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Angelman syndrome-associated point mutations in the Zn²⁺-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome.

ABSTRACT: Deregulation of the HECT ubiquitin ligase UBE3A/E6AP has been implicated in Angelman syndrome as well as autism spectrum disorders. We and others have previously identified the 26S proteasome as one of the major UBE3A-interacting protein complexes. Here, we characterize the interaction of UBE3A and the proteasomal subunit PSMD4 (Rpn10/S5a). We map the interaction to the highly conserved Zn²⁺-binding N-terminal (AZUL) domain of UBE3A, the integrity of which is crucial for binding to PSMD4. Interestingly, two Angelman syndrome point mutations that affect the AZUL domain show an impaired ability to bind PSMD4. Although not affecting the ubiquitin ligase or the estrogen receptor ?-mediated transcriptional regulation activities, these AZUL domain mutations prevent UBE3A from stimulating the Wnt/?-catenin signaling pathway. Taken together, our data indicate that impaired binding to the 26S proteasome and consequential deregulation of Wnt/?-catenin signaling might contribute to the functional defect of these mutants in Angelman syndrome.

SUBMITTER: Kuhnle S

PROVIDER: S-EPMC6254356 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome.

Project description:Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.

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The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region.

Project description:Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13. Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region. We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb. Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region. The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested. The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels. Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus. Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products.

| S-EPMC139148 | biostudies-literature

The role of whole exome sequencing in the UBE3A point mutation of Angelman Syndrome: A case report.

Project description:IntroductionAngelman Syndrome (AS) is a rare disorder with a relatively well-defined phenotype caused by lack of expression of the maternally inherited ubiquitin-protein ligase E3A (UBE3A) gene in the brain. This article describes the role of genetic testing using whole-exome sequencing (WES) in detecting rare AS variants, a point mutation in the UBE3A gene.Case presentationWe describe a rarely reported clinical presentation of AS in a two year and ten months old girl with severe developmental delay, movement and balance disorder, frequent smiling, apparent happy demeanor, speech impairment, absence of seizure, lack of sleep, and abnormal food-related behavior. Physical examination showed microcephaly, with facial characteristics of AS, ataxia gait, and truncal hypotonia. The electroencephalogram showed medium amplitude rhythmic 2-3c/s. Brain Magnetic Resonance Imaging revealed microcephaly, corpus callosum dysgenesis, and heterotopia grey matter on the bilateral lateral ventricle. WES was conducted to search pathogenic variants and showed a heterozygous mutation in exon 9 of the UBE3A gene, c.1513C > T (p.Arg505Ter).ConclusionAngelman syndrome is a neurodevelopmental disorder that has several underlying genetic etiologies. WES could detect a rare variant of Angelman syndrome, identified as the point mutation of the UBE3A gene, which cannot be seen with other modalities.

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Mutation analysis of UBE3A in Angelman syndrome patients.

Project description:Angelman syndrome (AS) is caused by chromosome 15q11-q13 deletions of maternal origin, by paternal uniparental disomy (UPD) 15, by imprinting defects, and by mutations in the UBE3A gene. UBE3A encodes a ubiquitin-protein ligase and shows brain-specific imprinting. Here we describe UBE3A coding-region mutations detected by SSCP analysis in 13 AS individuals or families. Two identical de novo 5-bp duplications in exon 16 were found. Among the other 11 unique mutations, 8 were small deletions or insertions predicted to cause frameshifts, 1 was a mutation to a stop codon, 1 was a missense mutation, and 1 was predicted to cause insertion of an isoleucine in the hect domain of the UBE3A protein, which functions in E2 binding and ubiquitin transfer. Eight of the cases were familial, and five were sporadic. In two familial cases and one sporadic case, mosaicism for UBE3A mutations was detected: in the mother of three AS sons, in the maternal grandfather of two AS first cousins, and in the mother of an AS daughter. The frequencies with which we detected mutations were 5 (14%) of 35 in sporadic cases and 8 (80%) of 10 in familial cases.

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Identification of small molecule activators of the ubiquitin ligase E6AP/UBE3A and Angelman syndrome-derived E6AP/UBE3A variants

Project description:Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10 percent of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical crosslinking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment.

2020-09-28 | PXD020602 | Pride

Ube3a imprinting impairs circadian robustness in Angelman syndrome models.

