The basis for selective E1-E2 interactions in the ISG15 conjugation system

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.     

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184–196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r1^1–183 (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1C^YGY) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1C^C93S-[¹⁵N]UB^K48R oxyester displayed two-state conformational exchange, whereas the Ube2r1C^C93S/YGY-[¹⁵N]UB^K48R oxyester showed predominantly one state. Together with NMR studies that compared UB^K48R oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity.     

Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μM in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ～3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.     

Mammalian E4 is required for cardiac development and maintenance of the nervous system

Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a(-/-) mouse embryonic fibroblasts. Furthermore, Ufd2a(+/-) mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.     

A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.     

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.     

A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a "non-removable" N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting the first example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.     

The ubiquitin-protein ligases Nedd4 and Nedd4-2 show similar ubiquitin-conjugating enzyme specificities

Nedd4 and Nedd4-2 are closely related HECT-type ubiquitin-protein ligases (E3) implicated in the regulation of a number of proteins and pathways. Given the close homology between these E3 enzymes it would be predicted that a conserved ubiquitin-conjugating enzyme (E2) specificity exists between the two proteins. However, E2 specificities for Nedd4 and Nedd4-2 are not well established. In the present studies we aimed at clarifying the E2-specificities of Nedd4 and Nedd4-2 using in vitro ubiquitination assays. We demonstrate strong substrate ubiquitination in the presence of UbcH5b by both Nedd4 and Nedd4-2. We also found that Ube2e3, an E2 previously shown to be used by Nedd4-2, is used less efficiently than UbcH5b. Our results suggest that for optimal ubiquitination Nedd4 and Nedd4-2 require the same E2 enzymes.     

Mulan E3 ubiquitin ligase interacts with multiple E2 conjugating enzymes and participates in mitophagy by recruiting GABARAP

Mulan is an E3 ubiquitin ligase embedded in the outer mitochondrial membrane (OMM) with its RING finger facing the cytoplasm and a large domain located in the intermembrane space (IMS). Mulan is known to have an important role in cell growth, cell death, and more recently in mitophagy. The mechanism of its function is poorly understood; but as an E3 ligase it is expected to interact with specific E2 ubiquitin conjugating enzymes and these complexes will bind and ubiquitinate specific substrates. The unique topology of Mulan can provide a direct link of communicating mitochondrial signals to the cytoplasm. Our studies identified four different E2 conjugating enzymes (Ube2E2, Ube2E3, Ube2G2 and Ube2L3) as specific interactors of Mulan. Each of these E2 conjugating enzymes was fused to the RING finger domain of Mulan and used in a modified yeast two-hybrid screen. Several unique interactors for each Mulan–E2 complex were isolated. One such specific interactor of Mulan–Ube2E3 was the GABARAP (GABA_A receptor-associated protein). GABARAP is a member of the Atg8 family of proteins that plays a major role in autophagy/mitophagy. The interaction of GABARAP with Mulan–Ube2E3 required an LC3-interacting region (LIR) located in the RING finger domain of Mulan as well as the presence of Ube2E3. The isolation of four different E2 conjugating enzymes, as specific partners of Mulan E3 ligase, suggests that Mulan is involved in multiple biological pathways. In addition, the interaction of GABARAP with Mulan–Ube2E3 supports the role of Mulan as an important regulator of mitophagy and provides a plausible mechanism for its function in this process.     

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.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

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.     

大鼠睾丸特异表达基因Ube1的分离鉴定及生物学特征

本研究采用抑制性消减杂交(suppression subtracfive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因Ube1 (GenBank登录号EF690356).该基因序列全长3433 bp,其中开放阅读框有3171 bp,编码一个含1057个氨基酸的蛋白质.Blast比对显示,Ube1与小鼠泛素激活酶基因Ubely1的同源性为93%,与人泛素激活酶基因UBE1的同源性为82%.Ube1基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点,这些位点也存在于人类和小鼠的泛素激活酶1中.RT-PCR分析显示,Ube1在睾丸中大量表达,而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达.荧光定量PCR分析不同生精细胞中Ube1的表达,显示Ube1在A型精原细胞中大量表达,在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达.以上结果提示,Ube1是大鼠睾丸特异表达基因,可能通过参与泛素/蛋白酶体途径来影响精子发生.     

The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.     

NMR assignments of ubiquitin fold domain (UFD) in SUMO-activating enzyme subunit 2 from rice

The small ubiquitin-related modifier (SUMO) is a ubiquitin-like post-translational modifier that alters the localization, activity, or stability of many proteins. In the sumoylation process, an activated SUMO is transferred from SUMO-activating enzyme E1 complex (SAE1/SAE2) to SUMO-conjugating enzyme E2 (Ubc9). Among the multiple domains in E1, a C-terminal ubiquitin fold domain (UFD) of SAE2 shows high affinity for Ubc9, implying that UFD will be functionally important. We report NMR chemical shift assignments of UFD in SAE2 from rice. Almost all the resonances of UFD were assigned uniquely, representing a single conformation of UFD in solution. This is a contrast to the previous report for the corresponding UFD of human SAE2 which shows two conformational states. The secondary structure prediction of UFD in rice SAE2 shows the similar overall structure to the crystal structures of UFD in other E1 proteins such as SAE2 of human and yeast, ubiquitin-activating enzyme of yeast, and NEDD8-activating enzyme E1 catalytic subunit of human. Concomitantly, differences in the length of helices, strands, and loops are observed, particularly in the binding region to E2, supposing the variation in the UFD-E2 binding mode which may play a critical role in determining E1-E2 specificity.     

The Ubiquitin-conjugating Enzyme (E2) Ube2w Ubiquitinates the N Terminus of Substrates

Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification, we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH₂ group as the site of conjugation. To confirm N-terminal ubiquitination, we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked, ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein Tau. Finally, we directly sequenced the N terminus of unmodified and ubiquitinated ataxin-3, demonstrating that Ube2w attaches ubiquitin to the N terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N terminus of substrates.