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Characterization of PUB22-interacting E2s. (A) SLCA of cLUC-PUB22 with nLUC-fused UBC1 (group III), UBC5 (group IV), UBC 9 (group VI), UBC12 (group VI), UBC17 (group VII), UBC26 (group XI), UBC28 (group VI), UBC30 (group VI), COP10, and UBC35 (group XV) fused to cLuc with nLuc-PUB22 transiently co-transformed in Arabidopsis mesophyll protoplasts. Transformation efficiencies were normalized by Renilla luciferase harboured in the vector containing the E2. Values indicate the average value of three independent biological experiments S.D. Statistically significant differences indicated by different letters were determined by one-way ANOVA and Tukey post-hoc test (p<0.05). (B) In vitro autoubiquitination assay with MBP-PUB22 in the presence of ubiquitin, Arabidopsis His-UBA1 and His-tagged E2s: UBC1 (group III), UBC5 (group IV), or UBC30 (group VI), as a positive control. Ubiquitination reactions were stopped after 2h. (C) In vitro autoubiquitination of MBP-PUB22 in the presence of ubiquitin, His-UBA1 and His-tagged UBC17 (group VII) or UBC30 (group VI), as a positive control. Ubiquitination reaction was incubated for 1h or overnight (o/n). (D) In vitro autoubiquitination of MBP-PUB22 in the presence of ubiquitin, His-UBA1 and Histagged UBC35 (group XV) with His-tagged Uev1D, or UBC30 (group VI), as a positive control. Ubiquitination reaction was stopped after 2h. Vertical line indicates high molecular weight species of polyubiquitinated PUB22. (B-D) Ubiquitination reactions were resolved in a 7% and 12% Tris-glycine PAGE. Arrowhead indicates expected size of the ubiquitin-E2 conjugate. CBB-Coomassie brilliant blue, Ububiquitin, IB-immunoblot.
Source publication
Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin-conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyse the attachment of ubiquitin and have emerged as ke...
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... confirmed the pairing between PUB22 and E2s that had been identified by BiFC, as well as COP10. By contrast, non-interacting PUB22-E2 pairs displayed low Luc activity, suggesting that they do not interact in vivo with PUB22 ( Figure 3A and S7A). ...
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... vitro autoubiquitination assays showed that UBC1, which did not interact with PUB22, was unable to mediate ubiquitination in vitro under conditions that resulted in strong UBC30- mediated autoubiquitination of PUB22 ( Figure 3B). UBC5 did not display any intrinsic activity in the control reaction without the E3. ...
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... presence of PUB22 resulted in the formation of free ubiquitin chains, but not autoubiquitination of PUB22. This effect was also observed with the Trp40Ala variant, which abrogates group VI E2- dependent activity of PUB22 ( Figure 3B). This may suggest that the Trp40Ala mutation still allows interaction of PUB22 with UBC5 and therefore, a distinct mode of interaction than with group VI E2s. ...
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... spite of interacting with PUB22 in vivo, UBC17 did not mediate in vitro autoubiquitination under the used conditions. However, the presence of PUB22 enhanced ubiquitin-bound form of UBC17 after 1h in support of interaction taking place in vitro ( Figure 3C). Overnight incubation resulted in similar levels of ubiquitin-bound form due to the inherent reactivity of UBC17 in the presence of the Trp40Ala variant ( Figure 3C). ...
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... the presence of PUB22 enhanced ubiquitin-bound form of UBC17 after 1h in support of interaction taking place in vitro ( Figure 3C). Overnight incubation resulted in similar levels of ubiquitin-bound form due to the inherent reactivity of UBC17 in the presence of the Trp40Ala variant ( Figure 3C). ...
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... to its homologues, UBC35 requires ubiquitin conjugating enzyme variant (Uev) as a cofactor for activity (34). In the presence of Uev1D, UBC35 generated free ubiquitin chains ( Figure 3D), as previously reported (34). However, in the presence of PUB22, UBC35 activity was stimulated resulting in the increased generation of high molecular chains ( Figure 3D). ...
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... the presence of Uev1D, UBC35 generated free ubiquitin chains ( Figure 3D), as previously reported (34). However, in the presence of PUB22, UBC35 activity was stimulated resulting in the increased generation of high molecular chains ( Figure 3D). ...
