Interaction Between Syntaxin 8 and HECTd3, a HECT Domain Ligase

Ubiquitination of proteins and their degradation within the proteasome has emerged as the major proteolytic mechanism used by mammalian cells to regulate cytosolic and nuclear protein levels. Substrate ubiquitylation is mediated by ubiquitin (Ub) ligases, also called E3 Ub ligases. HECT-E3 Ub ligases are characterized by the presence of a C-terminal HECT domain that contains the active site for Ub transfer onto substrates. Among the many E3 Ub ligases, the family homologous to E6-Ap C-terminus (HECT) E3 Ub ligases, which includes the yeast protein Rsp5p and the mammalian homolog NEDD4, AIP4/Itch, and Smurf, has been shown to ubiquitylate membrane proteins and, in some instances, to induce their degradation. In this report, we have identified Syntaxin 8 as a binding protein to a novel HECT domain protein, HECT domain containing 3 (HECTd3), by yeast two-hybrid screen. Besides HECT domain, HECTd3 contains an anaphase-promoting complex, subunit 10 (APC10) domain. Our co-immunoprecipitation experiments show that Syntaxin 8 directly interacts with HECTd3 and that the overexpression of HECTd3 promotes the ubiquitination of Syntaxin 8. Immunofluorescence results show that Syntaxin 8 and HECTd3 have similar subcellular localization.     

HECT类泛素连接酶NEDL1和NEDL2的功能研究进展

泛素化能够促使底物蛋白降解或调节其它生理过程,在生命活动中具有极其重要的作用。E3即泛素连接酶,在泛素化过程中决定底物分子的特异性,因此,E3的功能研究一直是蛋白质泛素化研究领域的一个热点。NEDL1和NEDL2是HECT类泛素连接酶NEDD4家族中同源性较高的两个成员。它们通过不同的方式分别增强p53和p73的转录活性。NEDL1又与多种肿瘤(如神经母细胞瘤、结直肠癌、乳腺癌)和神经退行性疾病(如脊髓侧索硬化病)的发生发展密切相关。因此,对NEDL1和NEDL2的研究对于揭示相关疾病机理具有非常重要的意义。     

The HECT Domain of the Ubiquitin Ligase Rsp5 Contributes to Substrate Recognition

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.     

HECT Domain-containing E3 Ubiquitin Ligase Nedd4 Interacts with and Ubiquitinates Sprouty2

Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and Spry2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The Spry2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser¹¹² and Ser¹²¹, on Spry2. The phospho-Ser^112/121 region on Spry2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate Spry2 via Lys⁴⁸ on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.     

