IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-α (tumour necrosis factor-α)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation.
IAP (inhibitor of apoptosis) proteins are an evolutionarily conserved class of multifunctional proteins . They originally gained their name for their ability to inhibit apoptosis, a form of programmed cell death. Indeed, IAPs are frequently over-expressed in tumours and have been implicated in oncogenesis, chemoresistance and poor patient survival . Recent evidence suggests that IAPs also play a wide range of roles in the development and homoeostasis of metazoans by modulating cellular processes other than apoptotic cell death . These proteins are characterized by the presence of highly conserved BIR (baculoviral IAP repeat) domains, a sequence of 70 amino acids that folds to three stranded β-sheets surrounded by four short α-helices to co-ordinate a zinc ion. So far, eight mammalian IAPs [XIAP (X-linked IAP), c-IAP1 (cellular IAP 1), c-IAP2 (cellular IAP2), NAIP (neuronal IAP), BRUCE (baculoviral IAP-repeat containing 6), ML-IAP (melanoma IAP), ILP2 (baculoviral IAP-repeat containing 8) and Survivin] are known. Apart from the BIR domains, some IAPs (XIAP, c-IAP1, c-IAP2, ML-IAP and ILP2) also carry a RING (really interesting new gene) domain with E3 ligase activity and a CARD (caspase activation recruitment domain) (c-IAP1 and c-IAP2), the function of which is currently unknown. NAIP is a unique member of this family as it possesses NOD (nucleotide-binding oligomerization domain) and LRRs (leucine-rich repeat domains) in addition to the BIR domains .
IAPs can directly bind to caspases, the key effector proteases of apoptosis, via the BIR domains and as such inhibit their activation and cell death. XIAP is considered to be the most potent and direct inhibitor of caspases and the mechanism of caspase inhibition by XIAPs has been worked out in great detail. The BIR3 domain of XIAP is responsible for the inhibition of the initiator caspase 9 and the BIR2 domain, in combination with a linker region immediately N-terminal to BIR2, is responsible for mediating inhibition of the effector caspases 3 and 7 [1,3]. Except XIAP and ILP2, the other IAPs lack the critical residues that confer direct inhibition of caspases; however, ILP2 is highly unstable and the endogenous expression of this protein has not yet been demonstrated . c-IAPs (c-IAP1 and 2) were identified as binding partners of TRAFs [TNF (tumour necrosis factor)-associated factors) 1 and 2. A large body of evidence suggested a crucial role for these IAPs in modulating NF-κB (nuclear factor κB) activation through both canonical and non-canonical pathways by modulating RIP1 (receptor-interacting protein 1) polyubiquitination and destabilizing NIK1 (NF-κB inducing kinase 1) respectively .
During apoptosis, the permeabilization of the mitochondrial outer membrane leads to the release of pro-apoptotic proteins Smac (second mitochondrial activator of caspases)/DIABLO and Omi/HtrA2 from the mitochondrial intermembrane space to the cytosol to antagonize IAPs so that caspases can be activated to accomplish apoptotic cell death. These natural IAP antagonists possess a so-called IBM (IAP-binding motif) characterized by the presence of a tetrapeptide (Ala-Val-Pro-Ile in human Smac and Ala-Val-Pro-Ser in human Omi) at their N-terminus, which is exposed upon cleavage of their targeting sequence following mitochondrial import . Following their leakage from the mitochondrial inter-membrane space, IAP-antagonists bind to the BIR2 and BIR3 domains of IAPs via their IBM and, thereby, prevent them from interacting with caspases. In addition, binding of Smac can also trigger auto-ubiquitination and degradation of c-IAPs, but not XIAP. Based on these observations, small molecular inhibitors that mimic the Smac N-terminal IBM were developed. As expected, Smac mimetics triggered auto-ubiquitination of c-IAPs and triggered cell death via activation of the extrinsic cell-death pathway. This is accomplished by NF-κB activation via the non-canonical pathway causing production of autocrine TNF-α, over-activation of TNF-R1 (TNF receptor 1) and subsequent induction of caspase 8 [5,6].
