Ubiquitin-protein ligase E3A (UBE3A) mediation of viral infection and human diseases.

The Ubiquitin-protein ligase E3A, UBE3A, also known as E6-associated protein (E6-AP), is known to play an essential role in regulating the degradation of various proteins by transferring Ub from E2 Ub conjugating enzymes to the substrate proteins. Several studies indicate that UBE3A regulates the stabilities of key viral proteins in the virus-infected cells and, thereby, the infected virus-mediated diseases, even if it were reported that UBE3A participates in non-viral-related human diseases. Furthermore, mutations such as deletions and duplications in the maternally inherited gene in the brain cause human neurodevelopmental disorders such as Angelman syndrome (AS) and autism. It is also known that UBE3A functions as a transcriptional coactivator for the expression of steroid hormone receptors. These reports establish that UBE3A is distinguished by its multitudinous functions that are paramount to viral pathology and human diseases. This review is focused on molecular mechanisms for such intensive participation of UBE3A in disease formation and virus regulation.

Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome.

Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.

Elevated ROS levels during the early development of Angelman syndrome alter the apoptotic capacity of the developing neural precursor cells.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder caused by the maternally inherited loss of function of the UBE3A gene. AS is characterized by a developmental delay, lack of speech, motor dysfunction, epilepsy, autistic features, happy demeanor, and intellectual disability. While the cellular roles of UBE3A are not fully understood, studies suggest that the lack of UBE3A function is associated with elevated levels of reactive oxygen species (ROS). Despite the accumulating evidence emphasizing the importance of ROS during early brain development and its involvement in different neurodevelopmental disorders, up to date, the levels of ROS in AS neural precursor cells (NPCs) and the consequences on AS embryonic neural development have not been elucidated. In this study we show multifaceted mitochondrial aberration in AS brain-derived embryonic NPCs, which exhibit elevated mitochondrial membrane potential (ΔΨm), lower levels of endogenous reduced glutathione, excessive mitochondrial ROS (mROS) levels, and increased apoptosis compared to wild-type (WT) littermates. In addition, we report that glutathione replenishment by glutathione-reduced ethyl ester (GSH-EE) corrects the excessive mROS levels and attenuates the enhanced apoptosis in AS NPCs. Studying the glutathione redox imbalance and mitochondrial abnormalities in embryonic AS NPCs provides an essential insight into the involvement of UBE3A in early neural development, information that can serve as a powerful avenue towards a broader view of AS pathogenesis. Moreover, since mitochondrial dysfunction and elevated ROS levels were associated with other neurodevelopmental disorders, the findings herein suggest some potential shared underlying mechanisms for these disorders as well.

A high-fidelity RNA-targeting Cas13 restores paternal Ube3a expression and improves motor functions in Angelman syndrome mice.

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function mutations in maternally expressed UBE3A. No gene-specific treatment is available for patients so far. Although intact and transcriptionally active, paternally inherited UBE3A is silenced by elongation of antisense long noncoding RNA UBE3A-ATS in neurons. Here, we demonstrated that RNA targeting of paternal Ube3a-ATS with a high-fidelity CRISPR-Cas13 (hfCas13x.1) system could restore Ube3a expression to similar levels as that of maternal Ube3a in the cultured mouse neurons. Furthermore, injection into lateral ventricles with neuron-specific hSyn1 promoter-driven hfCas13x.1 packaged in adeno-associated virus (AAV-PHP.eb) could restore paternal Ube3a expression in cortex and hippocampus of neonatal AS mice for up to 4 months after treatment. Behavioral tests showed that expression of paternal Ube3a significantly alleviated AS-related symptoms, including obesity and motor function. Our results suggested that hfCas13x.1-mediated suppression of the Ube3a-ATS lncRNA potentially serves as a promising targeted intervention for AS.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Improving Gene Therapy for Angelman Syndrome with Secreted Human UBE3A.

The rare genetic neurodevelopmental disease Angelman syndrome (AS) is caused by the loss of function of UBE3A, a ubiquitin ligase. The disease results in a lifetime of severe symptoms, including intellectual disability and motor impairments for which there are no effective treatments. One avenue of treatment for AS is the use of gene therapy to reintroduce a functional copy of the UBE3A gene. Our group had previously shown that recombinant adeno-associated virus (rAAV) expressing mouse Ube3a could rescue deficits in a mouse model of AS. Here, we expand on this work and show that this approach could be successfully replicated in a second AS model using the human UBE3A gene. Furthermore, we address the challenge of limited vector distribution in the brain by developing a novel modified form of UBE3A. This modified protein, termed STUB, was designed with a secretion signal and a cell-penetrating peptide. This allowed transduced cells to act as factories for the production of UBE3A protein that could be taken up by neighboring non-transduced cells, thus increasing the number of neurons receiving the therapeutic protein. Combining this construct with intracerebroventricular injections to maximize rAAV distribution within the brain, we demonstrate that this novel approach improves the recovery of behavioral and electrophysiological deficits in the AS rat model. More importantly, a comparison of rAAV-STUB to a rAAV expressing the normal human UBE3A gene showed that STUB was a more effective therapeutic. These data suggest that rAAV-STUB is a new potential approach for the treatment of AS.

