Ubiquitin Ligation Research Articles

Noncanonical assembly, neddylation and chimeric cullin–RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex

Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, ‘cullin–RING’ and ‘RBR’, are individually found in several hundred human E3 ligases, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin–RING and RBR domains. Here, cryo-EM, biochemistry and cellular assays elucidate a 1.8-MDa hexameric human CUL9–RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9–RBX1 in trans. Our data show CUL9 as unique among RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrates are recruited to various upstream domains, while ubiquitylation relies on both CUL9’s neddylated cullin and RBR domains achieving self-assembled and chimeric cullin–RING/RBR E3 ligase activity.

What have genetic studies of rare sequence variants taught us about the aetiology of schizophrenia?

With a population prevalence of 1%, schizophrenia is widespread, yet the aetiology of this psychiatric disorder remains elusive. There is an evident genetic component of schizophrenia, with heritability estimates lying at 60%-80%. While genome-wide association studies have identified 120 gene loci associated with schizophrenia risk, these involved common variants that confer only small effects on individual risk (median odds ratio < 1.2). The recent emergence of whole exome sequencing (WES) technologies has facilitated the identification of rare sequence variants, including some protein-truncating variants that have significant effects on risk. Three key large-scale WES studies have demonstrated that rare sequence variants in the genes SETD1A , CACNA1G , CUL1 , GRIA3 , GRIN2A , HERC1 , RB1CC1 , SP4 , TRIO , XPO7 , and AKAP11 confer substantial risk for schizophrenia. These genes are highly expressed in central nervous system neurons and their products participate in diverse molecular functions including synaptic transmission, transcriptional regulation, and ubiquitin ligation. The understanding of these functional roles illuminates putative molecular mechanisms which may lead to schizophrenia-like phenotypes. It will also be possible to develop model systems in which the effects of impaired function of these genes can be further explored. Genetic studies of rare variants to date suggest that glutamatergic system dysregulation, chromatin modification, and the ubiquitin-proteasome system play key roles in schizophrenia aetiology.

Structure of CRL7FBXW8 reveals coupling with CUL1–RBX1/ROC1 for multi-cullin-RING E3-catalyzed ubiquitin ligation

Most cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7FBXW8 assembly. CUL7’s exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1–FBXW8 to a neddylated CUL1–RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL–CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.

An integrative pan-cancer analysis revealing the difference in small ring finger family of SCF E3 ubiquitin ligases.

The SCF (Skp1-cullin-F-box proteins) complex is the largest family of E3 ubiquitin ligases that mediate multiple specific substrate proteins degradation. Two ring-finger family members RBX1/ROC1 and RBX2/RNF7/SAG are small molecular proteins necessary for ubiquitin ligation activity of the multimeric SCF complex. Accumulating evidence indicated the involvement of RBX proteins in the pathogenesis and development of cancers, but no research using pan-cancer analysis for evaluating their difference has been directed previously. We investigated RBX1/2 expression patterns and the association with clinicopathological features, and survivals of cancer patients obtained from the TCGA pan-cancer data. The binding energies of RBX1/2-CUL1 complexes were preliminarily calculated by using molecular dynamics simulations. Meanwhile, we assessed their immune infiltration level across numerous databases, including TISIDB and Timer database. High expression levels of RBX1/2 were observed in most cancer types and correlated with poor prognosis of patients analyzed. Nonetheless, exceptions were observed: RBX2 expression in KICH was higher than normal renal tissues and played a detrimental role in KICH. The expression of RBX1 was not associated with the prognostic risk of KICH. Moreover, the combination of RBX1 and CUL1 expression is more stable than that of RBX2 and CUL1. RBX1/2 expression showed their own specific characteristics in tumor pathological stages and grades, copy number variation and immune components. These findings not only indicated that the difference of RBX1/2 might result in varying degrees of tumor progression, but also suggested that they might serve as biomarkers for immune infiltration in cancers, shedding new light on therapeutics of cancers.