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... high signal levels were also observed for the pairing of PUB24 with UBC28, UBC35 and UBC17 ( Figure 4B). In contrast to PUB22 (Figure 3A), PUB20 did only display a weak signal with the tested group VI E2s UBC9, UBC12, UBC28, and UBC30 ( Figure 4A). ...
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... is conceivable that the ARM repeats, and/or their interacting proteins, including substrates, prevent the binding to some E2s by steric hindrance. In fact, most E2s that exclusively interacted with the U-box contain extended C-terminal tails in their amino acid sequence ( Figure S3). All E2s characterized so far recognize E3s through the L1 and L2 loops, as well as the N- terminal α helix 1 on the E2 surface ( Figure 2A and S3)(1). ...
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... SPA motif in loop 2 was shown to be required for binding of E2s to the U-box type E3 CHIP (53). PUB22-interacting E2s from groups VI, VII, XI and XV, contain the SPA motif ( Figure S3). However, E2s that were unable to interact with PUB22 (e.g. ...
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... E2s that were unable to interact with PUB22 (e.g. UBC9 and UBC12; Figure 1A) also contained the motif (Figure S3), suggesting that this feature is not critical for specificity, but generally required for interaction. ...
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... overall sequence conservation of the E2s belonging to group VI is very high, especially within the surfaces responsible for the interaction with the E3. Main difference in UBC9 is a 30 amino acids extension at its N-terminus ( Figure S3). In case of UBC12 the only striking feature is the replacement at position 16 of a conserved aspartic acid present in members of group VI by a histidine (3). ...
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... closest homologue in humans is UBE2W, which was shown to monoubiquitinate the N-terminus of substrates priming them for Lys63-linked polyubiquitination by UBE2N (54- 56). Accordingly, UBC17 did not support autoubiquitination of PUB22 in vitro ( Figure 3C). Nevertheless, the presence of PUB22 enhanced the levels of ubiquitin-bound UBC17, which was dependent on an intact U-box. ...
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... the tested U-box ligases most interacted with UBC35 ( Figure 3A and 4A to 4C), suggesting a general role in the modification of substrates with Lys63 chains. The exception was PUB13 that was reported to be involved in the regulation of receptor kinases (42,63). ...
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... confirmed the pairing between PUB22 and E2s that had been identified by BiFC, as well as COP10. By contrast, non-interacting PUB22-E2 pairs displayed low Luc activity, suggesting that they do not interact in vivo with PUB22 ( Figure 3A and S7A). ...
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... vitro autoubiquitination assays showed that UBC1, which did not interact with PUB22, was unable to mediate ubiquitination in vitro under conditions that resulted in strong UBC30- mediated autoubiquitination of PUB22 ( Figure 3B). UBC5 did not display any intrinsic activity in the control reaction without the E3. ...
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... presence of PUB22 resulted in the formation of free ubiquitin chains, but not autoubiquitination of PUB22. This effect was also observed with the Trp40Ala variant, which abrogates group VI E2- dependent activity of PUB22 ( Figure 3B). This may suggest that the Trp40Ala mutation still allows interaction of PUB22 with UBC5 and therefore, a distinct mode of interaction than with group VI E2s. ...
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... spite of interacting with PUB22 in vivo, UBC17 did not mediate in vitro autoubiquitination under the used conditions. However, the presence of PUB22 enhanced ubiquitin-bound form of UBC17 after 1h in support of interaction taking place in vitro ( Figure 3C). Overnight incubation resulted in similar levels of ubiquitin-bound form due to the inherent reactivity of UBC17 in the presence of the Trp40Ala variant ( Figure 3C). ...
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... the presence of PUB22 enhanced ubiquitin-bound form of UBC17 after 1h in support of interaction taking place in vitro ( Figure 3C). Overnight incubation resulted in similar levels of ubiquitin-bound form due to the inherent reactivity of UBC17 in the presence of the Trp40Ala variant ( Figure 3C). ...
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... to its homologues, UBC35 requires ubiquitin conjugating enzyme variant (Uev) as a cofactor for activity (34). In the presence of Uev1D, UBC35 generated free ubiquitin chains ( Figure 3D), as previously reported (34). However, in the presence of PUB22, UBC35 activity was stimulated resulting in the increased generation of high molecular chains ( Figure 3D). ...
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... the presence of Uev1D, UBC35 generated free ubiquitin chains ( Figure 3D), as previously reported (34). However, in the presence of PUB22, UBC35 activity was stimulated resulting in the increased generation of high molecular chains ( Figure 3D). ...
