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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.

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Related in: MedlinePlus

Outline array based, mating yeast two-hybrid matrix screen. (A) Organization of LexA–E2 fusions as three independent bacterial triplicate clones and an empty LexA vector. (B) LexA–E2 protein expression levels. Total protein lysates of yeast cells transformed with LexA–E2 fusions were resolved on SDS–PAGE, transferred to membranes and probed with either LexA antibody or yeast tubulin (TUB1). (C) Overview of experimental yeast two-hybrid screening procedure. EGY48α cells were transformed with 36 LexA–E2 fusions and EGY48a cells were transformed with 250 B42–RING-fusions. Screening for pair-wise E2–E3 interactions was carried out by standardized array-based mating between EGY48α and EGY48a cells on YPD for 24 h at 30°C, followed by diploid selection on SC HWU− for 48 h at 30°C. Interactions were visualized by transferring diploids on SC HWU− X-Gal (colorimetric selection) or on SC HWUL− (auxotrophic selection) under both B42-inducing (galactose as main carbon source) and repressive (glucose) conditions. Digital images of the interactions were taken at 3, 24-h interval time points. (D) Overview of standardized LexA–E2 array. Each spot represents EGY48α cells transformed with the indicated LexA–E2 fusion construct. Spots with a red square contained LexA–UBE2D2 and LexA–UBE2D2 K63E constructs and were mated throughout the entire procedure with EGY48a cells transformed with B42–CNOT4 N63 as positive and negative interaction controls, respectively. Spots with a green square were transformed with LexA–Caf40 and LexA–CNOT2-15 and served as auto-activating controls. Blue squared spots represent either EGY48a cells transformed with LexA-empty vector and an untransformed EGY48α strain, as mating/growing controls.
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f1: Outline array based, mating yeast two-hybrid matrix screen. (A) Organization of LexA–E2 fusions as three independent bacterial triplicate clones and an empty LexA vector. (B) LexA–E2 protein expression levels. Total protein lysates of yeast cells transformed with LexA–E2 fusions were resolved on SDS–PAGE, transferred to membranes and probed with either LexA antibody or yeast tubulin (TUB1). (C) Overview of experimental yeast two-hybrid screening procedure. EGY48α cells were transformed with 36 LexA–E2 fusions and EGY48a cells were transformed with 250 B42–RING-fusions. Screening for pair-wise E2–E3 interactions was carried out by standardized array-based mating between EGY48α and EGY48a cells on YPD for 24 h at 30°C, followed by diploid selection on SC HWU− for 48 h at 30°C. Interactions were visualized by transferring diploids on SC HWU− X-Gal (colorimetric selection) or on SC HWUL− (auxotrophic selection) under both B42-inducing (galactose as main carbon source) and repressive (glucose) conditions. Digital images of the interactions were taken at 3, 24-h interval time points. (D) Overview of standardized LexA–E2 array. Each spot represents EGY48α cells transformed with the indicated LexA–E2 fusion construct. Spots with a red square contained LexA–UBE2D2 and LexA–UBE2D2 K63E constructs and were mated throughout the entire procedure with EGY48a cells transformed with B42–CNOT4 N63 as positive and negative interaction controls, respectively. Spots with a green square were transformed with LexA–Caf40 and LexA–CNOT2-15 and served as auto-activating controls. Blue squared spots represent either EGY48a cells transformed with LexA-empty vector and an untransformed EGY48α strain, as mating/growing controls.

Mentions: It has been estimated earlier that within the human genome, 30–40 E2s are present (Pickart, 2001). To precisely annotate the complete human E2 superfamily, we searched human cDNA databases for candidates. Our search showed 52 E2 family members that contain the UBC-fold (Supplementary Table I). Of these 52 E2s, we identified 16 pseudogenes for which no expressed protein-coding mRNAs could be found. We also identified an additional E2-like enzyme, UBE2N-like, lacking the central ubiquitin-accepting cysteine. Among the remaining 35 enzymes, three E2s were annotated as putative E2s and no ubiquitin(-like)-conjugating activity has been reported for these (UBE2D4, UBE2W and UBE2U) so far. Recently, it has been shown that UBE2F has the ability to act as a second NEDD8 E2 and prefers interaction with the Rbx2 RING protein, adding divergence to the NEDDylation system (Huang et al, 2009). So at this moment, 31 E2 enzymes are described to be involved in the process of ubiquitin or ubiquitin-like protein conjugation. Among these E2s, there are well-known examples with biochemically active roles in protein ubiquitination, such as UBE2D2(UbcH5B) and UBE2L3(UbcH7) (Shimura et al, 2000; Ozkan et al, 2005). Apart from the wild-type (WT) E2s, we also included the UbcH5B(K63E) mutant, which has been shown earlier to fail to interact with the WT CNOT4 RING-finger, but showed an altered-specificity interaction with the D48K/E49K double mutated CNOT4 (Winkler et al, 2004). Using multiple sequence alignments, we determined the boundaries of the UBC-fold. These 36 UBC-folds were fused to the C-terminus of LexA, DNA sequence verified, transformed in MATα cells and arranged in a standardized array (BD–E2 array) (Figure 1B and D).