The NEL Family of Bacterial E3 Ubiquitin Ligases.

Some pathogenic or symbiotic Gram-negative bacteria can manipulate the ubiquitination system of the eukaryotic host cell using a variety of strategies. Members of the genera Salmonella, Shigella, Sinorhizobium, and Ralstonia, among others, express E3 ubiquitin ligases that belong to the NEL family. These bacteria use type III secretion systems to translocate these proteins into host cells, where they will find their targets. In this review, we first introduce type III secretion systems and the ubiquitination process and consider the various ways bacteria use to alter the ubiquitin ligation machinery. We then focus on the members of the NEL family, their expression, translocation, and subcellular localization in the host cell, and we review what is known about the structure of these proteins, their function in virulence or symbiosis, and their specific targets.

The ubiquitin ligation machinery in the defense against bacterial pathogens.

The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which—inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes—become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling. Remarkably, all these defense responses seem to emanate from a dense layer of ubiquitin which coats the invading pathogens. In this review, we focus on the diverse group of host cell E3 ubiquitin ligases that help to tailor this ubiquitin coat. In particular, we address how the divergent ubiquitin conjugation mechanisms of these ligases contribute to the complexity of the anti‐bacterial coating and the recruitment of different ubiquitin‐binding effectors. We also discuss the activation and coordination of the different E3 ligases and which strategies bacteria evolved to evade the activities of the host ubiquitin system.

Ubiquitin ligation to F-box protein targets by SCF\u2013RBR E3\u2013E3 super-assembly

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme’s true physiological substrate.

In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors.

Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA's activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA's modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA's substrate Rab33b. Prior to the identification and publication of SidJ's activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA's activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.

Cullin Ring Ubiquitin Ligases (CRLs) in Cancer: Responses to Ionizing Radiation (IR) Treatment.

Treatment with ionizing radiation (IR) remains the cornerstone of therapy for multiple cancer types, including disseminated and aggressive diseases in the palliative setting. Radiotherapy efficacy could be improved in combination with drugs that regulate the ubiquitin-proteasome system (UPS), many of which are currently being tested in clinical trials. The UPS operates through the covalent attachment of ATP-activated ubiquitin molecules onto substrates following the transfer of ubiquitin from an E1, to an E2, and then to the substrate via an E3 enzyme. The specificity of ubiquitin ligation is dictated by E3 ligases, which select substrates to be ubiquitylated. Among the E3s, cullin ring ubiquitin ligases (CRLs) represent prototypical multi-subunit E3s, which use the cullin subunit as a central assembling scaffold. CRLs have crucial roles in controlling the cell cycle, hypoxia signaling, reactive oxygen species clearance and DNA repair; pivotal factors regulating the cancer and normal tissue response to IR. Here, we summarize the findings on the involvement of CRLs in the response of cancer cells to IR, and we discuss the therapeutic approaches to target the CRLs which could be exploited in the clinic.