人泛素连接酶AREL1催化结构域的晶体结构

泛素化是一种重要的翻译后修饰,几乎调控着生命活动的所有方面.泛素连接酶是泛素化过程中唯一对底物蛋白质有特异性识别能力的一类酶,它们在泛素化过程中是不可或缺的,起到非常关键的作用.人抗凋亡E3泛素连接酶(AREL1)是HECT泛素连接酶家族成员之一,它能够泛素化促凋亡蛋白SMAC、HtrA2和ARTS,并通过蛋白酶体将它们降解,从而发挥抵抗细胞凋亡的作用.本文解析了3.2?分辨率的人AREL1蛋白催化结构域(AREL1^HECT)的晶体结构,并将其与HECT家族中其他成员的结构进行了比对.尺寸排阻色谱和X射线小角散射的结果表明,AREL1^HECT在溶液中是以多种聚集状态形式存在的,小角散射的3D模型进一步表明AREL1^HECT在溶液中会发生二聚化.这些结果将为AREL1^HECT与泛素复合物结构的解析及功能的分析提供坚实的结构基础,为揭示AREL1泛素化底物蛋白质的分子机制提供重要的依据.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.The tripeptide, γ-glutamylcysteinylglycine or GSH, is the most abundant intracellular nonprotein thiol. It serves multiple physiological functions, including maintenance of redox homeostasis, providing reducing equivalents for the elimination of reactive oxygen species, protection against electrophilic xenobiotics, maintenance of protein structure, and storage/transport of l-cysteine. GSH is synthesized de novo by two ATP-dependent consecutive reactions: ligation of l-glutamate to l-cysteine by the activity of glutamate-cysteine ligase (GCL²; EC 6.3.2.2), a rate-limiting step in the pathway, followed by the coupling of glycine to γ-glutamylcysteine by glutathione synthase (EC 6.3.2.3). The GCL holoenzyme is heterodimeric, consisting of a catalytic (GCLc) and a modifier (GCLm) subunit, each encoded by a unique gene. The intracellular ratios of GLCc and GCLm are, however, not necessarily equimolar and may be altered under conditions of oxidative stress. Although GCLc by itself is fully competent to catalyze the biosynthesis of GSH (1), dimerization with GCLm lowers the K_m for glutamate and also decreases sensitivity to feedback inhibition by GSH (2).Overexpression of GCL subunits in cultured cells has been reported to increase GSH production and confer enhanced protection against oxidative stress (3), apoptosis (4), and oxidant-induced DNA lesions (5). We have shown that overexpressions of GCLc or GCLm boost GSH biosynthesis and extend life span in Drosophila melanogaster (6). In contrast, inhibition of GCL activity results in decreased GSH levels, enhanced susceptibility to oxidative or nitrosative stress, increased DNA damage, and cell cycle arrest (7, 8). The homozygous knock-out for the catalytic GCLc subunit is embryonic lethal (9).GSH is predominantly synthesized in the cytoplasm, but its levels greatly vary among the different intracellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cytosol (10–12). Currently available evidence suggests the existence of two alternate mechanisms for the differential distribution of GSH in subcellular compartments: (i) GSH may first be synthesized in the cytoplasm and then transported into the organelles either actively or via passive diffusion; (ii) GSH may be synthesized in those organelles that display activities of GCL and glutathione synthase (13, 14).It has been demonstrated that GSH levels in the nucleus of proliferating cells are much higher than those in the confluent cells (15); however, the mechanism underlying this variation is presently unclear. In the present study, we provide evidence that GSH is synthesized in the nucleus and that this synthesis is dependent upon the shuttling of the GCLc, but not the GCLm, subunit from the cytoplasm to the nucleus. The ability of GCLc to migrate into the nucleus is due to the presence of a nuclear localization signal (NLS).     

Functional dissection of a HECT ubiquitin E3 ligase

Ubiquitination is one of the most prevalent protein post-translational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here we used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4, in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, demonstrated that an accumulation of Lys-63-linked polyubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally we showed that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.     

Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.     

The E3 Ubiquitin Ligase cIAP1 Binds and Ubiquitinates Caspase-3 and -7 via Unique Mechanisms at Distinct Steps in Their Processing

