localization and ligand specificity. The first group comprises receptors (TLR1, -2, -4, -5, -6 and -11) that localize at the cell surface and that

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mainly recognize constituents of microbial membranes, such as proteins, lipoproteins, and lipids. The second group is composed of receptors (TLR3, -7, -8 and -9) localized at the intracellular endosome/lysosome membranes that detect nucleic acids from microbe and host origins [49]. Depending on the receptor, signaling downstream of TLRs occurs either via recruitment of Myeloid Differentiation primary response protein 88 (MyD88) or Toll/IL-1 Receptor (TIR) domain-containing adaptor-inducing IFNβ (TRIF), with the exception of TLR4 that activates both MyD88-and TRIF-dependent responses [49, 50].

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TLR4, in complex with its co-receptor MD-2, recognizes bacterial lipopolysaccharide (LPS) -a constituent of the outer membrane of

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Gram-negative bacteria known to induce septic shock. Whereas the TLR4-induced MyD88-dependent response leads to the production of pro-inflammatory cytokines through activation of the NF-κB and MAPK pathways (Figure 2B), TLR4-induced TRIF-dependent signaling

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triggers production of type I IFNs via activation of IRFs [49, 50]. Recently, an in vitro study making use of cIAP1/2-depleted macrophages

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demonstrated a specific role of these IAPs in the regulation of MyD88-dependent MAPKs activation, and not of NF-κB or IRFs, by controlling the stability of a certain pool of TRAF3 [27] (Figure 2B). Upon TLR4 engagement, cIAP1 and cIAP2, as well as TRAF3 and TRAF6, are

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recruited to the MyD88-signaling complex. Activation of TRAF6 in the complex leads to its K63-linked auto-ubiquitylation and to

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TRAF6-dependent K63ubiquitylation of cIAP1/2, a modification that presumably activates their E3 activities. The Ub chains conjugated to TRAF6 allow recruitment and activation of the IKK complex that further activates the canonical NF-κB pathway. Activation of the TAB2/3-TAK1 complex, which is also recruited to TRAF6 Ub chains, further requires translocation of the signaling complex to the cytosol, a

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process inhibited by TRAF3 but positively regulated by cIAP1/2. Upon activation, these IAPs induce K48 ubiquitylation-dependent proteasomal degradation of TRAF3, which releases the translocation inhibition and leads to TAK1 and downstream MAPKs activation [27]. Importantly, the study of Tseng and colleagues provide molecular insights that may explain earlier findings on the resistance of cIAP1- and cIAP2-deficient mice to lethal doses of LPS [51, 52]. Apart from TLR4 signaling, a role for cIAP1/2 in MyD88-dependent MAPKs activation was also demonstrated downstream of TLR2 (a sensor of lipoproteins) [27].

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In contrast, TLR3 (a sensor of viral double-stranded RNA)-induced TRIF-dependent immune responses were shown not to depend on cIAP1/2

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[27]. Nevertheless, several earlier studies showed that cIAP1/2 regulate at least some aspects of TLR3 signaling, since depletion of cIAP1/2 in epithelial cells sensitized them to TLR3-mediated death [53-56]. Under these circumstances, cIAP1/2 were shown to prevent association of

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RIPK1 – recruited by TRIF [57] – with caspase-8 (and its subsequent activation), probably by regulating RIPK1 ubiquitylation status [53, 54].

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progression of virus-associated pathologies.

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This protective function of cIAP1/2 in TLR3-mediated death may be of particular relevance in the control of virus replication, and in the

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2.1.3. RIG-I signaling

RIG-I, the founding member of the cytosolic RIG-Like Receptor (RLR) family, plays a key role in the induction of antiviral innate immune responses by triggering activation of the IRF and NF-κB transcription factors [58]. RIG-I detects short cytosolic blunt-ended

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5’-triphosphate dsRNA of at least 20bp in length from various viral origins [59]. Upon viral RNA recognition, RIG-I binds to IFNβ Promoter Stimulation 1 (IPS1; also called VISA, MAVS or CARDIF), an adaptor protein localized at the mitochondrial membrane. IPS1 allows further recruitment of TRAF3 and of TRAF2/6 to the signaling complex, which are respectively believed to activate the IRF and NF-κB pathways. On the one hand, the conjugation of TRAF3 with K63-linked Ubs provides a docking site for the recruitment and activation of the TBK1 (IKK-related kinases TANK-Binding Kinase 1) and IKKε kinases, which subsequently activate IRF3 and IRF7 by phosphorylation [60-63]. On the other hand,

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TRAF6, possibly via a Ub-dependent mechanism, recruits and activates the IKK complex that activates p50/RelA NF-κB dimers [64].

