Biochemical and genetic studies of UBR3, a ubiquitin ligase with a function in olfactory and other sensory systems

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the approximately 70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express beta-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates.     

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.     

Structural dissection of a gating mechanism preventing misactivation of ubiquitin by NEDD8's E1

Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL's C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin's Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8's heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin's E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8's residue 72 and UBA3's residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3's preference for NEDD8's Ala72 appears to be indirect, due to proper positioning of UBA3's Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin's Arg72 and an E1's Gln. However, APPBP1-UBA3's failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3's Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.     

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1(-/-) UBR2(-/-) mice die as early embryos, the rescued UBR1(-/-) UBR2(-/-) fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a approximately 70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1(-/-) UBR2(-/-) fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.     

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.     

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.     

Sequential E2s drive polyubiquitin chain assembly on APC targets

The anaphase-promoting complex (APC), or cyclosome, is an E3 ubiquitin-protein ligase that collaborates with E2 ubiquitin-conjugating enzymes to assemble polyubiquitin chains on proteins important for cell-cycle progression. It remains unclear how the APC - or many other E3s - promotes the multiple distinct reactions necessary for chain assembly. We addressed this problem by analyzing APC interactions with different E2s. We screened all budding yeast E2s as APC coenzymes in vitro and found that two, Ubc4 and Ubc1, are the key E2 partners for the APC. These proteins display strikingly different but complementary enzymatic behaviors: Ubc4 supports the rapid monoubiquitination of multiple lysines on APC targets, while Ubc1 catalyzes K48-linked polyubiquitin chain assembly on preattached ubiquitins. Mitotic APC function is lost in yeast strains lacking both Ubc1 and Ubc4. E2-25K, a human homolog of Ubc1, also promotes APC-dependent chain extension on preattached ubiquitins. We propose that sequential E2 proteins catalyze K48-linked polyubiquitination and thus proteasomal destruction of APC targets.     

E3-independent monoubiquitination of ubiquitin-binding proteins

Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitinated targets and control numerous biological processes. They themselves undergo UBD-dependent monoubiquitination, which promotes intramolecular binding of the UBD to the attached Ub and leads to their inactivation. Here, we report that, in contrast to the established ubiquitination pathway, the presence of UBDs allows the ubiquitination of host proteins independently of E3 ligases. UBDs of different types, including UBA, UIM, UBM, NFZ, and UBZ, can directly cooperate with Ub-charged E2 enzymes to promote monoubiquitination. Using FRET and siRNA technologies, we verify that Ub-loaded E2 and substrates interact in cells and that E2 enzymes are essential for their monoubiquitination in vivo. This modification is mechanistically and functionally distinct from E3-mediated and growth factor-dependent monoubiquitination.     

The E4orf6/E1B55K E3 ubiquitin ligase complexes of human adenoviruses exhibit heterogeneity in composition and substrate specificity

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin α3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin α3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.     

Determinants of functionality in the ubiquitin conjugating enzyme family

The E2 enzymes are key enzymes in the ubiquitin and ubiquitin-like protein ligation pathways. To understand the functionality of the different E2 enzymes, we analyzed 190 protein sequences and 211 structures and electrostatic potentials. Key findings include: The ScUbc1 orthologs are defined by a C-terminal UBA domain. An N-terminal sequence motif that is highly conserved in all E2s except for Cdc34 orthologs is important for the stabilization of the L7 loop and is likely to be involved in E1 binding. ScUbc11p has a different electrostatic potential from E2-Cp and other proteins with which it has high sequence similarity but different functionality. All the E2s known to ubiquitinate histones have a negative potential. The members of the NCUBE family have a positive electrostatic potential, although its form is different from that of the SUMO conjugating E2s. The specificities of only the ScUbc4/Ubc5 and ScUbc1p orthologs are reflected in their L4 and L7 loops.     

The ubiquitin pathway is required for innate immunity in Arabidopsis

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 ( snc1 ) carries a point mutation in a Resistance ( R )-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1 -mediated constitutive resistance is modifier of snc1 5 ( mos5 ), which carries a 15-bp deletion in UBA1 , one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1 , suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1 -mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1 -related R -genes RPP4 and RPP5 , are dispensable in snc1 -mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.     