Inhibitor of apoptosis (IAP) proteins are widely expressed throughout nature and suppress cell death under a variety of circumstances. X-linked IAP, the prototypical IAP in mammals, inhibits apoptosis largely through direct inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once thought to also inhibit caspases, but more recent studies have suggested otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the proteolytic activities of effector caspases on fluorogenic or endogenous substrates. However, cIAP1 does bind to caspase-3 and -7 and does so, remarkably, at distinct steps prior to or following the removal of their prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif, AKPD, on the N terminus of the large subunit of fully mature caspase-7, whereas cIAP1 bound to partially processed caspase-3 in a manner that required its prodomain and cleavage between its large and small subunits but did not involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier protein/ubiquitin-conjugating enzymes), and in the case of caspase-3, differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric caspase-3 (bearing the AKPD motif) were degraded in vivo in a proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least in part, by facilitating the ubiquitination and turnover of active effector caspases in cells.Apoptosis is a programmed form of cell death that is generally executed through the activation of caspases,² cysteine proteases that exhibit an almost absolute preference for cleavage after aspartate residues. Caspases are synthesized as single-chain zymogens, containing a prodomain, as well as large and small subunits that include residues required for substrate recognition and cleavage (1). During death receptor or mitochondria-dependent apoptosis, the long prodomain-containing initiator caspase-8/10 and -9 are recruited via their adapter proteins, Fas-associated death domain and apoptotic protease-activating factor-1 (Apaf-1), to multimeric caspase-activating complexes known as the death-inducing signaling complex and the apoptosome, respectively (1, 2). In the latter case, mitochondrial outer membrane permeabilization (MOMP) is required to mediate the release of cytochrome c from the intermembrane space into the cytosol, where it stimulates dATP/ATP-dependent oligomerization of Apaf-1 into the apoptosome (2). Once recruited, all initiator caspases are concentrated within their respective complexes and are thought to be activated as a result of dimerization, with concomitant autocatalytic cleavage of the activation loops that separate their large and small subunits (1). However, unlike caspase-8 and -10, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity, so that in addition to promoting dimerization, the apoptosome may also induce conformational changes in caspase-9 that are necessary for its activation (3–6).In contrast to initiator caspases, effector caspases, such as caspase-3 and -7, contain short prodomains and exist normally as latent dimers, wherein their activation loops sterically hinder substrate access and hold the substrate binding pocket in an inactive conformation (1). Effector caspases are directly activated by caspase-8, -9, and -10, and following cleavage of caspase-3 between its large and small subunits, the two-chain p20/p12 form becomes a catalytically active heterotetramer and undergoes subsequent autocatalytic processing between its prodomain and large subunits to generate the fully mature p17/p12 form of the enzyme (7). Similarly, procaspase-7 is also activated following cleavage of its activation loop to generate its two-chain p22/p12 form; however, it remains unclear whether removal of its prodomain in cells (to generate its p19/p12 form) is accomplished primarily via autocatalysis, active caspase-3, or perhaps by serine proteases at a non-aspartate residue (8, 9). Caspase-3 and -7 exhibit significant sequence and structural homology, differing primarily in their short prodomains. Despite this fact, caspase-3 processes a wider array of protein substrates during apoptosis and is largely responsible for dismantling the cell (10). Thus, interesting questions remain regarding the physiological roles of caspase-7, whether caspase-7 activity is differentially regulated compared with caspase-3, and what structural features determine (and in some cases limit) its substrate specificity.Given the devastating consequences of unfettered caspase activation, cells have evolved mechanisms to regulate caspase activity. For example, IAPs, originally identified in baculoviruses, possess one or more baculovirus IAP repeat (BIR) domains, and at least one of the eight family members, XIAP, selectively inhibits the activities of caspase-9, -3, and -7 (1, 11). Mechanistically, the BIR3 domain in XIAP binds to an exposed IBM on the N terminus of the small subunit of processed caspase-9, situated directly above the active site, and limits the access of substrates (12, 13). By contrast, the linker region (located between the BIR1 and BIR2 domains in XIAP) lies across the active sites of caspase-3 and -7 and binds in a reverse orientation to substrates, thereby preventing cleavage of the linker while simultaneously preventing the access of substrates (14, 15). The BIR2 domain then stabilizes the linker-caspase-3 (and linker-caspase-7) interactions further by binding to an exposed IBM on the N terminus of the small subunit in the adjacent caspase dimer (14, 16). Importantly, IAP antagonists, such as Smac/DIABLO and Omi/HtrA2, are normally sequestered to the intermembrane space of mitochondria and are released (along with cytochrome c) into the cytoplasm during apoptosis. As IAP antagonists also possess IBMs, they bind to BIR domains and prevent or relieve the inhibition of caspases by IAPs (1).Previously, two additional IAP family members, cIAP1 and cIAP2, were also thought to inhibit caspases, but more recent studies suggest that these IAPs bind but do not inhibit caspases (17–19). Nevertheless, various studies have shown that cIAPs can protect cells from apoptosis, are overexpressed or mutated in some cancers, and can promote tumorigenesis (20–25), raising questions as to how these IAPs inhibit cell death or whether they have additional functions (26). XIAP, cIAP1, and cIAP2 possess C-terminal RING zinc finger domains with E3 ubiquitin (Ub) ligase activities capable of catalyzing the ubiquitination and subsequent proteasomal degradation of cellular targets, including themselves (27, 28). Moreover, cIAPs have been shown to ubiquitinate several factors, including TNF receptor-associated factor 2, the serine/threonine kinase NIK, receptor-interacting protein 1, and the IAP antagonist Smac (29–34). However, although there is some evidence to support a direct role for ubiquitination in the regulation of effector caspases by XIAP (35, 36), the role of cIAPs in this process remains unclear, particularly in vivo. We demonstrate herein that cIAP1 binds to caspase-3 and -7 at unique steps in their processing, prior to or following the removal of their prodomains, respectively. Moreover, rather than directly inhibiting these effector caspases, cIAP1 ubiquitinates them and targets them for proteasome-dependent degradation, thereby suppressing apoptosis.     