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In a study by Mao et al., the authors report that depletion of cIAP1 or cIAP2 by siRNA strongly decreases Sendai virus (SeV)-or transfected Poly(I:C)-induced type I IFNs as well as NF-κB activation [25]. They show that cIAP1 and cIAP2 interact with TRAF3, TRAF6

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and IPS1 following SeV infection, and suggest that the positive regulatory function of cIAP1/2 in the RIG-I pathway resides in their ability to

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conjugate TRAF3 and TRAF6 with K63-linked Ub chains [25]. The authors also indicate that cIAP1/2 depletion facilitates in vitro vesicular stomatitis virus (VSV) replication [25]. In another study, SeV infection was reported to induce TBK1/IKK-mediated phosphorylation of

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XIAP, resulting in its K48-ubiquitylation and subsequent proteasomal degradation [65]. In this case, the authors suggest that this mechanism

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would contribute to the anti-viral response by facilitating death of the infected cells, thereby limiting virus replication and spread. In marked contrast with these findings, three recent independent publications suggest no role for XIAP or cIAP1/2 in the intrinsic RIG-I antiviral responses against RNA viruses [66-68]. Indeed, in two of these studies, the authors show that SMAC mimetic treatment, which

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induces cIAP1/2 depletion and XIAP inhibition, does not impair IFN production in response to VSV infection [66, 67]. Rather, the Ub-ligases TRAF2/5 and 6 seemed to function redundantly to mediate IPS1-dependent IFN signaling [67]. In line with these results, SMAC mimetic treatment was shown not to alter the VSV infectivity and replication in vitro in cancer cells and in vivo in tumor-mouse models [66]. Accordingly, the study of Rodrigue-Gervais et al. showed that the antiviral immunity against Influenza A virus is normal in cIAP2-deficient mice, and the absence of a defect in RIG-I signaling in cIAP2-deficient BMDCs was further confirmed in vitro by measuring the production of IFNβ following SeV infection or 5’triphosphate dsRNA transfection [68]. Of note, cIAP2-deficient mice however exhibit increased susceptibility and mortality to influenza A virus infection due to enhanced death receptor-induced death of airway epithelial cells [68]. Similarly, SMAC mimetic treatment combined with oncolytic viral infection was shown to sensitize cancer cells to death receptor-induced Page 13 of 39

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death in several mouse models of cancer [66], highlighting the pro-survival role of IAPs downstream of death receptors under inflammatory

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conditions (see section 2.2.2.).

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Although it has been established that SeV, VSV and Influenza A virus infections can all be sensed by RIG-I [58], the absolute

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dependence on RIG-I for the induction of an antiviral response may vary between cell types, as previously reported [69]. It is therefore possible that the apparent contradictory findings on the role of IAPs in RIG-I signaling originate from the differential use in various cell types.

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Nevertheless, the recent in vivo studies seem to exclude any physiological role for IAPs in RIG-I signaling.

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2.1.4. The inflammasomes

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Contrary to the previously described PRRs, some members of the NLR family (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12 and NLRC4) as well as the cytosolic DNA sensor absent in melanoma 2 (AIM2) do not transduce signals leading to the transcriptional upregulation of pro-inflammatory mediators. Instead, these PRRs contribute to the inflammatory response by assembling into caspase-1-activating

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platforms, called inflammasomes, which regulate the proteolytic maturation of IL-1 and IL-18 [30, 34, 70]. In addition, inflammasomes initiate an inflammatory type of cell death, called pyroptosis. The maturation of IL-1 and IL-18 into their bioactive counterparts requires a two-step mechanism. The first step, referred to as “priming”, consists in the synthesis of the inactive precursors pro-IL-1β and pro-IL-18, and can be obtained by activation of various receptors (TLRs, NOD receptors, RLRs, TNFRs, or IL-1R). The second step involves the proteolytic removal of their pro-domain, which is, in the context of the inflammasome, accomplished by caspase-1-mediated cleavage [30, 34, 70].