519 Paranuclear cytokeratin aggregates help Merkel cell carcinoma evade extrinsic apoptosis

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Most MCC tumors are virus positive and express Merkel cell polyomavirus oncogenes, whereas virus negative MCC are associated with abundant UV signature mutations. The majority of MCC tumors express CK20. Staining for CK20 and other intermediate filaments in a paranuclear dot pattern is a histological feature used to diagnose MCC. The functional significances of these paranuclear protein aggregates is unknown. We immunostained native MCC tumors with a pan-CK antibody and found areas of paranuclear dot staining in 94% of tumors. Immunofluorescent microscopy (IF) of MCC cell lines showed that paranuclear dot proteins often extend into a lattice-like structure surrounding the nucleus. Electron microscopy revealed that the intermediate filaments in the paranuclear dot did not resided within any cellular organelles but formed a cytoplasmic inclusion surrounding the centrosome. Using IF we confirmed that in 100% of interphase cells the paranuclear dot colocalized with the centrosome. In contrast, mitotic MCC cells showed partial disaggregation of the paranuclear dot and stochastic segregation of dot proteins into daughter cells, suggesting the paranuclear dot is a dynamic structure that reforms with each cell division. Disruption of microtubules with nocodazole resulted in dot disaggregation, demonstrating that microtubules are essential for dot maintenance. In contrast, inhibition of ubiquitin ligation enhanced cytokeratin aggregation. Interestingly, IF staining showed colocalization of the paranuclear dot with Fas Associate Death Domain (FADD). Consistent with functional sequestration of FADD in the paranuclear dot, TNFα and Fas ligand failed to induce apoptosis in dot-positive MCC cell lines. Taken together, the paranuclear dot may allow MCC tumors to evade extrinsic apoptosis by sequestering FADD and disrupting death receptor signaling.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Abstract 630: Txlnb-Deficient Mice Reveal That Ubiquitin-Proteosome-System Dysfunction is Not Sufficient to Impair Cardiac Performance

Prior reports show that impaired cardiac function is associated with, if not caused by, Ubiquitin-Proteasome-System (UPS) dysfunction. Rodent loss-of-function studies for proteins involved in protein folding/ER stress responses, ubiquitin ligation, and proteasomal degradation are frequently associated with accumulation of misfolded/ubiquitinated proteins, decreased proteasomal activities, worse outcomes to cardiac stress, and premature death. By contrast, gain of function models that improve protein folding/trafficking or activate the proteasome prevent these molecular phenotypes and improve cardiac function. We have recently found an exception to this association while characterizing beta-taxilin (TXLNB), an understudied muscle- and heart-enriched protein with unknown function. In cardiomyocytes, TXLNB overexpression decreased ubiquitinated proteins and increased proteasome activity, whereas knockdown promotes proteasomal insufficiency. Similarly, hearts from TXLNB knockout (TXLNB-KO) mice show increased ubiquitinated proteins (>2-fold, n=8, p=.007) and decreased 26SB5 proteasome activity (~40%, n=8, p=.025), starting by 12 weeks of age and persisting through life. Despite robust cardiac proteasomal insufficiency, TXLNB-KO mice surprisingly exhibit normal cardiac function [echo/ekg measures done in several independent cohorts (n>7 per sex/genotype) up to ~24 months of age]. In addition, these mice do not show worse responses to mild pressure-overload induced by thoracic aortic constriction (n>13 per sex/genotype). While their cardiac function is normal, TXLNB-KO males do display slight reductions in heart growth with age (10 months, p=.023, n=7; 18 months, p=.07, n=11-17; 24 months, p=.026, n=3-4), suggesting that TXLNB function may interface with hypertrophic signaling. Together, our findings indicate that TXLNB serves to bolster cardiac UPS, and more importantly, that UPS dysfunction and proteasomal insufficiency is not sufficient to cause cardiac dysfunction in mice. Future studies will need to examine if the incongruency with prior reports relates to distinct proteosomal pools (e.g. sarcomeric vs. nuclear vs. ER-associated) and/or the specific identities of the accumulated ubiquitinated proteins.

Neofunctionalization of Mitochondrial Proteins and Incorporation into Signaling Networks in Plants.