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... high signal levels were also observed for the pairing of PUB24 with UBC28, UBC35 and UBC17 ( Figure 4B). In contrast to PUB22 (Figure 3A), PUB20 did only display a weak signal with the tested group VI E2s UBC9, UBC12, UBC28, and UBC30 ( Figure 4A). ...
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... is conceivable that the ARM repeats, and/or their interacting proteins, including substrates, prevent the binding to some E2s by steric hindrance. In fact, most E2s that exclusively interacted with the U-box contain extended C-terminal tails in their amino acid sequence ( Figure S3). All E2s characterized so far recognize E3s through the L1 and L2 loops, as well as the N- terminal α helix 1 on the E2 surface ( Figure 2A and S3)(1). ...
Context 24
... SPA motif in loop 2 was shown to be required for binding of E2s to the U-box type E3 CHIP (53). PUB22-interacting E2s from groups VI, VII, XI and XV, contain the SPA motif ( Figure S3). However, E2s that were unable to interact with PUB22 (e.g. ...
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... E2s that were unable to interact with PUB22 (e.g. UBC9 and UBC12; Figure 1A) also contained the motif (Figure S3), suggesting that this feature is not critical for specificity, but generally required for interaction. ...
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... overall sequence conservation of the E2s belonging to group VI is very high, especially within the surfaces responsible for the interaction with the E3. Main difference in UBC9 is a 30 amino acids extension at its N-terminus ( Figure S3). In case of UBC12 the only striking feature is the replacement at position 16 of a conserved aspartic acid present in members of group VI by a histidine (3). ...
Context 27
... closest homologue in humans is UBE2W, which was shown to monoubiquitinate the N-terminus of substrates priming them for Lys63-linked polyubiquitination by UBE2N (54- 56). Accordingly, UBC17 did not support autoubiquitination of PUB22 in vitro ( Figure 3C). Nevertheless, the presence of PUB22 enhanced the levels of ubiquitin-bound UBC17, which was dependent on an intact U-box. ...
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Citations
... Our assay could also be used with E3 and substrates from other plant species. Additionally, our assay can also be applied to single-subunit E3 ligases by replacing the SCF components with a monomeric E3 ligase, while considering the replacement of suitable E2 [41]. ...
Ubiquitination is one of the most important post-translational modifications in eukaryotes. The ubiquitination cascade includes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The E3 ligases, responsible for substrate recognition, are the most abundant and varied proteins in the cascade and the most studied. SKP1-CUL1-F-Box (SCF)-type E3 ubiquitin ligases are multi-subunit RING (Really Interesting New Gene) E3 ubiquitin ligases, composed of CUL1 (Cullin 1), RBX1 (RING BOX 1), SKP1 (S-phase Kinase-associated Protein 1), and F-box proteins. In vitro ubiquitination assays, used for studying the specific recognition of substrate proteins by E3 ubiquitin ligases, require the purification of all components involved in the cascade, and for assays with SCF-type E3 ligases, additional proteins (several SCF complex subunits). Here, the Duet expression system was used to co-express E1, E2, ubiquitin, ubiquitylation target (substrate), and the four subunits of a SCF-type E3 ligase in E. coli. When these proteins co-exist in bacterial cells, ubiquitination occurs and can be detected by Western Blot. The effectiveness of this bacterial system for detecting ubiquitination cascade activity was demonstrated by replicating both AtSCFTIR1-mediated and human SCFFBXO28-mediated ubiquitylation in bacteria. This system provides a basic but adaptable platform for the study of SCF-type E3 ubiquitin ligases.
... Along with these genes, three other genes related to the immune system were also found to show footprints of recent selection. For example, UBP13 (Fig. 5e) encodes a ubiquitin-specific protease that is responsible for initial pathogen perception 60 , and UBC36 encodes an E2 ubiquitin-conjugating enzyme involved in dampening immune signaling 61 . The wall-associated receptor-like kinase gene, WAK2, also plays an important role in disease resistance 62 . ...
