Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network

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.Protein ubiquitination is mediated by the sequential action of an E1 activating enzyme, an E2 conjugating enzyme, and a range of E3 proteins, which are thought to confer substrate specificity (Hershko and Ciechanover 1998). Two main forms of E3 proteins have been characterized: HECT domain ligases, which act as enzymatic intermediates in protein ubiquitination and E3-RING proteins, which appear to be nonenzymatic recognition factors, although their exact role in catalysis remains to be established (Ozkan et al. 2005; Petroski et al. 2006). Although the sequence of events that facilitate the addition of ubiquitin (Ub) or ubiquitin-like (Ubl) proteins is conserved in all eukaryotic cells, the extent and form of Ub and Ubl modification can be diverse, ranging from the addition of single Ub or Ubl residues at one or more sites within a target protein (mono- and multi-ubiquitination), to the assembly of a range of structurally distinct polyubiquitin chains (Peng et al. 2003), which may confer different functional properties (Welchman et al. 2005; Ikeda and Dikic 2008).Although E2 and E3 proteins are thought to work in a combinatorial manner to generate different forms of substrate modification (Weissman 2001; Christensen et al. 2007), very little is known about the specificity or combinatorial nature of human E2/E3-RING interactions. As such, the potential for redundancy or antagonism within the human ubiquitome remains unclear, as does the degree of connectivity between different network components. As Ub and Ubl proteins are known to play a key role in the regulation of both physiological and pathological processes, there is a growing need to develop a better understanding of the complex ways in which E2 and E3 proteins work together in human cells.While isolated biochemical studies and unbiased global interactome projects continue to provide invaluable and exciting data, coverage of the human ubiquitome, and, in particular, E2/E3 interactions, remains limited. To provide new insights into partner preferences and the degree of redundancy within this combinatorial process, the density of network coverage must be significantly increased using techniques that define or predict binary interactions. Analysis of data contained within the MINT, IntAct, BioGRID, and HPRD databases revealed 60 human E2/E3-RING interactions (Fig. 1A; Supplemental Files 3 and 4), including data from both binary and co-complex isolation studies. This lack of experimental data is compounded by the expansion in E2- and E3-RING protein numbers, which has occurred during eukaryotic evolution. Searches performed using data from the Inparanoid and Homologene databases show that 34 of the 39 human E2-related proteins have clearly identifiable orthologs in yeast, fly, or worm. However, 48% (146 out of 304) of E3-RING proteins do not (Supplemental File 1). Consequently, methods developed to predict interactions between orthologous proteins in different species (Interolog interactions) (Matthews et al. 2001; Lehner and Fraser 2004) cannot be used to assign combinatorial preferences for all human E2/E3-RING complexes.Open in a separate windowFigure 1.Binary human E2/E3-RING protein interaction networks. (A) Previously known interactions derived from the MINT, IntAct, BioGRID, and HPRD databases at the time of this study. (B) Predicted human E2/E3-RING interactions including Interologs (purple edges) or non-Interolog predicted interactions from Hi-map and IntNet databases (orange edges). (C) Increased coverage within the human E2/E3-RING interaction space as a result of this study. Novel interactions are shown as red edges. Bold edges represent interactions confirmed by our data. Blue nodes represent E2 ubiquitin conjugating enzymes, while yellow nodes represent E3-RING proteins. To aid network analysis and node identification, all networks are provided as ready-to-view Cytoscape files (Supplemental File 3).To address these problems we have assembled a high-density binary protein interaction map containing >1810 human E2- or E3-RING interactions. Initially, two stringent Y2H methods were used to investigate the spectrum of human E2 protein interactions and the combinatorial nature of human E2/E3-RING complexes. In addition, a structure-based true homology modeling method was also used to provide an independent prediction of interactions between 3180 human E2/E3-RING pairs. Finally, experimental data from this study were combined with known human E2- or E3-RING interactions and available Interolog data to generate a one-step extended map, which provides an initial insight into the gross topology and modular organization within the highly combinatorial human E2/E3-RING network.