Activation of UBC5 ubiquitin-conjugating enzyme by the RING finger of ROC1 and assembly of active ubiquitin ligases by all cullins

Protein ubiquitination plays an important role in regulating the abundance and conformation of a broad range of eukaryotic proteins. This process involves a cascade of enzymes including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). E1 and E2 represent two families of structurally related proteins and are relatively well characterized. In contrast, the nature and mechanism of E3, proposed to contain activities in catalyzing isopeptide bond formation (ubiquitin ligation) and substrate targeting, remains inadequately understood. Two major families of E3 ubiquitin ligases, the HECT (for homologous to E6-AP C terminus) family and the RING family, have been identified that utilize distinct mechanisms in promoting isopeptide bond formation. Here, we showed that purified RING finger domain of ROC1, an essential subunit of SKP1-cullin/CDC53-F box protein ubiquitin ligases, was sufficient to activate UBCH5c to synthesize polyubiquitin chains. The sequence flanking the RING finger in ROC1 did not contribute to UBCH5c activation, but was required for binding with CUL1. We demonstrated that all cullins, through their binding with ROC proteins, constituted active ubiquitin ligases, suggesting the existence in vivo of a large number of cullin-RING ubiquitin ligases. These results are consistent with the notion that the RING finger domains allosterically activate E2. We suggest that RING-E2, rather than cullin-RING, constitutes the catalytic core of the ubiquitin ligase and that one major function of the cullin subunit is to assemble the RING-E2 catalytic core and substrates together.     

Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1(-/-) mice, which lacked the pathway's ubiquitin ligase E3alpha, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2(-/-) mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2(-/-) genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2(-/-) females died as embryos, whereas UBR2(-/-) males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2(-/-) testes was normal and spermatogonia were intact as well, but UBR2(-/-) spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2(-/-) spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.     

Multiple forms of ubiquitin-activating enzyme E1 from wheat. Identification of an essential cysteine by in vitro mutagenesis.

Ubiquitin-activating enzyme, E1, directs the ATP-dependent formation of a thiol ester linkage between itself and ubiquitin. The energy in this bond is ultimately used to attach ubiquitin to various intracellular proteins. We previously reported the isolation of multiple E1s from wheat and the characterization of a cDNA encoding this protein (UBA1). We now report the derived amino acid sequence of two additional members of this gene family (UBA2 and UBA3). Whereas the amino acid sequence of UBA2 is nearly identical to UBA1, the sequence of UBA3 is significantly different. Nevertheless, the protein encoded by UBA3 catalyzes the ATP-dependent activation of ubiquitin in vitro. Comparison of derived amino acid sequences of genes encoding E1 from plant, yeast, and animal tissues revealed 5 conserved cysteine residues, with one potentially involved in thiol ester bond formation. To identify this essential residue, codons corresponding to each of the 5 cysteines in UBA1 were individually altered using site-directed mutagenesis. The mutagenized enzymes were expressed in Escherichia coli and assayed for their ability to activate ubiquitin. Only substitution of the cysteine at position 626 abolishes E1 activity, suggesting that this residue forms the thiol ester linkage with ubiquitin.     

Spatiotemporal regulation of c-Fos by ERK5 and the E3 ubiquitin ligase UBR1, and its biological role

c-Fos is regulated by phosphorylation and multiple turnover mechanisms. We found that c-Fos was ubiquitylated in the cytoplasm during IL-6/gp130 stimulation under MEK inhibition and sought the mechanisms involved in the regulation. We show that sustained ERK5 activity and the E3 ligase UBR1 regulate the stability and subcellular localization of c-Fos. UBR1, rapidly induced by STAT3, interacts with and ubiquitylates c-Fos in the cytoplasm for its accelerated degradation. ERK5 inhibits the nuclear export of c-Fos by phosphorylating Thr232 in the c-Fos NES(221-233) and disrupts the interaction of c-Fos with UBR1 by phosphorylating Ser32. Moreover, UBR1 depletion in HeLa cells, which constitutively express UBR1 at a high level, enhances both c-Fos expression and cell growth, whereas ERK5 depletion reduces both of them. Interestingly, an NES mutant of c-Fos, but not wild-type, substitutes ERK5 activity for HeLa cell proliferation. Thus, this spatiotemporal regulation of c-Fos by ERK5 and UBR1 is critical for the regulation of c-Fos/AP-1.     

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.