E3 Ligase - Enzymes
Ubiquitin modification of substrate proteins is achieved by the activity of E1 activating, E2 conjugating and E3 ligase enzymes. Substrate proteins can be modified with one (mono-ubiquitination) or many (poly-ubiquitination) molecules of ubiquitin, and each type of modification has distinct regulatory fates. E3 ligages number in the thousands and have the highest level of specificity for the target protein to be modified by ubiquitin or ubiquitin-like modifiers (UBL).
In addition, many E3s have been implicated in human disease and are attractive targets for drug discovery. However, currently no small molecule modulators for this class of enzymes have reached the clinic. Each E3 enzyme targets a small number of proteins for Ub modification but the exact substrates are mostly unknown and their identification continues to be a challenge. E3 ligase enzymes are a large (> 500) and complex super- family, many of which contain binding domains to interact with ubiquitin, E2 enzymes and substrate proteins. In addition to substrate ubiquitination, many E3 ligases can also self- or auto-ubiquitinate in the presence of an E2 enzyme, a property that may be used as an auto-regulatory mechanism to control its own intracellular levels. In general, the detailed molecular mechanism, stoichiometries and linkage site selection of E3 enzymes are poorly understood. As with ubiquitin E3 ligases, similar activities are also part of the final conjugation processes for other UBL proteins. There are E3 enzymes that are specific for targets that are modified SUMO, NEDD8, ISG15 and presumably also for FAT10 and UFM1.
Ubiquitin E3 enzymes are classified into two primary classes according to domain homology and mechanism of action. The subtypes include the HECT (homologous to E6-AP Cterminus) and RING (Really Interesting New Gene) proteins. The HECT E3 ligases contain a large domain (~ 350 residues) with a catalytic cysteine residue that transfers Ub to via a cognate E2 directly to the substrate. These enzymes interact directly with substrate target proteins to effect poly-ubiquitination. Examples of HECT proteins include the viral E6AP, ARB-BP1, Itch, NEDD4, Smurf2 and WWPI. The RING E3 ligases have two zinc ions coordinated by multiple CYS and His residues to form a globular E2-binding domain. In contrast to HECT E3s, RING E3s do not have the recognizable catalytic active sites that define “classical” enzymes. Instead, these E3s which have large binding interfaces and act as scaffold proteins that bring together the participant E2 and substrate proteins. Examples of RING-FINGER proteins include BRCA1, Cbl, Efp, Hdm2, MurF1, Parkin, SIAH, TRAF6, Rfn11 and XIAP. Two other RING-FINGER related domains, the U-box (UFD2-homology domain) and PHD (plant homeo domain) also confer E3 activity. U-box E3s (CHIP, UFD2, PRP19, UIP5, CYC4) have a similar RING-FINGER tertiary structure and may participate in protein quality control via their interaction with chaperones. PHD containing proteins (c- MIR, AIRE, MR1, MR2) have E3 activity that is PHD domain-dependent but it is not known if all PHD proteins function similarly. Another group of E3s are multi-component complexes such as the modular SCF (Skp1/Cullin/F-box/Rbx1/2) family. These complexes are exemplified by the Rbx RING-FINGER E3 Ligase Enzymes proteins in various combinations with at least three components including a Cullin protein, an adaptor (Sk1, Elongin B/C or a BTB) and a substrate binding protein (Fbox, SOCS or BTB). The APC (anaphase-promoting complex) is also a large multiprotein- E3 complex that regulates both entry and exit from mitosis via the ubiquitination of key cell cycle regulators such as cyclin B, and securin. This large complex (~ 11 subunits) is similar to SCF and contains a catalytic core cullin (Apc2) and a RING protein (Apc11).
E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2 approximately ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either mono-ubiquitinate a substrate lysine or synthesize poly-ubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.