Methylation analysis and developmental profile of two individuals with Angelman syndrome due to mosaic imprinting defects.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternally-inherited UBE3A on chromosome 15q11.2. In AS due to a chromosomal deletion that encompasses UBE3A, paternal uniparental disomy of chromosome 15, or imprinting defects (ImpD), the SNRPN locus is unmethylated, while in neurotypical individuals, it is ∼50% methylated. We present the developmental profile of two adults with mild AS assessed using standardized behavioral and neurodevelopmental measures. Both had intellectual disability with unusually advanced verbal communication skills compared to other individuals with AS. Methylation of the SNRPN locus was examined using Methylation Specific Quantitative Melt Analysis (MS-QMA) in different tissues at one time point for participant A (22 years) and two time points for participant B (T1: 22 years, T2: 25 years), and these levels were compared to a typical AS cohort. While participant A showed methylation levels comparable to the typical AS cohort, participant B showed methylation mosaicism in all tissues at both time points and changes in methylation levels from T1 to T2. AS should be considered in individuals with intellectual disability and verbal speech who may not have the typical symptoms of AS.

Expanding the clinico-molecular spectrum of Angelman syndrome phenotype with the GABRG3 gene: Evidence from methylation and sequencing studies.

Angelman syndrome (AS) (OMIM#105830) is an imprinting disorder caused due to alterations in the maternal chr 15q11-13 region. Majority of cases can be diagnosed by methylation-specific polymerase chain reaction (MS-PCR) of SNRPN gene and by UBE3A sequencing, however, about 10% of cases with AS phenotype remain undiagnosed. Differential diagnoses of AS can be detected by chromosomal microarray (CMA) and clinical exome sequencing (CES). In this study, 30 cases with AS features were evaluated by MS-PCR, CMA, and CES. SNRPN MS-PCR confirmed AS in eight (26%), CMA and CES diagnosed nine (30%) cases. One case was identified with a novel variant c.1125C > T in GABRG3, located at 15q12 region, which is currently not associated with any syndrome. The GABRG3 gene is also speculated to be imprinted, a MS-PCR assay was designed to confirm its differential parental methylation status. This assay identified another case with altered GABRG3 methylation. The two cases with GABRG3 alteration-sequence change and methylation indicate that GABRG3 may be associated with a subtype of AS or a new related syndrome. Performing GABRG3 MS-PCR and sequencing of a larger group of patients with AS phenotype and normal SNPRN and UBE3A status will help in establishing exact genotype-phenotype correlation.

Therapies in preclinical and clinical development for Angelman syndrome.

Introduction: Angelman syndrome is a rare genetic neurodevelopmental disorder, caused by deficiency or abnormal function of the maternal ubiquitin protein-ligase E3A, known as UBE3A, in the central nervous system. There is no disease-modifying treatment available, but the therapeutic pipeline of Angelman syndrome includes at least 15 different approaches at preclinical or clinical development. In the coming years, several clinical trials will be enrolling patients, which prompted this comprehensive review.Areas covered: We summarize and critically review the different therapeutic approaches. Some approaches attempt to restore the missing or nonfunctional UBE3A protein in the neurons via gene replacement or enzyme replacement therapies. Other therapies aim to induce expression of the normal paternal copy of the UBE3A gene by targeting a long non-coding RNA, the UBE3A-ATS, which interferes with its own expression. Another therapeutic category includes compounds that target molecular pathways and effector proteins known to be involved in Angelman syndrome pathophysiology.Expert opinion: We believe that by 2022-2023, more than five disease-modifying treatments will be simultaneously at clinical testing. However, the are several challenges with regards to safety and efficacy, which need to be addressed. Additionally, there is still a significant unmet need for clinical trial readiness.

Functional rescue in an Angelman syndrome model following treatment with lentivector transduced hematopoietic stem cells.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.

Bioinformatics analyses show dysregulation of calcium-related genes in Angelman syndrome mouse model.