Project description:The paternal allele of Ube3a is silenced by imprinting in neurons, and Angelman syndrome (AS) is a disorder arising from a deletion or mutation of the maternal Ube3a allele, which thereby eliminates Ube3a neuronal expression. Sleep disorders such as short sleep duration and increased sleep onset latency are very common in AS.We found a unique link between neuronal imprinting of Ube3a and circadian rhythms in two mouse models of AS, including enfeebled circadian activity behavior and slowed molecular rhythms in ex vivo brain tissues. As a consequence of compromised circadian behavior, metabolic homeostasis is also disrupted in AS mice. Unsilencing the paternal Ube3a allele restores functional circadian periodicity in neurons deficient in maternal Ube3a but does not affect periodicity in peripheral tissues that are not imprinted for uniparental Ube3a expression. The ubiquitin ligase encoded by Ube3a interacts with the central clock components BMAL1 and BMAL2. Moreover, inactivation of Ube3a expression elevates BMAL1 levels in brain regions that control circadian behavior of AS-model mice, indicating an important role for Ube3a in modulating BMAL1 turnover.Ube3a expression constitutes a direct mechanistic connection between symptoms of a human neurological disorder and the central circadian clock mechanism. The lengthened circadian period leads to delayed phase, which could explain the short sleep duration and increased sleep onset latency of AS subjects. Moreover, we report the pharmacological rescue of an AS phenotype, in this case, altered circadian period. These findings reveal potential treatments for sleep disorders in AS patients.

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Ube3a reinstatement mitigates epileptogenesis in Angelman syndrome model mice.

Project description:Angelman syndrome (AS) is a neurodevelopmental disorder in which epilepsy is common (~90%) and often refractory to antiepileptics. AS is caused by mutation of the maternal allele encoding the ubiquitin protein ligase E3A (UBE3A), but it is unclear how this genetic insult confers vulnerability to seizure development and progression (i.e., epileptogenesis). Here, we implemented the flurothyl kindling and retest paradigm in AS model mice to assess epileptogenesis and to gain mechanistic insights owed to loss of maternal Ube3a. AS model mice kindled similarly to wild-type mice, but they displayed a markedly increased sensitivity to flurothyl-, kainic acid-, and hyperthermia-induced seizures measured a month later during retest. Pathological characterization revealed enhanced deposition of perineuronal nets in the dentate gyrus of the hippocampus of AS mice in the absence of overt neuronal loss or mossy fiber sprouting. This pro-epileptogenic phenotype resulted from Ube3a deletion in GABAergic but not glutamatergic neurons, and it was rescued by pancellular reinstatement of Ube3a at postnatal day 21 (P21), but not during adulthood. Our results suggest that epileptogenic susceptibility in AS patients is a consequence of the dysfunctional development of GABAergic circuits, which may be amenable to therapies leveraging juvenile reinstatement of UBE3A.

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UBE3A reinstatement as a disease-modifying therapy for Angelman syndrome.

Project description:Half a century ago, Harry Angelman reported three patients with overlapping clinical features, now well known as Angelman syndrome. Angelman syndrome is caused by mutations affecting the maternally inherited UBE3A gene, which encodes an E3-ubiquitin ligase that is critical for typical postnatal brain development. Emerging evidence indicates that UBE3A plays a particularly important role in the nucleus. However, the critical substrates that are controlled by UBE3A remain elusive, which hinders the search for effective treatments. Moreover, given the multitude of signalling mechanisms that are derailed, it is unlikely that targeting a single pathway is going to be very effective. Therefore, expectations are very high for approaches that aim to restore UBE3A protein levels. A particular promising strategy is an antisense oligonucleotide approach, which activates the silenced paternal UBE3A gene. When successful, such treatments potentially offer a disease-modifying therapy for Angelman syndrome and several other neurodevelopmental disorders. What this paper adds Loss of UBE3A affects multiple signalling pathways in the brain. Emerging evidence suggests that UBE3A plays a critical role in the cell nucleus. Trials using antisense oligonucleotides to restore UBE3A levels are continuing.

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Truncation of Ube3a-ATS unsilences paternal Ube3a and ameliorates behavioral defects in the Angelman syndrome mouse model.

Project description:Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.

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The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/?-catenin pathway by inhibiting the proteasome.

Project description:UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear ?-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

| S-EPMC5535025 | biostudies-literature