A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: changing species specificity by substitution of a single residue

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.     

Plant ubiquitylation and proteolytic systems, the key elements in hormonal signaling pathways

The general function of the ubiquitylation systems is to conjugate ubiquitin to lysine residues within substrate proteins, thus targeting them for degradation by the proteasome. In Arabidopsis thaliana more than 1300 genes (approximately 5% of the proteome) encode components of the ubiquitin/26S proteasome pathway. Approximately 90% of these genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity to the ubiquitin/26S proteasome pathway. The plant E3 ubiquitin ligases comprise a large and diverse family of proteins or protein complexes containing either a HECT domain, a RING-finger or U-box domain. The SCF class of E3 ligases is the most thoroughly studied in plants because some of them participate in regulation of hormone signaling pathways. The role of the SCF is to ubiquitylate repressors of hormone response (auxin, gibberellins), whereas in response to ethylene, abscisic acid and brassinosteroids the SCF participate in degradation of positive regulators in the absence of the hormone.     

The U-box protein family in plants

The U-box is a highly conserved domain recently identified at the C terminus of yeast UFD2, an E4 ubiquitination factor. In yeast, UFD2 is the only U-box-containing protein, but there are two UFD2 homologs and several other proteins containing a U-box domain in humans. Intriguingly, a database search revealed 37 predicted proteins containing a U-box in Arabidopsis. The plant U-box (PUB) proteins form five distinct subclasses, suggesting that they play diverse roles. The ARC1 gene from Brassica, required for self-incompatibility, is currently the only PUB gene functionally characterized. Here, we discuss the characteristics and possible functions of the PUB gene family.     

Structure and biochemical function of a prototypical Arabidopsis U-box domain

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the betabetaalphabeta fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal alpha-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal alpha-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal alpha-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitin-dependent protein degradation is a prevalent regulatory mechanism in plants.     

Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis

Approximately 5% of the Arabidopsis (Arabidopsis thaliana) proteome is predicted to be involved in the ubiquitination/26S proteasome pathway. The majority of these predicted proteins have identity to conserved domains found in E3 ligases, of which there are multiple types. The RING-type E3 is characterized by the presence of a cysteine-rich domain that coordinates two zinc atoms. Database searches followed by extensive manual curation identified 469 predicted Arabidopsis RING domain-containing proteins. In addition to the two canonical RING types (C3H2C3 or C3HC4), additional types of modified RING domains, named RING-v, RING-D, RING-S/T, RING-G, and RING-C2, were identified. The modified RINGs differ in either the spacing between metal ligands or have substitutions at one or more of the metal ligand positions. The majority of the canonical and modified RING domain-containing proteins analyzed were active in in vitro ubiquitination assays, catalyzing polyubiquitination with the E2 AtUBC8. To help identity regions of the proteins that may interact with substrates, domain analyses of the amino acids outside the RING domain classified RING proteins into 30 different groups. Several characterized protein-protein interaction domains were identified, as well as additional conserved domains not described previously. The two largest classes of RING proteins contain either no identifiable domain or a transmembrane domain. The presence of such a large and diverse number of RING domain-containing proteins that function as ubiquitin E3 ligases suggests that target-specific proteolysis by these E3 ligases is a complex and important part of cellular regulation in Arabidopsis.     

An E3 ubiquitin ligase,ERECT LEAF1, functions in brassinosteroid signaling of rice

A spontaneous rice mutant, erect leaf1 (elf1–1), produced a dwarf phenotype with erect leaves and short grains. Physiological analyses suggested that elf1–1 is brassinosteroid-insensitive, so we hypothesized that ELF1 encodes a positive regulator of brassinosteroid signaling. ELF1, identified by means of positional cloning, encodes a protein with both a U-box domain and ARMADILLO (ARM) repeats. U-box proteins have been shown to function as E3 ubiquitin ligases; in fact, ELF1 possessed E3 ubiquitin ligase activity in vitro. However, ELF1 itself does not appear to be polyubiquitinated. Mutant phenotypes of 2 more elf1 alleles indicate that the entire ARM repeats is indispensable for ELF1 activity. These results suggest that ELF1 ubiquitinates target proteins through an interaction mediated by ARM repeats. Similarities in the phenotypes of elf1 and d61 mutants (mutants of brassinosteroid receptor gene OsBRI1), and in the regulation of ELF1 and OsBRI1 expression, imply that ELF1 functions as a positive regulator of brassinosteroid signaling in rice.     

The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases.     

