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A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system.

van Wijk SJ, de Vries SJ, Kemmeren P, Huang A, Boelens R, Bonvin AM, Timmers HT - Mol. Syst. Biol. (2009)

Bottom Line: Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others.For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B).Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Genetics, Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2-E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2-E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

Show MeSH
Quality of the E2–E3 network. Physical E2–E3 interactions in relation to literature-curated, functional E2–E3 pairs. Hub node E3s were selected and interactions with E2s were scored when biochemically tested in in vitro ubiquitination assays as reported in literature.
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f5: Quality of the E2–E3 network. Physical E2–E3 interactions in relation to literature-curated, functional E2–E3 pairs. Hub node E3s were selected and interactions with E2s were scored when biochemically tested in in vitro ubiquitination assays as reported in literature.

Mentions: Comparing the E2–E3 interactions obtained in this screen with E2–E3 literature-curated interactions evaluates the quality of the interactions in the E2–E3 network. Physical interactions between E2 and E3 enzymes are required for efficient ubiquitination in vivo and in vitro and therefore for their catalytic function. Besides this, comparing the novel E2–E3 pairs generated by our screen with annotated functional E2–E3 pairs provides insight into the functionality of the E2–E3 pairs. We first selected the top 10% interacting E3s and compared their physical E2 interaction patterns with information obtained by low-throughput E2–E3 experiments. As shown in Figure 5, 25 hub E3s showed a total number of 209 E2 interactions covering 60% of the total number of E2–E3 two-hybrid interactions. For the published literature we recovered 64 E2–E3 pairs for the 25 hub E3s; 35 of the 64 pairs were positive for in vitro or in vivo ubiquitination activity and 29 were tested negative. Of the 35 positive literature interactions we confirmed 33 in our screen, which we regard as true positives (Figure 5). We found only two pairs that were biochemically reported positive, but did not occur in the Y2H screen (false negatives). The observed literature interactions were mostly concerning the members of the UbcH5 family and UBE2E1 and UBE2E3. To investigate the possibility that these E2s are more versatile in ubiquitination reactions, we compared reported E2–E3 combinations that were negative in biochemical reactions. Of the 29 E2–E3 interactions reported negative we recovered 24 in our dataset (true negatives). We also found five pairs that were reported negative in ubiquitination assays, but showed an interaction in the Y2H screen (false positives). In conclusion, this indicates a specificity of 83% (24/(5+24)) and a sensitivity of 94% (33/(2+33)) between biochemically active E2–E3 pairs and pairs found interacting in our screen. In the above analysis we focussed on the top 10% of interactors. Analysis of sensitivity and specificity of the complete data set is confounded by uncertainty of proper folding and/or localization of the Y2H proteins (Supplementary Table VI), which are potential pitfalls of Y2H screening. Therefore, we focussed on the E2 and E3s, which display at least one interaction. This criterion retains 20 E2s and 104 E3s from our screen. Literature inspection of this set showed 118 E2–E3 interactions of which 83 scored positive and 35 negative. Of the 83 positive interactions we recovered 48 interactions in our dataset. Of the 35 negative interactions 30 were also scored in Y2H interaction. This yields a sensitivity of 86% and a specificity of 58%. The verification rate of our screen (true positives plus true negatives/total interactions) is 66%, which compares well to global Y2H screens reported earlier (Li et al, 2004; Rual et al, 2005). On the basis of these data, we conclude that the detected E2 interactions for the hub E3s are in agreement with functional E2–E3 pairs reported in the literature, emphasizing the quality of interactions found in our screen.


A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system.

van Wijk SJ, de Vries SJ, Kemmeren P, Huang A, Boelens R, Bonvin AM, Timmers HT - Mol. Syst. Biol. (2009)

Quality of the E2–E3 network. Physical E2–E3 interactions in relation to literature-curated, functional E2–E3 pairs. Hub node E3s were selected and interactions with E2s were scored when biochemically tested in in vitro ubiquitination assays as reported in literature.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2736652&req=5

f5: Quality of the E2–E3 network. Physical E2–E3 interactions in relation to literature-curated, functional E2–E3 pairs. Hub node E3s were selected and interactions with E2s were scored when biochemically tested in in vitro ubiquitination assays as reported in literature.
Mentions: Comparing the E2–E3 interactions obtained in this screen with E2–E3 literature-curated interactions evaluates the quality of the interactions in the E2–E3 network. Physical interactions between E2 and E3 enzymes are required for efficient ubiquitination in vivo and in vitro and therefore for their catalytic function. Besides this, comparing the novel E2–E3 pairs generated by our screen with annotated functional E2–E3 pairs provides insight into the functionality of the E2–E3 pairs. We first selected the top 10% interacting E3s and compared their physical E2 interaction patterns with information obtained by low-throughput E2–E3 experiments. As shown in Figure 5, 25 hub E3s showed a total number of 209 E2 interactions covering 60% of the total number of E2–E3 two-hybrid interactions. For the published literature we recovered 64 E2–E3 pairs for the 25 hub E3s; 35 of the 64 pairs were positive for in vitro or in vivo ubiquitination activity and 29 were tested negative. Of the 35 positive literature interactions we confirmed 33 in our screen, which we regard as true positives (Figure 5). We found only two pairs that were biochemically reported positive, but did not occur in the Y2H screen (false negatives). The observed literature interactions were mostly concerning the members of the UbcH5 family and UBE2E1 and UBE2E3. To investigate the possibility that these E2s are more versatile in ubiquitination reactions, we compared reported E2–E3 combinations that were negative in biochemical reactions. Of the 29 E2–E3 interactions reported negative we recovered 24 in our dataset (true negatives). We also found five pairs that were reported negative in ubiquitination assays, but showed an interaction in the Y2H screen (false positives). In conclusion, this indicates a specificity of 83% (24/(5+24)) and a sensitivity of 94% (33/(2+33)) between biochemically active E2–E3 pairs and pairs found interacting in our screen. In the above analysis we focussed on the top 10% of interactors. Analysis of sensitivity and specificity of the complete data set is confounded by uncertainty of proper folding and/or localization of the Y2H proteins (Supplementary Table VI), which are potential pitfalls of Y2H screening. Therefore, we focussed on the E2 and E3s, which display at least one interaction. This criterion retains 20 E2s and 104 E3s from our screen. Literature inspection of this set showed 118 E2–E3 interactions of which 83 scored positive and 35 negative. Of the 83 positive interactions we recovered 48 interactions in our dataset. Of the 35 negative interactions 30 were also scored in Y2H interaction. This yields a sensitivity of 86% and a specificity of 58%. The verification rate of our screen (true positives plus true negatives/total interactions) is 66%, which compares well to global Y2H screens reported earlier (Li et al, 2004; Rual et al, 2005). On the basis of these data, we conclude that the detected E2 interactions for the hub E3s are in agreement with functional E2–E3 pairs reported in the literature, emphasizing the quality of interactions found in our screen.

Bottom Line: Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others.For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B).Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Genetics, Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2-E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2-E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

Show MeSH