Because of their symbiotic origin, many mitochondrial proteins are well conserved across eukaryotic kingdoms. It is however less obvious how specific lineages have obtained novel nuclear-encoded mitochondrial proteins. Here, we report a case of mitochondrial neofunctionalization in plants. Phylogenetic analysis of genes containing the Domain of Unknown Function 295 (DUF295) revealed that the domain likely originated in Angiosperms. The C-terminal DUF295 domain is usually accompanied by an N-terminal F-box domain, involved in ubiquitin ligation via binding with ASK1/SKP1-type proteins. Due to gene duplication, the gene family has expanded rapidly, with 94 DUF295-related genes in Arabidopsis thaliana alone. Two DUF295 family subgroups have uniquely evolved and quickly expanded within Brassicaceae. One of these subgroups has completely lost the F-box, but instead obtained strongly predicted mitochondrial targeting peptides. We show that several representatives of this DUF295 Organellar group are effectively targeted to plant mitochondria and chloroplasts. Furthermore, many DUF295 Organellar genes are induced by mitochondrial dysfunction, whereas F-Box DUF295 genes are not. In agreement, several Brassicaceae-specific DUF295 Organellar genes were incorporated in the evolutionary much older ANAC017-dependent mitochondrial retrograde signaling pathway. Finally, a representative set of DUF295 T-DNA insertion mutants was created. No obvious aberrant phenotypes during normal growth and mitochondrial dysfunction were observed, most likely due to the large extent of gene duplication and redundancy. Overall, this study provides insight into how novel mitochondrial proteins can be created via “intercompartmental” gene duplication events. Moreover, our analysis shows that these newly evolved genes can then be specifically integrated into relevant, pre-existing coexpression networks.

Purification and functional characterization of the DUB domain of SdeA.

Intracellular pathogens like Legionella pneumophila hijack the host ubiquitination network in order to create a facultative niche for their survival by means of effector molecules secreted into the host cell. Some of these effectors function as ubiquitin ligases or deubiquitinases, among other types of enzymes. Deubiquitinating enzymes (DUBs) remove ubiquitin or ubiquitin-like modifiers from conjugated substrates to regulate various cellular processes. Members of the SidE effector family from the L. pneumophila pathogen harbor multiple functional domains that possess discrete biochemical activities impinging on host ubiquitin signaling. At the N-terminal end of these ~1500-residue proteins is a ~200-residue conserved DUB domain capable of recognizing both ubiquitin and the NEDD8 Ubl. SdeA, a member of the SidE family, plays an important role in intracellular bacterial replication. Downstream domains in this protein also catalyze substrate ubiquitination via a phosphoribosyl linkage. Several mammalian Rab proteins (Rab1, Rab30, and Rab33) have been shown to be targeted. The novel mechanism is independent of the classical E1 and E2 ubiquitin ligation machinery and does not require ATP. The N-terminal DUB domain, which does not appear to affect this ubiquitination activity, but it catalyzes cleavage of three different types of polyubiquitination chains (K11, K48, and K63) commonly found in host cells. This chapter describes methods, including purification of recombinant SdeA (full-length and DUB domain alone), and enzymatic assays that have been utilized to characterize the deubiquitination activity of SdeA.

Methods to study phosphoribosylated ubiquitin ligation and removal.

Ubiquitination is a prevalent protein modification catalyzed by E1, E2, and E3 enzymes that activate, conjugate, and ligate, respectively, the ubiquitin protein to substrate protein. In order to establish a mutualistic or parasitic relationship with their eukaryotic hosts, many microorganisms hijack different aspects of the ubiquitination machinery using bacterial proteins that function as E3 ligases or as enzymes that modify E2s or ubiquitin. Recently, the SidE family of effector proteins (SidEs) from the intracellular bacterial pathogen Legionella pneumophila was found to catalyze ubiquitination by a mechanism unrelated to the classical three-enzyme cascade. Instead of utilizing ATP, SidEs-catalyzed ubiquitination reactions are energized by nicotinamide adenine dinucleotide (NAD). Ubiquitin is first activated by ADP-ribosylation at residue Arg42 to form ADP-ribosylated ubiquitin (ADPR-Ub). ADPR-Ub is then cleaved by an activity conferred by a phosphodiesterase (PDE)-related domain also embedded in the SidE family proteins. ADPR-Ub cleavage is coupled to covalent attachment of phosphoribosylated ubiquitin to serine residues of target proteins and the release of AMP. Furthermore, SidE-induced ubiquitination can be reversed by SidJ, another virulence factor from L. pneumophila. Here, we describe the experimental details for SdeA-induced ubiquitination of the small GTPase Rab33b and its reversal by SidJ.