Urban greening provides important ecosystem services and ideal places for urban recreation and is a serious consideration for municipal decision-makers. Among the tree species cultivated in urban green spaces, Robinia pseudoacacia stands out due to its attractive flowers, fragrances, high trunks, wide adaptability, and essential ecosystem services. However, the genomic basis and consequences of its wide-planting in urban green spaces remains unknown. Here, we report the chromosome-level genome assembly of R. pseudoacacia, revealing a genome size of 682.4 Mb and 33,187 protein-coding genes. More than 99.3% of the assembly is anchored to 11 chromosomes with an N50 of 59.9 Mb. Comparative genomic analyses among 17 species reveal that gene families related to traits favoured by urbanites, such as wood formation, biosynthesis, and drought tolerance, are notably expanded in R. pseudoacacia. Our population genomic analyses further recover 11 genes that are under recent selection. Ultimately, these genes play important roles in the biological processes related to flower development, water retention, and immunization. Altogether, our results reveal the evolutionary forces that shape R. pseudoacacia cultivated for urban greening. These findings also present a valuable foundation for the future development of agronomic traits and molecular breeding strategies for R. pseudoacacia.
... A0A173FEH2 is an E3 ubiquitin ligase SCF complex subunit SKP1/ASK1 family protein and an ortholog of ARABIDOPSIS SKP-LIKE 2 (ASK2); this protein forms a complex with TIR1 in the SCFTIR1 complex and is required for the auxin response in Arabidopsis thaliana [41,42]. A0A1D5STP8 is a ubiquitin-conjugating enzyme E2 whose Arabidopsis ortholog UBC35, together with UBC36, positively regulates plant auxin responses [43]. Therefore, our data imply that auxin synthesis and signaling were repressed in the wheat seedling roots under relatively high-B stress. ...
Background
Boron (B) is a trace element that is essential for normal wheat development, such as root growth. In wheat, roots are important organs that absorb nutrients and water. However, at present, there is insufficient research on the molecular mechanism underlying how short-term B stress affects wheat root growth.
Methods and results
Here, the optimal concentration of B for wheat root growth was determined, and the proteomic profiles of roots under short-term B deficiency and toxicity were analyzed and compared by the isobaric tag for relative and absolute quantitation (iTRAQ) technique. A total of 270 differentially abundant proteins (DAPs) that accumulated in response to B deficiency and 263 DAPs that accumulated in response to B toxicity were identified. Global expression analysis revealed that ethylene, auxin, abscisic acid (ABA), and Ca²⁺ signals were involved in the responses to these two stresses. Under B deficiency, DAPs related to auxin synthesis or signaling and DAPs involved in calcium signaling increased in abundance. In striking contrast, auxin and calcium signals were repressed under B toxicity. Twenty-one DAPs were detected under both conditions, including RAN1 that played a core role in the auxin and calcium signals. Overexpression of RAN1 was shown to confer plant resistance to B toxicity by activating auxin response genes, including TIR and those identified by iTRAQ in this research. Moreover, growth of the primary roots of tir mutant was significantly inhibited under B toxicity.
Conclusion
Taken together, these results indicate that some connections were present between RAN1 and the auxin signaling pathway under B toxicity. Therefore, this research provides data for improving the understanding of the molecular mechanism underlying the response to B stress.
... For example, RGLG1/2 ubiquitin E3 ligase can interact with StUBC13 to form a E2-E3 complex in potato using Y2H, SLC, and BiFC technology assays, and the complex is involved in the iron deficiency response of Arabidopsis roots [30]. In this regard, future research will focus on how StUBC13-StUEV1s conjugates different E3s to form Lys-63-linked poly-Ub chains and influences target proteins activities to regulate multiple cellular processes, such as [8,[31][32][33][34][35]. ...
Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.
... Moreover, PAMP treatment leads to an increase in PUB4 accumulation, which promotes its role in the stabilization of activated BIK1. Both accumulation of PUB4 and phosphorylation of residues in the hinge region after immunostimulation are reminiscent of the stabilization mechanism first described for PUB22, in which phosphorylation inhibits autoubiquitination and degradation (Furlan et al, 2017), and may also contribute to regulate association to E2s (Kowarschik et al, 2018;Turek et al, 2018). While we reveal here that PUB4 positively regulates activated BIK1 accumulation, the E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B were recently shown to promote BIK1 activation , thus illustrating that distinct BIK1 ubiquitination events positively regulate both its accumulation and activation. ...
Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.
... UBE2V2 (also known as MMS2) is a variant protein of the E2 ubiquitin conjugation enzyme; it forms an E2 complex with UBE2N (Ubc13) that then interacts with certain E3 ubiquitin ligases to catalyze poly-ubiquitination [37]. As the fate of a protein modified by ubiquitination is determined by the specific type of ubiquitin linkage, lysine 63 (K63)linked ubiquitination was reported to regulate protein functions, such as activating proteins and promoting protein-protein interactions, and K48-linked ubiquitination generally targets substrate proteins for degradation via the proteasome [33,38]. ...