U-box proteins as a new family of ubiquitin ligases

Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families. The U-box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. We recently showed that mammalian U-box proteins, in conjunction with an E1 and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3. U-box proteins have thus been defined as a third family of E3s. We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins.     

C-terminal armadillo repeats are essential and sufficient for association of the plant U-box armadillo E3 ubiquitin ligase SAUL1 with the plasma membrane

Ubiquitination plays important roles in plant growth and development. Whereas ubiquitin-dependent protein degradation and modulation in the cytoplasm and nucleus are well established in plants, ubiquitination events mediated by E3 ubiquitin ligases at the plasma membrane are largely unknown. Here, it is demonstrated that the suppressor of premature senescence and cell death SENESCENCE-ASSOCIATED UBIQUITIN LIGASE 1 (SAUL1), a plant U-box armadillo repeat (PUB-ARM) E3 ubiquitin ligase, localizes at the plasma membrane. Among the members of the PUB-ARM protein family, this localization is unique to SAUL1 and its two closest homologues. A novel armadillo repeat domain was identified at the SAUL1 C-terminus that directs specific association with the plasma membrane and is crucial for SAUL1 function in vivo. The data suggest that a small subgroup of PUB-ARM proteins including SAUL1 have functions at the plasma membrane probably by modifying target proteins by ubiquitination.     

Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity

The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.     

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.     

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.     

Arabidopsis AtCUL3a and AtCUL3b form complexes with members of the BTB/POZ-MATH protein family

The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RING-finger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.     

Genome-Wide Survey and Expression Analysis of Chlamydomonas reinhardtii U-box E3 Ubiquitin Ligases (CrPUBs) Reveal a Functional Lipid Metabolism Module

E3 ubiquitin ligases determine the substrate specificity of ubiquitination. Plant U-box (PUB) E3 ligases, with a typical 70-amino acid U-box domain, participate in plant developmental processes and environmental responses. Thus far, 64 PUB proteins have been identified in Arabidopsis and 77 PUB proteins have been identified in Oryza. However, detailed studies on U-box genes in the model microalgae Chlamydomonas reinhardtii are lacking. Here, we present a comprehensive analysis of the genes encoding U-box family proteins in C. reinhardtii. Following BLASTP analysis, 30 full-length U-box genes were identified in the C. reinhardtii genome sequence. Bioinformatics analyses of CrPUB genes were performed to characterize the phylogenetic relationships, chromosomal locations and gene structures of each member. The 30 identified CrPUB proteins are clustered into 3 distinct subfamilies, and the genes for these proteins are unevenly distributed among 14 chromosomes. Furthermore, the quantitative real-time RT-PCR or semi-quantitative RT-PCR analysis of 30 CrPUB mRNA abundances under nitrogen starvation showed that 18 CrPUB genes were induced by N starvation and that 7 genes were repressed in the N-poor environment. We selected five CrPUB genes exhibiting marked changes in expression under N-free conditions for further analysis in RNAi experiments and examined the oil content of these gene-silenced transgenic strains. The silencing of CrPUB5 and CrPUB14, which are typically down-regulated under N starvation, induced 9.8%-45.0% and 14.4%-61.8% lipid accumulation, respectively. In contrast, the silencing of CrPUB11, CrPUB23 and CrPUB28, which are markedly up-regulated under N-free conditions, decreased the lipid content by 5.5%-27.8%, 8.1%-27.3% and 6.6%-27.9%, respectively. These results provide a useful reference for the identification and functional analysis of this gene family and fundamental information for microalgae lipid metabolism research.     

Six amino acid changes confined to the leucine-rich repeat beta-strand/beta-turn motif determine the difference between the P and P2 rust resistance specificities in flax

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of approximately 150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G-->E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted beta-strand/beta-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.     

NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

Interaction of the ring finger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes

The U-box domain has been suggested to be a modified RING finger motif where the metal-coordinating cysteines and histidines have been replaced with other amino acids. Known U-box-containing proteins have been implicated in the ubiquitin/proteasome system. In a search for proteins interacting with the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-box containing protein, termed UIP5, that is exclusively found in the nucleus as part of a nuclear dot-like structure. Interaction between UbcM4 and UIP5 was observed in vivo and in vitro with bacterially expressed proteins. In addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analysis showed that the U-box, like the RING finger in other proteins, forms the physical basis for the interaction with E2 enzymes. Further support for the structural similarity between U-box and RING finger comes from the observation that, in both cases, the same regions within the UbcM4 molecule are required for interaction. Our results establish at the molecular level a link between the U-box and the ubiquitin conjugating system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.