A Bifunctional Role for the UHRF1 UBL Domain in the Control of Hemi-methylated DNA-Dependent Histone Ubiquitylation

DNA methylation patterns regulate gene expression programs and are maintained through a highly coordinated process orchestrated by the RING E3 ubiquitin ligase UHRF1. UHRF1 controls DNA methylation inheritance by reading epigenetic modifications to histones and DNA to activate histone H3 ubiquitylation. Here, we find that all five domains of UHRF1, including the previously uncharacterized ubiquitin-like domain (UBL), cooperate for hemi-methylated DNA-dependent H3 ubiquitin ligation. Our structural and biochemical studies, including mutations found in cancer genomes, reveal a bifunctional requirement for the UBL in histone modification: (1) the UBL makes an essential interaction with the backside of the E2 and (2) the UBL coordinates with other UHRF1 domains that recognize epigenetic marks on DNA and histone H3 to direct ubiquitin to H3. Finally, we show UBLs from other E3s also have a conserved interaction with the E2, Ube2D, highlighting a potential prevalence of interactions between UBLs and E2s.

A MALDI-TOF Approach to Ubiquitin Ligase Activity

In this issue of Cell Chemical Biology,De Cesare etal. (2018) report the development of a high-throughput assay that measures E2/E3 enzyme activity by MALDI-TOF mass spectrometry and apply this to screen for small molecule E3 inhibitors. This assay potentially accelerates the drug discovery for the ubiquitin ligation pathway.

PO-228 Regulation of GDE2 membrane localization and trafficking

IntroductionGlycerophosphodiester phosphodiesterase 2 (GDE2) is a multi-pass membrane protein that promotes neuronal differentiation through the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins at the cell surface. High GDE2 expression is associated with favourable outcome in neuroblastoma, while loss of GDE2 in mice leads to neuronal pathologies similar to human neurodegenerative diseases. Thus, enhancing GDE2 activity could be an attractive therapeutic strategy for neuroblastoma and related pathologies. However, the regulation of GDE2 is poorly understood.Material and methodsWe employed TIRF microscopy to study GDE2 subcellular localization in neuronal cell lines. Membrane internalisation of GDE2 was detected by confocal microscopy, and confirmed biochemically by biotin labelling assays. To determine the nature of the GDE2-containg intracellular compartments, we examined co-localization of GDE2 with endosome markers by both confocal microscopy and immunoprecipitation assays.Results and discussionsWhen expressed at relatively low levels in N1E-115, Neuro2A and SH-SY5Y neuronal cell lines, GDE2 localises to discrete membrane microdomains, as well as in high-turnover intracellular vesicles. We corroborated this intracellular trafficking by biotin labelling assays, which showed that GDE2 undergoes constitutive endocytosis and recycling back to the plasma membrane in a serum-independent manner.In addition, GDE2 was found to co-localise with well-established early-endosome markers, namely EEA1 and Rab5, indicating that GDE2 internalises from the plasma membrane. GDE2 also localised partially to Rab7-positive late endosomes, but was hardly detected in lysosomes (LAMP1, LysoTracker). Furthermore, GDE2 localised to Rab11-positive recycling endosomes, pinpointing the long recycling pathway.When co-expressed with labelled ubiquitin, GDE2 was heavily poly-ubiquitinated, which takes place non-specifically in the four cytosolic lysine residues of GDE2. Lastly, sequential truncations in the N- and C-terminal cytosolic tails of GDE2 highlighted a 10-amino acid stretch that potentially regulates intracellular trafficking.ConclusionHere we report that, in neuronal cells, GDE2 is constitutively internalised and undergoes endocytic recycling along both the short and, in particular, the long recycling pathways. This process appears to be regulated by a stretch of 10 residues in the cytosolic C-terminal tail, which could be related to non-specific ubiquitin ligation.

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays invitro, and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways.