Simultaneous substrate and ubiquitin modification recognition by bispecific antibodies enables detection of ubiquitinated RIP1 and RIP2.

Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.

Inhibition of RIP1 improves immune reconstitution and reduces GVHD mortality while preserving graft-versus-leukemia effects.

Graft-versus-host disease (GVHD) remains the major cause of morbidity and nonrelapse mortality (NRM) after hematopoietic cell transplantation (HCT). Inflammatory cytokines mediate damage to key GVHD targets such as intestinal stem cells (ISCs) and also activate receptor interacting protein kinase 1 (RIP1; RIPK1), a critical regulator of apoptosis and necroptosis. We therefore investigated the role of RIP1 in acute GVHD using samples from HCT patients, modeling GVHD damage in vitro with both human and mouse gastrointestinal (GI) organoids, and blocking RIP1 activation in vivo using several well-characterized mouse HCT models. Increased phospho-RIP1 expression in GI biopsies from patients with acute GVHD correlated with tissue damage and predicted NRM. Both the genetic inactivation of RIP1 and the RIP1 inhibitor GNE684 prevented GVHD-induced apoptosis of ISCs in vivo and in vitro. Daily administration of GNE684 for 14 days reduced inflammatory infiltrates in three GVHD target organs (intestine, liver, and spleen) in mice. Unexpectedly, GNE684 administration also reversed the marked loss of regulatory T cells in the intestines and liver during GVHD and reduced splenic T cell exhaustion, thus improving immune reconstitution. Pharmacological and genetic inhibition of RIP1 improved long-term survival without compromising the graft-versus-leukemia (GVL) effect in lymphocytic and myeloid leukemia mouse models. Thus, RIP1inhibition may represent a nonimmunosuppressive treatment for GVHD.

XIAP deletion sensitizes mice to TNF-induced and RIP1-mediated death.

XIAP is a caspase-inhibitory protein that blocks several cell death pathways, and mediates proper activation of inflammatory NOD2-RIP2 signaling. XIAP deficiency in patients with inflammatory diseases such as Crohn's disease, or those needing allogeneic hematopoietic cell transplantation, is associated with a worse prognosis. In this study, we show that XIAP absence sensitizes cells and mice to LPS- and TNF-mediated cell death without affecting LPS- or TNF-induced NF-κB and MAPK signaling. In XIAP deficient mice, RIP1 inhibition effectively blocks TNF-stimulated cell death, hypothermia, lethality, cytokine/chemokine release, intestinal tissue damage and granulocyte migration. By contrast, inhibition of the related kinase RIP2 does not affect TNF-stimulated events, suggesting a lack of involvement for the RIP2-NOD2 signaling pathway. Overall, our data indicate that in XIAP's absence RIP1 is a critical component of TNF-mediated inflammation, suggesting that RIP1 inhibition could be an attractive option for patients with XIAP deficiency.

A solid-phase approach for the synthesis of muramyl dipeptide conjugates for detection of NOD2.

Proper recognition of invading pathogens and prompt initiation of host defense mechanisms are instrumental for the maintenance of organismal homeostasis. Nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs) serve as pathogen-recognition receptors that specifically recognize bacterial peptidoglycans. NOD2 detects muramyl dipeptide (MDP) through its carboxy-terminal leucine rich repeats (LRRs), which enables the activation of downstream inflammatory signaling. Synthesis of MDP conjugates based on solution phase chemistry have been previously reported. Our solid phase approach synthetically provides a facile approach for the conjugation of biological probes to MDP, with the advantage of minimal functional/protecting group manipulation, and reduction in the laborious process of intermediate purification and isolation. MDP conjugates that we generated using solid phase synthesis allow detection of NOD2 is cell lysates and NOD2 subcellular localization by immunofluorescence microscopy. MDP-PEG6-Cyanine5.5 conjugate selectively colocalized with WT NOD2 but not NOD2 variant found in Crohn's disease, which lacks carboxy-terminal end and cannot bind MDP. Overall, these data indicate that distinct solid phase-produced MDP conjugates can be used to examine biological properties of NOD2 and could potentially facilitate further development of NOD2 targeting agents.

Immunoblot Analysis of the Regulation of TNF Receptor Family-Induced NF-κB Signaling by c-IAP Proteins.

Proper maintenance of organismal homeostasis, development, and immune defense requires precise regulation of survival and signaling pathways. Inhibitor of apoptosis (IAP) proteins are evolutionarily conserved regulators of cell death and immune signaling that impact numerous cellular processes. Although initially characterized as inhibitors of apoptosis, the ubiquitin ligase activity of IAP proteins is critical for modulating various signaling pathways (e.g., NF-κB, MAPK) and cell survival. Cellular IAP1 and 2 regulate the pro-survival canonical NF-κB pathway by ubiquitinating RIP1 and themselves thus enabling recruitment of kinase (IKK) and E3 ligase (LUBAC) complexes. On the other hand, c-IAP1 and c-IAP2 are negative regulators of noncanonical NF-κB signaling by promoting ubiquitination and consequent proteasomal degradation of the NF-κB-inducing kinase NIK. Here we describe the involvement of c-IAP1 and c-IAP2 in NF-κB signaling and provide detailed methodology for examining functional roles of c-IAPs in these pathways.