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Recently, two independent groups have reported contrasting findings on the role of IAPs in caspase-1 activation and pro-IL-1β

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maturation [24, 28]. In the first study, Labbe et al. provided in vitro and in vivo evidence for non-redundant positive regulatory functions of cIAP1/2 in caspase-1 activation and pro-IL-1β processing. At the molecular level, the authors suggested a model in which cIAP1/2 facilitate

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caspase-1 activation by conjugating it with K63-linked Ubs, a modification that would favors its efficient activation by the NLRP3 and NLRC4

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inflammasomes [24]. Conversely, Vince et al. demonstrated a negative regulatory function of XIAP, cIAP1 and cIAP2 in pro-IL1β maturation [28]. In that second study, the authors showed that only the triple genetic deletion of XIAP, cIAP1, and cIAP2, or their simultaneous inhibition

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by the use of small molecule IAP antagonist (SMAC mimetics) [4], led to NLRP3 inflammasome activation, indicating at least partial

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redundancy in their inhibitory functions [28]. Of note, Vince et al. reported that pro-IL-1β processing triggered by IAPs depletion does not only rely on caspase-1 activation, but also on RIPK3-dependent ROS generation and caspase-8 activation. In line with these findings, a recent study indicated that the caspase-8 inhibitor cFLIP inhibits NLRP3 inflammasome activation and IL-1β generation induced by SMAC mimetic

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treatment [71]. Together, these data therefore support earlier studies implicating caspase-8 in pro-IL-1β processing [72, 73]. Intriguingly, dendritic cells deficient in caspase-8 were recently reported to be hyper-responsive to RIPK1/RIPK3-dependent NLRP3 inflammasome assembly [74], which adds another level of complexity in the regulation of pro-IL-1β processing by caspase-8. Finally, in a model of spinal cord injury-induced processing of pro-IL-1β, XIAP was shown to interact with ASC and NLRP1, but the role of this interaction in the maturation of pro-IL-1β was not evaluated [75]. Further studies are therefore greatly needed in order to reconcile or exclude some aspects of the opposite models proposed so far concerning the role of IAPs in the regulation of inflammasome activation, and in the production of bioactive IL-1β.

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2.2. Regulation of TNFR1-dependent outcomes by IAP proteins

At the site of infection, pro-inflammatory cytokines such as TNF regulate the inflammatory response by inducing further production of

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cytokines and chemokines and by regulating the eventual death of the inflammatory cells, as well as that of other cell types [31].

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2.2.1. Positive regulatory function of cIAP1/2 in TNFR1-mediated MAPKs and canonical NF-B activation

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TNF signals by binding and activating two cell surface receptors, TNFR1 and TNFR2, but most of its biological activities have been associated with TNFR1 signaling [76]. In most cell types, TNFR1 activation does not induce cell death but instead leads to the transcriptional upregulation of genes encoding pro-survival and pro-inflammatory molecules. Engagement of TNFR1 by TNF triggers the formation of a

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plasma membrane-bound signaling complex, referred to as TNFR1 complex I [77], that contains TRADD, TRAF2, RIPK1, cIAP1/2, and LUBAC [26, 78] (Figure 3). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains. These Ub chains act as scaffolds for the recruitment and activation of the TAB2/3-TAK1 complex and the IKK complex, which subsequently leads to the activation of the MAPKs and canonical NF-B signaling pathways that drive expression of pro-survival and pro-inflammatory molecules.

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Several studies demonstrated that cIAP1/2 positively regulate TNF-induced MAPKs and canonical NF-κB activation by promoting

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RIPK1 ubiquitylation [13, 14, 16] (Figure 3). The addition of K63-Ub chains to RIPK1 was reported to create a platform for the recruitment of the TAB-TAK1 and IKK-IKK-NEMO complexes, implying specificity of TABs and NEMO UBDs for K63 Ub linkages [79]. However,

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although cIAP1/2 were shown to directly conjugate RIPK1 with K63-Ub chains [13, 16], more recent studies indicated that K63-Ub linkages

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might not be essential for TNF-induced NF-B activation, and that NEMO possesses high affinity for other Ub chains, including K11-and linear chains [7, 39, 80, 81]. Consistently, mass spectrometry analysis revealed that RIPK1 is conjugated with K11-, K48-, K63-and linear Ub

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linkages in the TNFR complex I [44, 80], and that cIAP1 also mediates K11-and potentially linear-ubiquitylation of RIPK1 [42, 80, 82]. Taken

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together, these studies therefore indicate that cIAP1/2 positively regulate TNFR1-dependent signaling by conjugating RIPK1, and possibly other proteins, with Ub-linkages of various types. Interestingly, and in contrast with NOD1/2 signaling, recruitment of LUBAC to TNFR1

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complex I was shown to depend on cIAP1/2 ubiquitin ligase activities [45].

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2.2.2. Negative regulatory function of cIAP1/2 in TNFR1-mediated RIPK1dependent cell death

When the NF- B response is inhibited, the outcome of TNFR1 engagement switches from a pro-survival/pro-inflammatory response to

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a pro-apoptotic response. This switch occurs via a mechanism that involves internalization of complex I and assembly of a cytoplasmic caspase-8-inducing death complex, known as TNFR1 complex IIa, that contains TRADD, FADD and procaspase-8 [77, 83, 84] (Figure 3). As

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mentioned above, cIAP1/2 are required for TNF-induced canonical NF-κB activation [13, 14, 16, 85]. Consequently, their depletion also

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induces a switch to TNF-induced caspase-8-dependent apoptosis [13, 15, 84, 86-88]. However, in absence of cIAP1/2, TNF-induced apoptosis

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was shown to rely on RIPK1 kinase activity and not on TRADD, indicating that cIAP1/2 additionally regulate an NF-κB independent cell death checkpoint in the TNFR1 pathway [13, 84, 86, 89, 90]. To discriminate the RIPK1-dependent cytosolic death complex from complex IIa, it was defined as complex IIb [76, 83]. The molecular mechanism accounting for the differential assembly of complex IIa versus IIb is poorly

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understood, but suggested to rely on the differential ubiquitylation status of RIPK1 in complex I, allowing or not, inactivation of RIPK1 by TAK1 and/or NEMO recruitment [84, 90-92]. Of note, cIAP1/2 were also reported to negatively regulate activation of a TNFR1 complex IIb-like complex, called the Ripoptosome, which assembles independently of TNFR activation [54, 93]. Finally, in addition to apoptosis, TNF signaling also induces an inflammatory type of cell death, called programmed necrosis or necroptosis, which prevails in caspase-8 inhibited conditions [18]. TNF-mediated necroptosis relies on the assembly of another cytosolic death complex, known as the necrosome, which contains RIPK1 and RIPK3 [94] (Figure 3). RIPK1 triggers necroptosis through a phosphorylationdriven cascade involving RIPK3 as a partner and Mixed Lineage Kinase Like (MLKL) as a downstream cell death executioner [95, 96]. In vitro studies have shown that cIAP1/2 depletion sensitizes cells to TNF-induced necroptosis, presumably by affecting RIPK1 ubiquitylation status at Page 19 of 39

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the receptor complex [97, 98]. In accordance with this idea, de-ubiquitylation of RIPK1 by the de-ubiquitylase CYLD was shown to positively

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regulate the formation of the necrosome and the induction of necroptosis [99, 100]. In addition, cIAP1/2 may also protect cells from necroptosis by regulating RIPK3 expression at a post-translational level [101], possibly via K48-ubiquitylation [42]. Importantly, several recent

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in vivo studies have confirmed a physiological role for cIAP1/2 in promoting host survival during pathogenic infection by preventing

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necroptotic death of immune and non-immune cells triggered by inflammatory cytokines like TNF, FasL, TRAIL as well as IFN, which are

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produced upon sensing of the pathogens by the PRRs [68, 101-103].

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According to the high sensitivity of cIAP1/2-depleted immune cells to cytokine-induced necroptosis, it will therefore be of great

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importance to clearly establish the “infectious” state of the patient when using SMAC mimetics for the treatment of cancer. Nevertheless, the induction of cancer cell death by the combined use of SMAC mimetics with oncolytic viral infecting agents has very promising therapeutic

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potential [66].

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2.3. The role of cIAP1/2 in non-canonical NF-B activation

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The family of NF-B transcription factors plays a central role in orchestrating immune and inflammatory responses [104]. The

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activation of NF-B occurs by two distinct signaling pathways: the canonical and non-canonical pathways [41]. As described in the previous sections, the canonical pathway is essential for innate immunity and relies on the canonical IKK complex (composed of IKKα, IKK, and

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NEMO) for the degradation of IκBα and downstream activation and nuclear translocation of the p50/RelA NF-B dimers (Figure 2 and 3). In

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contrast, the non-canonical pathway is initiated by NIK activation, and is important for the development and maintenance of lymphoid organs and for adaptive immunity [41, 104]. In unstimulated cells, NIK is maintained at very low level through continuous proteasomal degradation,

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but activation of a subset of TNFR superfamily members, such as CD40, BAFF receptor (BAFFR), Fn14 (TWEAKR) and LTβR, induces its

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stabilization and activation (Figure 4). Upon activation, NIK phosphorylates and activates an IKKcomplex that in turn phosphorylates the NF-B2 precursor protein p100, leading to its processing into p52 and to the release and nuclear translocation of the p52/RelB NF-B dimers [104, 105].

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Whereas cIAP1/2 positively regulate activation of the canonical NF-B pathway downstream of the above-mentioned PRRs and TNFR1 (Figure 2 and 3), they fulfill a dual and opposite function in the regulation of the non-canonical pathway (Figure 4). Indeed, studies have shown that cIAP1/2 suppress constitutive activation of the non-canonical pathway, but are required for its receptor-induced activation [87, 106-111] (Figure 4). In a complex with TRAF2 and TRAF3, cIAP1/2 promote constitutive degradation of NIK by conjugating it with K48-Ub chains. However, upon receptor activation, the TRAF2/3-cIAP1/2 complex is recruited to the receptor, which induces cIAP1/2-mediated Ub-dependent proteasomal degradation of TRAF3 and/or lysosomal/proteasomal degradation of all complex components [108111]. In either case, the consequence is the stabilization and activation of NIK and of the non-canonical NF- B pathway.

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3. Conclusion and future perspectives

Although best known for their ability to suppress cell death through caspase inhibition, recent studies have highlighted the critical role

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of cIAP1, cIAP2 and XIAP as major regulators of inflammation. This newly revealed function was shown to entirely rely on their Ub-ligase

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activity, which allows them not only to regulate NFκB, MAPK or IRF pathways downstream of various PRRs and TNFRs but also to control inflammasome activation. Knowing the importance of inflammation in the development of many human diseases, these findings open exciting

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perspectives for the future treatment of these diseases by targeting IAPs. Nevertheless, caution should be taken as the regulatory function(s) of

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these IAPs is complex and still poorly understood. Indeed, they were shown to positively regulate activation of the MAPK, IRF and canonical NF-B pathways but to repress constitutive activation of the non

canonical NF-B pathway. In addition, their positive or negative regulatory role(s) in inflammasome assembly and/or activity is still a question of debate. Additional in vitro and in vivo studies are therefore greatly needed to better define the specific

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contribution of each IAP in the regulation of inflammatory responses, and to evaluate their potential as targets for the treatment of inflammatory diseases.

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Acknowledgements

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M.B. has a tenure track position within the Multidisciplinary Research Program of Ghent University (GROUP-ID). Research in his unit, headed by Prof. Vandenabeele, is supported by a Methusalem grant (BOF09/01M00709), European grants (Euregional PACT II), Belgian grants

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(Interuniversity Attraction Poles, IAP 7/32), Flemish grants (Research Foundation Flanders, FWO G.0875.11, FWO G.0973.11, FWO

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G.0A45.12N, FWO G.0172.12N, FWO G0787.13N), Ghent University grants (MRP, GROUP-ID consortium) and grants from VIB.

