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  • br Acknowledgments This work was supported

    2020-11-24


    Acknowledgments This work was supported by Grants from the National Research Foundation (2010-0000782) and the National Center for GM Crops (PJ008152) of the Next Generation BioGreen 21 Program funded by the Rural Development Administration, Republic of Korea, to W.T.K.
    Introduction Ubiquitylation is a posttranslational modification that regulates many events in eukaryotic cells. Most famously, ubiquitylation is involved in directing proteins for degradation by the 26S proteasome (Varshavsky, 2012). Attachment of the 8.5kDa protein ubiquitin to target proteins involves three classes of enzymes: (i) ubiquitin activating E1 enzymes that activate ubiquitin in an ATP-dependent process; (ii) ubiquitin conjugating (UBC) E2 enzymes, which are charged with ubiquitin by the E1; and (iii) the ubiquitin E3 ligases, which provide specificity for the final transfer of ubiquitin to a substrate Lys or N-terminal Met residue (Pickart, 2001). Of the >600 E3 ligases encoded in the human genome most can be classified into one of two families based on the presence of either, a homologous to the E6AP carboxyl terminus (HECT) domain, or a really interesting new gene (RING) domain. The HECT family of ligases is involved in catalysis as they receive ubiquitin from the E2 and transfer it to the substrate (Kee & Huibregtse, 2007). In contrast, the RING family bring the ubiquitin conjugated E2 enzyme (E2~Ub) and the substrate together, allowing transfer of ubiquitin directly from the E2 to the substrate (Deshaies & Joazeiro, 2009). However, recently the RING-in-between-RING (RBR) E3 proteins have been shown to utilise a hybrid RING/HECT mechanism (Wenzel, Lissounov, Brzovic, & Klevit, 2011). These proteins depend on a RING domain for E2~Ub recruitment, but ubiquitin is transferred to a cysteine in the E3 before attachment to the substrate. The Inhibitor of APoptosis (IAP) proteins were first identified as gene products of baculovirus that inhibited apoptosis of infected insect MCC950 sodium (Clem et al., 1991, Crook et al., 1993). Subsequently, the mammalian IAP family was identified (Rothe et al., 1995, Uren et al., 1996). All members of the IAP family possess at least one baculovirus IAP repeat (BIR) domain, a protein–protein interaction module. Many IAPs also contain a RING domain that confers on them the ability to promote their own ubiquitylation (autoubiquitylation), as well as the ubiquitylation of substrate proteins (Mace et al., 2010, Vaux and Silke, 2005). Ubiquitin transfer by IAPs is central to their ability to modulate signaling pathways. For example, cIAP1 promotes the addition of nondegradative ubiquitin signals on RIPK1, and these are required for assembly of the prosurvival signaling complex (Bertrand et al., 2008). In contrast, small molecule compounds that bind to the BIR domains promote cell death by enhancing cIAP autoubiquitylation and subsequent degradation (Varfolomeev et al., 2007, Vince et al., 2007). As well as modulating cell survival, important roles for IAPs in innate immune signaling have also been established (Vandenabeele & Bertrand, 2012), and the RING domain-mediated E3 ligase activity of XIAP is required for nucleotide-binding and oligomerization domain signaling (Damgaard et al., 2012, Damgaard et al., 2013). The ligase domain in IAPs resembles other RING domains and, as expected, it has a conserved flat surface that interacts with E2 enzymes (Mace et al., 2008). Ubiquitin transfer by IAPs depends on both the integrity of the E2-binding site and RING dimerization (Feltham et al., 2011). Disruption of the dimer abrogates activity and although the isolated RING domain from all IAP proteins forms a stable dimer, the longer forms of some IAPs, such as cIAP1, are largely monomeric and ubiquitin transfer is impeded. The structure of the autoinhibited monomeric form of cIAP1 showed that the RING dimer interface is occluded due to interactions with the third BIR domain (Dueber et al., 2011). Remarkably, the interaction interface on the BIR domain includes the pocket to which a number of proteins and small molecule compounds bind, and this structure also explained why addition of small molecule BIR-binding compounds promotes RING dimerization and autoubiquitylation of cIAP1 (Dueber et al., 2011).