br Conclusion In the last
Conclusion In the last year, significant advances in our understanding of ubiquitin transfer by IAPs and related E3 ligases have been made. The three crystal structures of the RING-bound E2~Ub show that the conjugate is bound by the RING domain in the closed conformation, where the I44-centered face of ubiquitin makes extensive contacts with the E2 (Dou et al., 2012, Dou et al., 2013, Plechanovova et al., 2012). This conformation is stabilized by RING–ubiquitin interactions; either an aromatic residue on the C terminus of the dimerized RING, or a region N-terminal to the ligase domain. These interactions place ubiquitin in a conformation that is thought to prime the thioester bond for nucleophilic attack by a substrate Lys/N-terminal Met. This model accounts for much of the prior biochemical data, and explains why RING dimerization and an aromatic residue at the dimer interface are required for ubiquitin transfer of IAPs (Dou et al., 2012, Nakatani et al., 2013). Despite advances in our understanding of the overall topology of the RING-E2~Ub complex (Dou et al., 2012, Dou et al., 2013, Plechanovova et al., 2012), and the importance of reducing conformational freedom for catalysis (Berndsen et al., 2013), a detailed mechanistic understanding of ubiquitin transfer from the E2 to a substrate Lys remains uncertain. In addition, although many features of the mode of binding and mechanism of transfer are likely to be conserved in most E2s, the RING-bound structure of a conjugate is only available for highly similar UbcH5 family members. Do other less similar E2~Ub conjugates adopt an analogous configuration when they are bound by their cognate RING domains? Likewise, only a handful of RING pdgfr have been characterized, and it seems likely that residues outside the core RING domain will be important for stabilization of the closed conformation in some cases (Dou et al., 2013). A molecular understanding of the complexes formed by other E2~Ub conjugates and RING E3s is eagerly awaited. Over the last 5 years, it has become clear that proper regulation of apoptosis is dependent on appropriate ubiquitylation of various cellular components (Vucic, Dixit, & Wertz, 2011). Here, we have focused on IAP proteins that contain a RING domain, but other E3 ligases such as MDM2, which is the primary E3 that regulates the abundance of the tumor suppressor protein p53, also have critical roles in regulating apoptosis. The RING domains from MDM2 and IAPs form similar dimers (Kostic et al., 2006, Linke et al., 2008, Mace et al., 2008), and it is anticipated that their mechanism of action will be comparable. However, other structurally distinct E3s, such as linear ubiquitin chain assembling complex (Stieglitz et al., 2013) also play important roles in regulating apoptosis and a detailed understanding of their interactions with the E2~Ub conjugate will be of considerable interest. Recent studies have also uncovered additional roles for E2 enzymes and E2~Ub conjugates in modulating the activity of deubiquitinating enzymes (DUBs), such as OTUB1 (Juang et al., 2012, Wiener et al., 2013, Wiener et al., 2012). Interestingly, OTUB1 has also been shown to associate and modulate the stability of cIAP1 (Goncharov et al., 2013). Together, these studies highlight the central role of E2~Ub conjugates in regulating cell death and it will be of considerable interest to understand the factors that influence the balance between E3-E2~Ub and DUB-E2~Ub interactions. We anticipate that biochemically defined assays that require preparations of pure reagents, such as those described here, will be essential for this understanding.
Introduction Sumoylation is an essential transient posttranslational modification that is predominantly detected in the nucleus and has key functions in many cellular pathways, including transcription, chromatin regulation, DNA replication, DNA damage responses, RNA splicing, cell cycle regulation, protein degradation, and intracellular trafficking (Droescher, Chaugule, & Pichler, 2013; Flotho & Melchior, 2013; Zhao, 2018).