As much of the IAP research has been focussed on their BIR and RING domains, two recent publications add a new twist to IAP biology: they unveiled an evolutionarily conserved UBA (ubiquitin-associated domain) in several IAPs [c-IAPs, XIAP, ILP2 and DIAP2 (Drosophila IAP)] [7,8]. This domain is localized in the linker region between the third BIR domain and the RING finger of XIAP and c-IAPs, as well as ILP-2 (Figure 1) [7,8]. The UBA belongs to the class of ubiquitin-binding domains present in a variety of cellular proteins known as ubiquitin receptors . The UBA forms a compact three α-helical bundle, which interacts with the Ile44 patch of ubiquitin via a conserved MGF/Y hydrophobic loop . While most UBA-containing proteins seem to bind to Lys48-linked chains, several UBAs bind to mono-ubiquitin and/or Lys63 chains . A systematic investigation of the binding propensities of more than 30 different UBAs indicate that their binding specificities are highly variable . In this issue of the Biochemical Journal Blankenship et al.  demonstrate that the UBA of IAPs (in particular c-IAP1) binds to both mono- and poly-ubiquitin chains (Lys48- and Lys63-linked) with low micromolar affinities. Moreover, by using multiple methods (NMR spectroscopy, isothermal titration calorimetry and surface-plasmon resonance) they conclude that UBAs of IAPs do not have particular selectivity for mono-ubiquitin or ubiquitin chains in vitro. In contrast, Gyrd-Hansen et al.  used GST (glutathione transferase) pulldown assays to demonstrate that XIAP, c-IAP1, c-IAP2 and DIAP2 readily bind to ubiquitin chains and do not associate with mono-ubiquitin . While c-IAP1 and DIAP2 bound equally well to Lys48- and Lys63-linkeages, XIAP and c-IAP2 appeared to prefer Lys63-linked chains and linear tetra-ubiquitins . Both studies indicated that the characteristic MGF motif in UBAs of IAPs is essential for ubiquitin binding since mutation in these critical residues (MF/AA) impairs the binding of IAPs to the ubiquitin message. The observed discrepancies between these two studies is probably due to the different methods and/or constructs used. Given that the affinities for ubiquitin binding are relatively low, small differences in experimental design may favour binding to ubiquitin chains over mono-ubiquitin, as was shown for the ubz (ubiquitin-binding ZnF) domain of Wrnip1 . An additional aspect of ubiquitin binding by ubiquitin receptors, which is often neglected, is that their in vivo specificities are often influenced by secondary interactions with additional partners.
Schematic representation of XIAP demonstrating the functional domains
It is obviously important to know, what is the role of UBAs in modulating IAP-mediated cellular functions? Blankenship et al.  demonstrate that the UBA is not required for TNF-α receptor protein complex assembly or for binding to TRAF2 and Smac. On the other hand, the authors indicate possible involvement of UBAs for c-IAP-mediated apoptosis resistance in multiple myeloma cells and for the recruitment of c-IAPs to the proteasome machinery. Consistently, mutations in this domain prevent proteasomal degradation, but not auto-ubiquitination of c-IAPs by Smac mimetic compounds; however, a role for UBAs for modulating c-IAP-mediated NF-κB activation has been ruled out, as mutations in the ubiquitin-binding region failed to impair c-IAP-mediated NIK1 degradation or NF-κB activation. On the other hand, Gyrd-Hansen et al.  demonstrated a requirement of the UBA for modulating IAP-mediated NF-κB activation, oncogenesis and cell survival. An in vivo role for the UBA in c-IAPs and the c-IAP2/MALT1 (mucosa-associated lymphoid tissue protein 1) fusion protein in mediating NF-κB activation was evident in their experiments. The physiological importance of the UBA in c-IAP1 was further exemplified by its requirement for endothelial cell survival in zebrafish as well as for the progression of liver cancer in nude mice . Interestingly, both studies conclude that although the UBA in c-IAP1 is not required for its E3 ligase activity and auto-ubiquitination, it seems to be important for providing resistance to TNF-α-mediated apoptosis .
Despite the discrepancies which need to be addressed in the future, these studies have clearly opened a new page in IAP biology as ‘ubiquitin catchers’ and pose several interesting questions. What are the ubiquitylated proteins IAPs bind to? How do these interactions contribute to the functionality of IAPs? What are the interacting proteins during cellular processes such as apoptosis? Gyrd-Hansen et al.  propose that the fusion protein cIAP2/MALT1 mediates constitutive activation of NF-κB by interacting with polyubiquitinated NEMO (NF-κB essential modulator). As c-IAPs mediate the polyubiquitination of RIP1, it is tempting to speculate that RIP1 might also bind to c-IAPs in a UBA-dependent manner. Other interesting candidates are IAPs themselves as they auto/cross-ubiquitinate and interact with each other [13,14]. For instance, c-IAP1, via its UBA might promote self-ubiquitination or ubiquitination of associated c-IAP2, thus regulating their in vivo functions. It was recently shown that proteins containing UBAs can mediate self-ubiquitination in an E3-independent manner  and that subsequent intra- and possibly inter-molecular binding leads to conformational changes preventing their capacity to interact with other targets in trans . Although the UBA seems to be dispensable for XIAP-mediated caspase inhibition, it is important for c-IAP1-mediated protection from TNF-α-induced cell death. It would be interesting to know how c-IAPs resist TNF-α-mediated apoptosis, especially if the UBA is not required for activation of NF-κB (as suggested by Blankenship et al. ). Since IAPs control various cellular processes, like mammary development and cellular differentiation, future studies will have to address the importance of the UBA in these processes. As with other UBA-containing proteins, IAPs probably exhibit preferential affinity to mono- or poly-ubiquitinated proteins in vivo that in turn could be influenced by signalling stimuli. Understanding the dynamics and functionality of these interactions will keep the IAP researchers busy and excited for years to come.
As the famous philosopher Friedrich Nietsche once said, “talking much about oneself may be a way of concealing oneself”. Lois Miller and colleagues cloned the first IAPs from baculoviruses 15 years ago as genes which protect insect cells from virus-induced cell death . Swiftly, cellular homologues have been identified in various metazoans and two structural moieties emerged: the BIR domains and the RING finger domains. The importance of these domains in mediating apoptosis resistance was highly recognized and has been the centre of all the discussion on IAPs as caspase inhibitors. The current discovery of an ‘ubiquitin catching’ domain in IAPs adds to its repertoire and reveals a thus far ‘concealed’ other side, which may be equally important and interesting.