Circulating neutrophils are activated shortly after stroke and in turn affect the fate of ischemic brain tissue, and microRNAs (miRNA) participate in regulating neuroinflammation. We probed the role of neutrophilic miRNA in ischemic stroke. miR-193a-5p was decreased in circulating neutrophils of acute ischemic stroke (AIS) patients and healthy controls. In another set of AIS patients treated with recombinant tissue plasminogen activator, higher neutrophilic miR-193a-5p levels were associated with favorable outcomes at 3 months and non-symptomatic intracerebral hemorrhage. An experimental stroke model and human neutrophil-like HL-60 cells were further transfected with agomiR-193a-5p/antagomiR-193a-5p or ubiquitin-conjugating enzyme V2 (UBE2V2)-siRNA prior to model induction for in vivo and in vitro studies. Results of 2,3,5-triphenyl tetrazolium chloride staining and neurological function evaluations at post-experimental stroke showed that intravenous agomiR-193a-5p transfusion protected against ischemic cerebral injury in the acute stage and promoted neurological recovery in the subacute stage. This protective role was suggested to correlate with neutrophil N2 transformation based on the N2-like neutrophil proportions in the bone marrow, peripheral blood, and spleen of the experimental stroke model and the measurement of neutrophil phenotype-associated molecule levels. Mechanistically, analyses indicated that UBE2V2 might be a target of miR-193a-5p. Cerebral injury and neuroinflammation aggravated by miR-193a-5p inhibition were reversed by UBE2V2 silencing. In conclusion, miR-193a-5p protects against cerebral ischemic injury by restoring neutrophil N2 phenotype-associated neuroinflammation suppression, likely, in part, via UBE2V2 induction.
... The dimeric E3s RNF4 and Birc7, contact the E2 via a single protomer, and Ub is folded back onto the E2 contacting both E3 RING domains to induce the 'closed state' [33,44]. Increased reactivity of E2s, in the presence of their cognate E3, is as expected conserved in plants [45]. The closed conformation has emerged as a hallmark of activation for ubiquitin, SUMO and Nedd8 E2s [25]. ...
... Combined with the conserved features in RING/U-box and HECT E3 ligases, it clarifies how E2s interact with several E3s. Identification of physiological E2-E3 pairs has remained one of the key challenges to elucidate ubiquitination mechanisms [9,40,[45][46][47]. ...
... However, both approaches miss important cellular determining factors, such as intracellular localization, interacting cofactors, post-translational regulation, as well as other competing E2s, which may be a defining feature. An in vivo screen using U-box E3s, confirmed that one E3 interacts with various E2s, and may therefore, modify substrates with different chain types [45]. Moreover, while the U-box itself confers pairing selectivity, the substrate-interaction domain adds an additional layer of selectivity by restricting the number of interacting E2s. ...
Most research in the field of ubiquitination has focused on E3 ubiquitin ligases because they are the specificity determinants of the ubiquitination process. Nevertheless, E2s are responsible for the catalysis during ubiquitin transfer, and are therefore, at the heart of the ubiquitination process. Arabidopsis has 37 ubiquitin E2s with additional ones mediating the attachment of ubiquitin-like proteins (e.g. SUMO, Nedd8 and ATG8). Importantly, E2s largely determine the type of ubiquitin chain built, and therefore, the type of signal that decides over the fate of the modified protein, such as degradation by the proteasome (Lys48-linked ubiquitin chains) or relocalization (Lys63-linked ubiquitin chains). Moreover, new regulatory layers impinging on E2s activity, including post-translational modifications or cofactors, are emerging that highlight the importance of E2s.
... For instance, OsUBC47 showed a potential association with D3 in LC-MS analysis and formed DTT-sensitive thioester bonds with OsUb in vitro, but it could not facilitate formation of the polyubiquitination chain when incubated with the SCF D3-GFP complex purified from transgenic calli (supplemental Table 1 and supplemental Figure 4C and 4D). The Arabidopsis OsUBC47 orthologs AtUBC35/36 need to be combined with a UEV counterpart to work sufficiently in chain formation (Kowarschik et al., 2017;Turek et al., 2018), which provides new clues to further investigate the activity of OsUBC47 in coordinating with SCF D3 E3 ligase. ...
Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes. Detecting E3 ligase activity in vitro is important for exploring the molecular mechanism of protein ubiquitination. However, in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve, especially in plants, mainly due to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs. In this study, we characterized components of the rice SCFDWARF3 (SCFD3) E3 ligase, screened the coordinated E2, and reconstituted active SCFD3 E3 ligase in vitro. We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1 (eSCR) protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53, which is the key transcriptional repressor in strigolactone signaling. Finally, we replaced D3 with other F-box proteins from rice and humans, and reconstituted active eSCF E3 ligases, including eSCFGID2, eSCFFBXL18 and eSCFCDC4 E3 ligases. Together, this work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins, providing a powerful platform to study the mechanisms of multisubunit SCF E3 ligases in eukaryotes.
... Hence, the physiological significance of physical interaction between AtUbc13 and AtCPR1 re-mains to be elucidated. AtUbc13 has been found to interact with two U-box E3 ligases, AtPUB22 and AtPUB24 (Turek et al., 2018), that are involved in plant immune responses (Trujillo et al., 2008). Whether these two Ub ligases serve as cognate E3s for AtUbc13 in plant immunity and whether this process requires K63-linked polyubiquitination need to be explored. ...
... tomato (Pst) DC3000 (Song et al., 2015). Interestingly, PARylation and K63-linked ubiquitination coordinately regulate pathogen-associated molecular pattern-triggered immunity (Turek et al., 2018;Yao et al., 2021). Both ubc13a,b and parp1,2 double mutants not only display enhanced disease susceptibility but also are compromised in flg22-or SA-induced resistance to Pst DC3000. ...
Ubiquitination is one of the best known post-translational modifications in eukaryotes, in which different linkage types of polyubiquitination result in different outputs of the target proteins. Distinct from the well-characterized K48-linked polyubiquitination that usually serves as a signal for the target protein degradation, K63-linked polyubiquitination often requires a unique E2 heterodimer Ubc13-UEV and alters the target protein activity instead of degradation. This review focuses on recent advances on roles of Ubc13-UEV mediated K63-linked polyubiquitination in plant growth, development and response to environmental stresses.
... The priming mechanism can require the formation of a dimer, in which each protomer contacts ubiquitin to position it for catalysis (104). In line with this, an E2 mutant that irreversibly binds ubiquitin displays stronger interaction with PUB22, suggesting that ubiquitin contributes to E2-E3 pairing (127). However, yeast and human UFD2s are monomeric and utilize an allosteric mechanism for conformational restriction (105). ...
... Studies using in vitro autoubiquitination activity had previously shown a pairing specificity between E2 and E3s (21,65,66). A pairwise screen to identify E2s that interact with PUB22 in vivo detected 11 UBCs belonging to 4 different groups, out of 37 tested Arabidopsis E2s (127). Interaction specificity was dictated by both the U-box as well as the ARM repeats. ...
... Further analyses with a subset of E2s showed that PUB22 and the closely related PUB20 and PUB24, as well as the UND-containing PUB4, interact with UBC35 in vivo. Because UBC35 is dedicated to building Lys63-linked chains, these E3s most likely mediate the modification of substrates with this chain type (107,127). By contrast, PUB13, which was shown to control FLS2 levels and mediate BRI1 internalization, did not interact with UBC35 (127), suggesting that it may pair with other E2s to mediate endocytosis or may require activation. ...
Posttranslational modifications add complexity and diversity to cellular proteomes. One of the most prevalent modifications across eukaryotes is ubiquitination, which is orchestrated by E3 ubiquitin ligases. U-box-containing E3 ligases have massively expanded in the plant kingdom and have diversified into plant U-box proteins (PUBs). PUBs likely originated from two or three ancestral forms, fusing with diverse functional subdomains that resulted in neofunctionalization. Their emergence and diversification may reflect adaptations to stress during plant evolution, reflecting changes in the needs of plant proteomes to maintain cellular homeostasis. Through their close association with protein kinases, they are physically linked to cell signaling hubs and activate feedback loops by dynamically pairing with E2-ubiquitin-conjugating enzymes to generate distinct ubiquitin polymers that themselves act as signals. Here, we complement current knowledge with comparative genomics to gain a deeper understanding of PUB function, focusing on their evolution and structural adaptations of key U-box residues, as well as their various roles in plant cells.
Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