BACKGROUND: Angelman syndrome (AS) is a genetic neurodevelopmental disorder caused by the loss of function of the UBE3A protein in the brain. In a previous study, we showed that activity-dependent calcium dynamics in hippocampal CA1 pyramidal neurons of AS mice is compromised, and its normalization rescues the hippocampal-dependent deficits. Therefore, we expected that the expression profiles of calcium-related genes would be altered in AS mice hippocampi. METHODS: We analyzed mRNA sequencing data from AS model mice and WT controls in light of the newly published CaGeDB database of calcium-related genes. We validated our results in two independent RNA sequencing datasets from two additional different AS models: first one, a human neuroblastoma cell line where UBE3A expression was knocked down by siRNA, and the second, an iPSC-derived neurons from AS patient and healthy donor control. FINDINGS: We found signatures of dysregulated calcium-related genes in AS mouse model hippocampus. Additionally, we show that these calcium-related genes function as signatures for AS in other human cellular models of AS, thus strengthening our findings. INTERPRETATION: Our findings suggest the downstream implications and significance of the compromised calcium signaling in Angelman syndrome. Moreover, since AS share similar features with other autism spectrum disorders, we believe that these findings entail meaningful data and approach for other neurodevelopmental disorders, especially those with known alterations of calcium signaling. FUNDING: This work was supported by the Angelman Syndrome Foundation and by the Israel Science Foundation, Grant Number 248/20.

Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.

Bioinformatics Analyses of the Transcriptome Reveal Ube3a-Dependent Effects on Mitochondrial-Related Pathways.

The UBE3A gene encodes the ubiquitin E3-ligase protein, UBE3A, which is implicated in severe neurodevelopmental disorders. Lack of UBE3A expression results in Angelman syndrome, while UBE3A overexpression, due to genomic 15q duplication, results in autism. The cellular roles of UBE3A are not fully understood, yet a growing body of evidence indicates that these disorders involve mitochondrial dysfunction and increased oxidative stress. We utilized bioinformatics approaches to delineate the effects of murine Ube3a deletion on the expression of mitochondrial-related genes and pathways. For this, we generated an mRNA sequencing dataset from mouse embryonic fibroblasts (MEFs) in which both alleles of Ube3a gene were deleted and their wild-type controls. Since oxidative stress and mitochondrial dysregulation might not be exhibited in the resting baseline state, we also activated mitochondrial functioning in the cells of these two genotypes using TNFα application. Transcriptomes of the four groups of MEFs, Ube3a+/+ and Ube3a-/-, with or without the application of TNFα, were analyzed using various bioinformatics tools and machine learning approaches. Our results indicate that Ube3a deletion affects the gene expression profiles of mitochondrial-associated pathways. We further confirmed these results by analyzing other publicly available human transcriptome datasets of Angelman syndrome and 15q duplication syndrome.

An overview of health issues and development in a large clinical cohort of children with Angelman syndrome.

This study presents a broad overview of health issues and psychomotor development of 100 children with Angelman syndrome (AS), seen at the ENCORE Expertise Center for AS in Rotterdam, the Netherlands. We aimed to further delineate the phenotype of AS, to evaluate the association of the phenotype with genotype and other determinants such as epilepsy and to get insight in possible targets for intervention. We confirmed the presence of a more severe phenotype in the 15q11.2-q13 deletion subtype. Novel findings were an association of (early onset of) epilepsy with a negative effect on development, a high occurrence of nonconvulsive status epilepticus, a high rate of crouch gait in the older children with risk of deterioration of mobility, a relatively low occurrence of microcephaly, a higher mean weight for height in all genetic subtypes with a significant higher mean in the nondeletion children, and a high occurrence of hyperphagia across all genetic subtypes. Natural history data are needed to design future trials. With this large clinical cohort with structured prospective and multidisciplinary follow-up, we provide unbiased data on AS to support further intervention studies to optimize outcome and quality of life of children with AS and their family.

A rapid and accurate methylation-sensitive high-resolution melting analysis assay for the diagnosis of Prader Willi and Angelman patients.

BACKGROUND: Prader Willi (PWS) and Angelman (AS) syndromes are rare genetic disorders characterized by deletions, uniparental disomy, and imprinting defects at chromosome 15. The loss of function of specific genes caused by genetic alterations in paternal allele causes PWS while the absence in maternal allele results AS. The laboratory diagnosis of PWS and AS is complex and demands molecular biology and cytogenetics techniques to identify the genetic mechanism related to the development of the disease. The DNA methylation analysis in chromosome 15 at the SNURF-SNRPN locus through MS-PCR confirms the diagnosis and distinguishes between PWS and AS. Our study aimed to establish the MS-PCR technique associated with High-Resolution Melting (MS-HRM) in PWS and AS diagnostic with a single pair of primers. METHODS: We collected blood samples from 43 suspected patients to a cytogenetic and methylation analysis. The extracted DNA was treated with bisulfite to perform comparative methylation analysis. RESULTS: MS-HRM and MS-PCR agreed in 100% of cases, identifying 19(44%) PWS, 3(7%) AS, and 21(49%) Normal. FISH analysis detected four cases of PWS caused by deletions in chromosome 15. CONCLUSION: The MS-HRM showed good performance with a unique pair of primers, dispensing electrophoresis gel analysis, offering a quick and reproducible diagnostic.

The role of long non-coding RNAs in the pathogenesis of hereditary diseases.

BACKGROUND: Thousands of long non-coding RNA (lncRNA) genes are annotated in the human genome. Recent studies showed the key role of lncRNAs in a variety of fundamental cellular processes. Dysregulation of lncRNAs can drive tumorigenesis and they are now considered to be a promising therapeutic target in cancer. However, how lncRNAs contribute to the development of hereditary diseases in human is still mostly unknown. RESULTS: This review is focused on hereditary diseases in the pathogenesis of which long non-coding RNAs play an important role. CONCLUSIONS: Fundamental research in the field of molecular genetics of lncRNA is necessary for a more complete understanding of their significance. Future research will help translate this knowledge into clinical practice which will not only lead to an increase in the diagnostic rate but also in the future can help with the development of etiotropic treatments for hereditary diseases.

Sex-Dependent Sensory Phenotypes and Related Transcriptomic Expression Profiles Are Differentially Affected by Angelman Syndrome.

Angelman syndrome (AS) is a genetic disorder which entails autism, intellectual disability, lack of speech, motor deficits, and seizure susceptibility. It is caused by the lack of UBE3A protein expression, which is an E3-ubiquitin ligase. Despite AS equal prevalence in males and females, not much data on how sex affects the syndrome was reported. In the herein study, we thoroughly characterized many behavioral phenotypes of AS mice. The behavioral data acquired was analyzed with respect to sex. In addition, we generated a new mRNA sequencing dataset. We analyzed the coding transcriptome expression profiles with respect to the effects of genotype and sex observed in the behavioral phenotypes. We identified several neurobehavioral aspects, especially sensory perception, where AS mice either lack the male-to-female differences observed in wild-type littermates or even show opposed differences. However, motor phenotypes did not show male-to-female variation between wild-type (WT) and AS mice. In addition, by utilizing the mRNA sequencing, we identified genes and isoforms with expression profiles that mirror the sensory perception results. These genes are differentially regulated in the two sexes with inverse expression profiles in AS mice compared to WT littermates. Some of these are known pain-related and estrogen-dependent genes. The observed differences in sex-dependent neurobehavioral phenotypes and the differential transcriptome expression profiles in AS mice strengthen the evidence for molecular cross talk between Ube3a protein and sex hormone receptors or their elicited pathways. These interactions are essential for understanding Ube3a deletion effects, beyond its E3-ligase activity.

A mouse model of Angelman syndrome imprinting defects.

Angelman syndrome, Prader-Will syndrome and Dup15q syndrome map to a cluster of imprinted genes located at 15q11-q13. Imprinting at this domain is regulated by an imprinting control region consisting of two distinct elements, the Angelman syndrome imprinting center (AS-IC) and the Prader-Willi syndrome imprinting center (PWS-IC). Individuals inheriting deletions of the AS-IC exhibit reduced expression of the maternally expressed UBE3A gene and biallelic expression of paternal-only genes. We have previously demonstrated that AS-IC activity partly consists of providing transcription across the PWS-IC in oocytes, and that these transcripts are necessary for maternal imprinting of Snrpn. Here we report a novel mouse mutation that truncates transcripts prior to transiting the PWS-IC and results in a domain-wide imprinting defect. These results confirm a transcription-based model for imprint setting at this domain. The imprinting defect can be preempted by removal of the transcriptional block in oocytes, but not by its removal in early embryos. Imprinting defect mice exhibit several traits often found in individuals with Angelman syndrome imprinting defects.

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

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 α-mediated transcriptional regulation activities, these AZUL domain mutations prevent UBE3A from stimulating the Wnt/ß-catenin signaling pathway. Taken together, our data indicate that impaired binding to the 26S proteasome and consequential deregulation of Wnt/ß-catenin signaling might contribute to the functional defect of these mutants in Angelman syndrome.