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Figure Legend

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Figure legends

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Fig. 1: The ubiquitylation system. Ubiquitylation involves the covalent attachment of ubiquitin (Ub) to a substrate protein by the coordinated action of a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2) and a Ub-ligase (E3). This process can lead to the conjugation of one Ub

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molecule (mono-ubiquitylation) or to the addition of various Ub chains (polyubiquitylation) to the substrate. Ubiquitin polymers are generated

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by isopeptide bonds between an internal lysyl residue of one Ub and the C-terminal glycyl residue of another. As Ub contains seven lysines (K6, K11, K27, K29, K33, K48 and K63), seven different Ub-Ub linkages are possible. An additional linkage, known as the linear linkage, can also be generated by attachment of the C-terminal glycine to the N-terminal methionyl residue. A simplistic view is to consider the existence of

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8 possible Ub chains, each generated with only one type of Ub-Ub linkage, but the reality may be more complex and involves the existence of Ub chains generated by different Ub-Ub linkages. The type of Ub linkage determines its consequences through the recruitment of specific Ub binding domain (UBD)-containing proteins. Deubiquitylating enzymes (DUB) negatively regulate ubiquitylation. PPi, inorganic diphosphate.

Fig. 2: Model of IAP-dependent regulation of the NOD1/2 pathway (A) and of the TLR4-MyD88 pathway (B). A) Following NOD1 or NOD2 stimulation, cIAP1, cIAP2, and XIAP are recruited to the receptor complex, where they all contribute to RIPK2 ubiquitylation. Whereas cIAP1/2 directly mediate K63-ubiquitylation of RIPK2, the type of Ub chains conjugated by XIAP remains undefined. RIPK2 ubiquitylation Page 32the of 39 allows recruitment of the TAB2/3-TAK1 and IKKα-IKKβ-NEMO (IKK) complexes. Activated TAK1 then activates by phosphorylation

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downstream MAPKs and IKKβ kinases. Activated IKKβ phosphorylates IκBα which triggers its K48-ubiquitylation and subsequent

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degradation by the proteasome, leading to the nuclear translocation of the p50/RelA NF-κB dimers that drive gene transcription (canonical NF-B pathway). The activation of the IKK complex is also facilitated by LUBAC, whose recruitment was shown to rely on XIAP E3 activity.

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B) TLR4 engagement by LPS results in the recruitment of MyD88 and in the formation of a receptor-associated signaling complex that

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contains TRAF6, cIAP1, cIAP2 and TRAF3. TRAF6 auto-ubiquitylation (K63-Ub chains) then allows recruitment of the TAB2/3-TAK1 and IKK complexes, and activation of the canonical NF-B pathway. Activation of TAK1 and of the downstream MAPKs further requires

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translocation of the signaling complex to the cytosol, a process inhibited by TRAF3, but positively regulated by cIAP1/2-dependent K48-

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ubiquitylation of TRAF3.

Page 33 of 39

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Fig. 3: Regulation of TNFR1 signaling by cIAP1 and cIAP2 Binding of TNF to TNFR1 triggers the formation of a receptor-associated

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signaling complex known as complex I. Within this complex, RIPK1 is conjugated with Ub chains (of various types) by cIAP1/2. These Ub chains act as scaffolds for the recruitment of the TAB2/3-TAK1 and the IKK complex, which subsequently lead to the activation of the

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MAPKs pathways and of the canonical NF-B signaling pathways that drive transcription of pro-inflammatory genes, and genes that prevent

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cell death. When the NF-B response is inhibited, by the use of the general translation inhibitor cycloheximide (CHX) or by ectopic expression of a non-degradable IB mutant, TNFR1 ligation switches from a pro-survival to a

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RIPK1-independent pro-apoptotic response by assembly of the cytoplasmic TNFR1 complex IIa. In contrast, when the NF-B response is

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inhibited by cIAP1/2, TAK1 or NEMO depletion, TNF stimulation induces RIPK1 kinase-dependent apoptosis by assembly of the cytosolic death complex IIb. However, when caspase-8 is inhibited, RIPK1 and RIPK3 associate to form the necrosome, which triggers MLKL-dependent necroptosis, or programmed necrosis.