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury.

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.

Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death.

Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1K376R/K376R mice, Ripk1K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1K376R/cko mice was tolerated. However, adult Ripk1K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Ubiquitin Ligases cIAP1 and cIAP2 Limit Cell Death to Prevent Inflammation.

Cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 ubiquitinate nuclear factor κB (NF-κB)-inducing kinase (NIK) to suppress non-canonical NF-κB signaling and substrates such as receptor interacting protein kinase 1 (RIPK1) to promote cell survival. We investigate how these functions contribute to homeostasis by eliminating cIap2 from adult cIap1-deficient mice. cIAP1 and cIAP2 (cIAP1/2) deficiency causes rapid weight loss and inflammation, with aberrant cell death, indicated by cleaved caspases-3 and -8, prevalent in intestine and liver. Deletion of Casp8 and Ripk3 prevents this aberrant cell death, reduces the inflammation, and prolongs mouse survival, whereas Ripk3 loss alone offers little benefit. Residual inflammation in mice lacking cIap1/2, Casp8, and Ripk3 is reduced by inhibition of NIK. Loss of Casp8 and Mlkl (mixed lineage kinase domain-like), but not Mlkl loss alone, also prevents cIAP1/2-deficient mice from dying around embryonic day 11. Therefore, a major function of cIAP1/2 in vivo is to suppress caspase-8-dependent cell death.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death.

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia-reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia-reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

Roles of c-IAP proteins in TNF receptor family activation of NF-κB signaling.

Precise regulation of survival and signaling pathways is essential for proper maintenance of organismal homeostasis, development, and immune defense. Inhibitor of apoptosis (IAP) proteins are evolutionarily conserved regulators of cell death and immune signaling that impact numerous cellular processes. Initially characterized as inhibitors of apoptosis, the ubiquitin ligase activity of IAP proteins is critical for modulating various signaling pathways (e.g., NF-κB, MAPK) and cellular fate. Cellular IAP1 and IAP2 regulate the pro-survival canonical NF-κB pathway by ubiquitinating RIP1 and enabling recruitment of kinase (IKK) and E3 ligase (LUBAC) complexes. On the other hand, c-IAP1 and c-IAP2 are negative regulators of noncanonical NF-κB signaling by promoting ubiquitination and consequent degradation of the NF-κB-inducing kinase NIK. In this article, we describe the involvement of c-IAP1 and c-IAP2 in NF-κB signaling and provide detailed methodology for examining how c-IAPs exert their functional roles.

Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment.

Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.

OTUB1 modulates c-IAP1 stability to regulate signalling pathways.

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Cellular inhibitors of apoptosis are global regulators of NF-κB and MAPK activation by members of the TNF family of receptors.

Tumor necrosis factor (TNF) family members are essential for the development and proper functioning of the immune system. TNF receptor (TNFR) signaling is mediated through the assembly of protein signaling complexes that activate the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in a ubiquitin-dependent manner. The cellular inhibitor of apoptosis (c-IAP) proteins c-IAP1 and c-IAP2 are E3 ubiquitin ligases that are recruited to TNFR signaling complexes through their constitutive association with the adaptor protein TNFR-associated factor 2 (TRAF2). We demonstrated that c-IAP1 and c-IAP2 were required for canonical activation of NF-κB and MAPK by members of the TNFR family. c-IAPs were required for the recruitment of inhibitor of κB kinase ß (IKKß), the IKK regulatory subunit NF-κB essential modulator (NEMO), and RBCK1/Hoil1-interacting protein (HOIP) to TNFR signaling complexes and the induction of gene expression by TNF family members. In contrast, TNFRs that stimulated the noncanonical NF-κB pathway triggered translocation of c-IAPs, TRAF2, and TRAF3 from the cytosol to membrane fractions, which led to their proteasomal and lysosomal degradation. Finally, we established that signaling by B cell-activating factor receptor 3 induced the cytosolic depletion of TRAF3, which enabled noncanonical NF-κB activation. These results define c-IAP proteins as critical regulators of the activation of NF-κB and MAPK signaling pathways by members of the TNFR superfamily.

TRAIL receptor signaling regulation of chemosensitivity in vivo but not in vitro.

BACKGROUND: Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear. METHODOLOGY/PRINCIPAL FINDINGS: To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model. CONCLUSIONS: These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs as well as drugs that directly target the TRAIL receptors.

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Orally bioavailable antagonists of inhibitor of apoptosis proteins based on an azabicyclooctane scaffold.

A series of IAP antagonists based on an azabicyclooctane scaffold was designed and synthesized. The most potent of these compounds, 14b, binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domain of c-IAP1 with K(i) values of 140, 38, and 33 nM, respectively. These compounds promote degradation of c-IAP1, activate caspases, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Finally, compound 14b inhibits tumor growth when dosed orally in a breast cancer xenograft model.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.