The Structure of the Catalytic Domain of a Plant Cellulose Synthase and Its Assembly into Dimers

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.     

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family

There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.     

Promiscuous Interactions of gp78 E3 Ligase CUE Domain with Polyubiquitin Chains

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Highlights? The ubiquitin ligase gp78 recruits ubiquitinated substrates via its CUE domain ? gp78CUE:Ub complex reveals a large set of specific interactions ? gp78CUE binds to both distal and proximal moities of diubiquitin in a similar fashion ? The gp78CUE domain binds to K48- and K63-linked diubiquitin equally well     

Differential Affinity and Catalytic Activity of CheZ in E. coli Chemotaxis

Push–pull networks, in which two antagonistic enzymes control the activity of a messenger protein, are ubiquitous in signal transduction pathways. A classical example is the chemotaxis system of the bacterium Escherichia coli, in which the kinase CheA and the phosphatase CheZ regulate the phosphorylation level of the messenger protein CheY. Recent experiments suggest that both the kinase and the phosphatase are localized at the receptor cluster, and Vaknin and Berg recently demonstrated that the spatial distribution of the phosphatase can markedly affect the dose–response curves. We argue, using mathematical modeling, that the canonical model of the chemotaxis network cannot explain the experimental observations of Vaknin and Berg. We present a new model, in which a small fraction of the phosphatase is localized at the receptor cluster, while the remainder freely diffuses in the cytoplasm; moreover, the phosphatase at the cluster has a higher binding affinity for the messenger protein and a higher catalytic activity than the phosphatase in the cytoplasm. This model is consistent with a large body of experimental data and can explain many of the experimental observations of Vaknin and Berg. More generally, the combination of differential affinity and catalytic activity provides a generic mechanism for amplifying signals that could be exploited in other two-component signaling systems. If this model is correct, then a number of recent modeling studies, which aim to explain the chemotactic gain in terms of the activity of the receptor cluster, should be reconsidered.     

HECT Domain-containing E3 Ubiquitin Ligase NEDD4L Negatively Regulates Wnt Signaling by Targeting Dishevelled for Proteasomal Degradation

Wnt signaling plays a pivotal role in embryogenesis and tissue homeostasis. Dishevelled (Dvl) is a central mediator for both Wnt/β-catenin and Wnt/planar cell polarity pathways. NEDD4L, an E3 ubiquitin ligase, has been shown to regulate ion channel activity, cell signaling, and cell polarity. Here, we report a novel role of NEDD4L in the regulation of Wnt signaling. NEDD4L induces Dvl2 polyubiquitination and targets Dvl2 for proteasomal degradation. Interestingly, the NEDD4L-mediated ubiquitination of Dvl2 is Lys-6, Lys-27, and Lys-29 linked but not typical Lys-48-linked ubiquitination. Consistent with the role of Dvl in both Wnt/β-catenin and Wnt/planar cell polarity signaling, NEDD4L regulates the cellular β-catenin level and Rac1, RhoA, and JNK activities. We have further identified a hierarchical regulation that Wnt5a induces JNK-mediated phosphorylation of NEDD4L, which in turn promotes its ability to degrade Dvl2. Finally, we show that NEDD4L inhibits Dvl2-induced axis duplication in Xenopus embryos. Our work thus demonstrates that NEDD4L is a negative feedback regulator of Wnt signaling.     

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5′-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis.     

E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.