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Fig. 4: Regulation of the non-canonical NF-κB pathway by cIAP1 and cIAP2 At the steady state level (A), TRAF3 constitutively binds NIK and mediates the recruitment of TRAF2, cIAP1 and cIAP2. cIAP1/2 directly promote K48ubiquitylation of NIK, which leads to its constitutive degradation by the proteasome and hence prevents the activation of the non-canonical NF-κB pathway. Upon receptor (for example CD40, Fn14 or LTβR) activation (B), the TRAF2/3-cIAP1/2 complex is recruited to the receptor. Then, two situations have been reported: either cIAP1/2 mediate TRAF3 degradation through its K48-ubiquitylation, or the complex components translocate to membrane fractions where they undergo proteasomal and/or lysosomal degradation. In both cases, this leads to NIK accumulation, which triggers IKKα-dependent p100 phosphorylation and subsequent ubiquitylation leading to the degradation of p100 into p52. The generated NF-κB RelB/p52 heterodimer then Page 34 of 39

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ep te

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translocates to the nucleus and activates the transcription of target genes.

Page 35 of 39

Figure 1 A E2

E1

E3

Ub

E1

Ub

E1

Substrate

E2

AMP+PPi

DUB

ed

Substrate

K48 UbUb Ub Ub

ce pt

Substrate

Ub

Substrate K63, K11, linear

Ub Ub Ub Ub

Substrate

Substrate

Ub Ub Ub Ub

Ub

K6, K11, K27

K29, K33

Mono-Ub

Ub

Ub

Ub

Ub

Ac

ATP

E3

M an

Ub

E2 +

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Substrate +

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i

Figure

• Proteosomal degradation

• Signal transduction • DNA repair • Endocytosis

• DNA repair • Proteosomal degradation • Lysosomal degradation

• Endocytosis • Endosomal sorting • DNA repair, virus budding, nuclear export

Page 36 of 39

B

Extracellular

DAP/MDP

cIAP1/2 RIPK2

XIAP Undefined Ub chain

NEMO Tab2/3 P

P

IKKβ

IKKα

LUBAC

TAK1

p50 P

RelA

IRAKs TRAF3

NEMO

IKKβ

cIAP1/2

IκBα

P AP1

K48 Ub chain

Tab2/3

IKKα

TRAF3 degradation TAK1

IRAKs TRAF6 P

p50

RelA

p50

RelA

Pro-inflammatory cytokines, chemokines, anti-microbial peptides

cIAP1/2

Tab2/3

K48-Ub chain

NF-κB

Myd88

Cytosolic complex

IκBα

P

Ac

MAPKs

P

IκBα

Proteasomal degradation

ce pt

P

TRAF6

K63-Ub chain

P

Myd88

TIRAP

ed

K63/undefined Ub chain

TLR4

Intracellular

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NOD1/NOD2

TRAF

Extracellular

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Intracellular

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A

XIAP

LPS

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Figure 2

TAK1

P MAPKs

p50

P RelA

AP1

Pro-inflammatory cytokines, chemokinesPage 37 of 39

Extracellular

TNFR1

Intracellular

Complex I

Complex IIb

cIAP1/2

RIPK1 FADD

CASP8

Various Ub chains

Necrosome

ed

Tab2/3

P

RIPK1 RIPK3

ce pt

FADD

TRADD

i

TRAF2/5

M an

TRAF2/5

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TNF

cr

Figure 3

RIPK1

P

Complex IIa

cIAP1/2 LUBAC NEMO IKKβ

Linear Ub chain

IKKα

TAK1 P

P MAPKs

CHX

IκBα

p50

RelA

Apoptosis

Necroptosis

Ac

Apoptosis MLKL

TRADD FADD CASP8

P AP1

p50

RelA Survival genes

Pro-inflammatory cytokines, chemokines

Page 38 of 39

i

Figure 4

cr

Ligand

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B

Extracellular Intracellular

TRAF3

Proteasomal/lysosomal degradation

TRAF2

M an

K48 Ub chain

cIAP1/2

TRAF3 degradation

NIK accumulation

IKKα

cIAP1/2

K48 Ub chain

NIK degradation

IKKα

ce pt

NIK

NIK

TRAF2

Ac

TRAF3

ed

A

RelB

P P

P

p100 K48 Ub chain

RelB p52

RelB p52 Target genes Page 39 of 39