Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

Abstract

In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a ;93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations.

RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…Our data also suggest that Ubc6e might cooperate with RNF185 in targeting CFTR to degradation. However, our data also show that this functional interaction is not exclusive and that E2s from the UbcH5 family can also cooperate with RNF185, which is in agreement with the identification of the E2 enzymes as RNF185 interacting partners, along with other E2 enzymes (64). The effect of UbcH5 knockdown on the RNF185-stimulated degradation of CFTR is, however, hard to interpret as UbcH5 depletion also impacts CHIP-dependent CFTR degradation (42).…”

UBE2O negatively regulates TRAF6-mediated NF-κB activation by inhibiting TRAF6 polyubiquitination

“…The putative E2 enzyme UBE2O has previously been reported to interact with the signaling mediators TRAF4, TRAF5 and TRAF6 in yeast two-hybrid assays [24]. We first verified this interaction by expressing UBE2O-Myc together with Flag-tagged TRAF1, 2, 3, 4, 5, or 6.…”

“…However, the function of UBE2O was previously unknown. Recently, more than one hundred E3 ubiquitin ligases were found to interact with UBE2O in yeast two-hybrid screens [24], including TRAF6, a mediator of TLR/IL-1R-induced NF-κB activation. As an important part of the host defense system, TLR/IL-1R-induced signaling needs to be tightly controlled to maintain immune homeostasis and avoid detrimental responses.…”

“…The function of UBE2O remains mostly unknown. By using yeast two-hybrid screens, Markson et al [24] identified more than 100 E3 ubiquitin ligases interacting with UBE2O, including TRAF6. However, it is as yet unknown whether UBE2O plays a role in TRAF6-mediated signaling.…”

Proteomic techniques to probe the ubiquitin landscape

“…Bioinformatic studies are already using the extensive data collected in the large scale studies [132,133]. New studies are now starting to combine the information of ubiquitination with other post-translational modifications like phosphorylation in order to construct more complex networks [134].…”