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)

Outline array based, mating yeast two-hybrid matrix screen. (A) Organization of LexA–E2 fusions as three independent bacterial triplicate clones and an empty LexA vector. (B) LexA–E2 protein expression levels. Total protein lysates of yeast cells transformed with LexA–E2 fusions were resolved on SDS–PAGE, transferred to membranes and probed with either LexA antibody or yeast tubulin (TUB1). (C) Overview of experimental yeast two-hybrid screening procedure. EGY48α cells were transformed with 36 LexA–E2 fusions and EGY48a cells were transformed with 250 B42–RING-fusions. Screening for pair-wise E2–E3 interactions was carried out by standardized array-based mating between EGY48α and EGY48a cells on YPD for 24 h at 30°C, followed by diploid selection on SC HWU− for 48 h at 30°C. Interactions were visualized by transferring diploids on SC HWU− X-Gal (colorimetric selection) or on SC HWUL− (auxotrophic selection) under both B42-inducing (galactose as main carbon source) and repressive (glucose) conditions. Digital images of the interactions were taken at 3, 24-h interval time points. (D) Overview of standardized LexA–E2 array. Each spot represents EGY48α cells transformed with the indicated LexA–E2 fusion construct. Spots with a red square contained LexA–UBE2D2 and LexA–UBE2D2 K63E constructs and were mated throughout the entire procedure with EGY48a cells transformed with B42–CNOT4 N63 as positive and negative interaction controls, respectively. Spots with a green square were transformed with LexA–Caf40 and LexA–CNOT2-15 and served as auto-activating controls. Blue squared spots represent either EGY48a cells transformed with LexA-empty vector and an untransformed EGY48α strain, as mating/growing controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Outline array based, mating yeast two-hybrid matrix screen. (A) Organization of LexA–E2 fusions as three independent bacterial triplicate clones and an empty LexA vector. (B) LexA–E2 protein expression levels. Total protein lysates of yeast cells transformed with LexA–E2 fusions were resolved on SDS–PAGE, transferred to membranes and probed with either LexA antibody or yeast tubulin (TUB1). (C) Overview of experimental yeast two-hybrid screening procedure. EGY48α cells were transformed with 36 LexA–E2 fusions and EGY48a cells were transformed with 250 B42–RING-fusions. Screening for pair-wise E2–E3 interactions was carried out by standardized array-based mating between EGY48α and EGY48a cells on YPD for 24 h at 30°C, followed by diploid selection on SC HWU− for 48 h at 30°C. Interactions were visualized by transferring diploids on SC HWU− X-Gal (colorimetric selection) or on SC HWUL− (auxotrophic selection) under both B42-inducing (galactose as main carbon source) and repressive (glucose) conditions. Digital images of the interactions were taken at 3, 24-h interval time points. (D) Overview of standardized LexA–E2 array. Each spot represents EGY48α cells transformed with the indicated LexA–E2 fusion construct. Spots with a red square contained LexA–UBE2D2 and LexA–UBE2D2 K63E constructs and were mated throughout the entire procedure with EGY48a cells transformed with B42–CNOT4 N63 as positive and negative interaction controls, respectively. Spots with a green square were transformed with LexA–Caf40 and LexA–CNOT2-15 and served as auto-activating controls. Blue squared spots represent either EGY48a cells transformed with LexA-empty vector and an untransformed EGY48α strain, as mating/growing controls.
Mentions: It has been estimated earlier that within the human genome, 30–40 E2s are present (Pickart, 2001). To precisely annotate the complete human E2 superfamily, we searched human cDNA databases for candidates. Our search showed 52 E2 family members that contain the UBC-fold (Supplementary Table I). Of these 52 E2s, we identified 16 pseudogenes for which no expressed protein-coding mRNAs could be found. We also identified an additional E2-like enzyme, UBE2N-like, lacking the central ubiquitin-accepting cysteine. Among the remaining 35 enzymes, three E2s were annotated as putative E2s and no ubiquitin(-like)-conjugating activity has been reported for these (UBE2D4, UBE2W and UBE2U) so far. Recently, it has been shown that UBE2F has the ability to act as a second NEDD8 E2 and prefers interaction with the Rbx2 RING protein, adding divergence to the NEDDylation system (Huang et al, 2009). So at this moment, 31 E2 enzymes are described to be involved in the process of ubiquitin or ubiquitin-like protein conjugation. Among these E2s, there are well-known examples with biochemically active roles in protein ubiquitination, such as UBE2D2(UbcH5B) and UBE2L3(UbcH7) (Shimura et al, 2000; Ozkan et al, 2005). Apart from the wild-type (WT) E2s, we also included the UbcH5B(K63E) mutant, which has been shown earlier to fail to interact with the WT CNOT4 RING-finger, but showed an altered-specificity interaction with the D48K/E49K double mutated CNOT4 (Winkler et al, 2004). Using multiple sequence alignments, we determined the boundaries of the UBC-fold. These 36 UBC-folds were fused to the C-terminus of LexA, DNA sequence verified, transformed in MATα cells and arranged in a standardized array (BD–E2 array) (Figure 1B and D).

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
Related in: MedlinePlus