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  • br Multi subunit RINGs There are


    Multi-subunit RINGs There are RING-type E3s that exist as multi-subunit assemblies (see Fig. 3B). A striking example is the Cullin RING Ligase (CRL) superfamily [35], which exhibits enormous plasticity in substrate specificity. Each CRL subfamily is characterized by a cullin protein (Cul-1, 2, 3, 4a, 4b, 5, or 7), a small RING protein (in most cases Rbx1/Roc1/Hrt1), and either an adaptor protein(s) that binds interchangeable substrate recognition elements or, in the case of CRL3, proteins that bind both to the cullin protein (Cul-3) and to substrate [36]. The CRL superfamily is exemplified by the well-studied Skp1-Cul1-F-box protein (SCF) family (Fig. 3B), in which one of ~69 (in humans) interchangeable F-box proteins can potentially recognize substrates [37] (reviewed in this issue by Bassermann et al.). Exchange of F-box proteins within the SCF scaffold takes place through a complex cycle that includes dynamic attachment and removal of the ubiquitin-like modifier, Nedd8 [38]. While the CRL superfamily overwhelmingly exhibits the greatest range of substrate recognition, other multi-subunit E3s exhibit even greater structural complexity. The mdm2 inhibitor promoting complex/cyclosome (APC/C) is a highly complex E3 that in humans contains 13 core subunits including a cullin-like protein and a small RING protein. It also has two interchangeable co-activator subunits, Cdc20 and Cdh1, which recognize distinct substrates and are active during different phases of the cell cycle [39] (reviewed in this issue by Bassermann et al.). Another multi-subunit RING-containing ligase is the Fanconi anemia (FANC) E3. There are at least 13 complementation groups associated with this disease, and proteins corresponding to eight complementation groups are components of the FANC ubiquitin ligase, including a RING-type protein (FancL). This E3 is recruited to sites of DNA damage to effect translesional repair. Despite its complexity, the role of the FANC E3, as we currently understand it, is limited to monoubiquitination of two associated proteins that are subsequently deubiquitinated as part of the DNA repair process. Degradation of a key FANC component, FANCM, via SCFβTrCP is responsible for inactivating the function of the FANC E3 during mitosis, thereby preventing chromosomal abnormalities [3], [5], [40]. Some multi-subunit RING-type E3s contain multiple RING proteins. The yeast GID (glucose-induced degradation deficient) complex, which targets fructose-1, 6 bisphosphatase for ubiquitination in response to glucose, consists of seven subunits including two interacting RINGs [41]. The yeast PEX ubiquitin ligase, which mono-ubiquitinates the peroxisome receptor, Pex5p, and possibly other substrates, includes three distinct RING proteins as part of a multi-subunit complex [42], [43]. The specific function of each of the RINGs in such complexes is currently unknown. Finally, there are single proteins that contain multiple RINGs. Mindbomb, involved in Notch signaling, has three RINGs in its C-terminal region, although to date only the most C-terminal of these has been studied and shown to be required for activity [44]. RING–IBR–RING (RBR) proteins are a class of ~13 proteins (in humans) that include a RING consensus sequence (RING1) followed by a Cys-rich ‘in between RING’ (IBR) region and a third domain, the RING2, that was originally characterized as a second RING-like domain. Although RBR proteins were thought to function as canonical RING E3s, recent studies have shown that they employ a RING–HECT hybrid mechanism [45], [46], [47], [48], [49]. The RING1 domain binds E2 (similar to the RING mechanism) but ubiquitin is transferred to a specific Cys within RING2 before being transferred to substrates (similar to the HECT mechanism). Well-known members of this family include Parkin, HHARI, HOIP, and HOIL-1L. The latter two are subunits of the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L, and Sharpin (a non-RING-containing protein). This complex plays critical roles in NF-κB activation (reviewed in this issue by Kazuhiro et al).