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  • Like all other cellular processes ubiquitination


    Like all other cellular processes, ubiquitination is also tightly regulated; more so, as myriads of pathways are controlled via this conjugation and dysregulation of ubiquitination has been implicated in various diseases including cancer [16], [17]. Quite a few of such regulatory mechanisms that control activities of various E3 ligases [18], [19] and affect their substrate recognitions have been deciphered. On the other hand, the existence of diverse mechanisms that regulate E2 activity is appreciated only recently [20], [21], [22]. All the E2 enzymes across all the species, including ~35 coded by the human genome, share a structurally conserved ubiquitin-conjugating UBC domain of ~150 residues harboring the catalytic cysteine. Despite this structural conservation, UBC cores from different E2s show significant sequence diversity justifying differences in their E3 selectivity and catalytic activity [23]. Besides the UBC domain, some of the E2s also contain extended regions in their C-terminal end (Class II E2s) or in their N-terminal end (Class III E2s), or both (Class IV E2s). The importance of such extensions in Ube2C (UbcH10) and Ube2R1 (Cdc34) has been well established [24], [25]. The N-terminal extended region of Ube2C plays a role in substrate selectivity by its cognate E3, the anaphase-promoting complex (APC), and the C-terminal extension of Ube2R1 (Cdc34) affects the processivity of the UBC core. However, roles of such extended regions have not been understood for most of the other E2s. Ube2D and Ube2E Ketorolac synthesis of E2s share a highly conserved UBC domain and seem to interact with the same subset of E3 ligases [26]. However, Ube2Ds consist of just the UBC domain whereas Ube2Es have 40- to 60-residue-long disordered N-terminal extensions apart from the core. While studying the E2 preference of the RING E3 ligase RNF4 (RING finger protein 4), we had observed limited activity of Ube2E1 (Datta et al., unpublished data) similar to the observations with the heterodimeric E3 ligase BRCA1-BARD1 [27]. It has also been demonstrated that the N-terminal extensions of Ube2Es limit the chain-building capability of the UBC domain, though the molecular mechanism behind this remained unclear [28]. We probed further into this N-terminal mediated activity reduction and show that not the mere presence but the E3-catalyzed intramolecular auto-ubiquitination (self-ubiquitination) of the disordered N-terminal extensions acts as the mechanism restricting activity of Ube2E class of the E2 enzymes.
    Discussion The Ube2E group of ubiquitin-conjugating enzymes, classified as the Class III E2s owing to their N-terminal extensions, is considered as “hub” E2s due to their capability of interacting with a large number of E3 ligases [26], [33]. The “E3 interactome” of these E2s is second only to that of the Ube2Ds and shares a significant overlap. Presumably, the resemblance between the UBC domain of these two E2 classes (~86% similar) results in their similarity in choosing cognate E3s. However, unlike Ube2Ds, the activity of Ube2E E2s has been observed to be limited with most of the interacting E3s including BRCA1-BARD1 [27]. Our results demonstrate that the limited activity of Ube2E1 is a direct consequence of ubiquitination at its flexible N-terminal extension. Thus, either the deletion of the entire N-terminus or mutating the Lys residues in that region to Arg relieves the attenuated activity of Ube2E1. The only exception to this effect of the N-terminal extension was the partial enhancement observed with Ube2E1∆N20 . Upon further investigation, we found that Ube2E1∆N20, unlike Ube2E1wt or Ube2E1∆N46, was unstable and invariably contained a contaminating degradation product of smaller molecular weight matching the “core domain” due to non-specific proteolysis of this truncated E2 construct. Thus, we concluded that the partial enhancement of Ube2E1∆N20 activity resulted from the contaminating core domain.