Ube3a Antibody
Description
Introduction to UBE3A Antibody
UBE3A antibodies are immunological reagents designed to detect and bind specifically to UBE3A protein, a HECT domain E3 ubiquitin ligase that targets substrate proteins, including itself, for proteasomal degradation . These antibodies serve as critical tools for investigating UBE3A's abundance and subcellular distribution, which have been linked to various neurodevelopmental disorders including Angelman syndrome, Dup15q syndrome, and certain forms of autism .
The development and evaluation of UBE3A antibodies have become increasingly important as researchers seek to understand the complex biology of UBE3A and its role in both normal physiology and pathological conditions. UBE3A antibodies are available in various formats, including polyclonal and monoclonal options, each with specific applications in research and potentially diagnostic settings .
Significance in Neurodevelopmental Research
UBE3A antibodies have gained particular significance due to the protein's neuronal imprinting pattern. While UBE3A is expressed from both parental alleles in most tissues, it is expressed exclusively from the maternal allele in neurons due to tissue-specific paternal imprinting . This unique expression pattern means that mutations or deletions affecting the maternal UBE3A allele can lead to a complete loss of functional UBE3A in neurons, resulting in Angelman syndrome .
UBE3A Protein Background
Before delving into the specifics of UBE3A antibodies, it is essential to understand the target protein itself. UBE3A is a multifunctional E3 ubiquitin ligase that plays crucial roles in cellular protein quality control and several signaling pathways .
Structure and Function
UBE3A accepts ubiquitin from E2 ubiquitin-conjugating enzymes and transfers it to substrate proteins, marking them for degradation by the 26S proteasome . Several substrates have been identified, including BMAL1, ARC (activity-regulated cytoskeleton-associated protein), LAMTOR1, RAD23A, RAD23B, MCM7, annexin A1, PML tumor suppressor, and the cell cycle regulator CDKN1B .
Beyond its ubiquitin ligase activity, UBE3A also functions as a transcriptional coactivator of steroid hormone receptors, particularly the progesterone receptor (PGR) . This dual functionality underscores the complex role of UBE3A in cellular processes.
Regulation and Expression
Research has revealed that UBE3A expression is regulated by neuronal activity. Quantitative real-time PCR experiments have shown that UBE3A mRNA expression increases significantly following membrane depolarization or glutamate receptor activation . Conversely, blocking neuronal activity using APV (NMDA receptor inhibitor), TTX (sodium channel blocker), and NBQX (AMPA receptor inhibitor) results in decreased UBE3A mRNA expression .
The transcription factor MEF2 has been identified as a direct regulator of UBE3A, binding to conserved consensus sites in UBE3A promoters 1 and 3 . This activity-dependent regulation suggests that UBE3A plays a critical role in experience-driven neuronal development and synaptic plasticity.
UBE3A Antibody Types and Manufacturers
Various commercial sources produce UBE3A antibodies with different characteristics and applications. These antibodies are generated using different immunogens and are available in different formats.
Monoclonal vs. Polyclonal UBE3A Antibodies
Commercial UBE3A antibodies are available in both monoclonal and polyclonal formats. Monoclonal antibodies, such as Cell Signaling's D10D3 Rabbit mAb (#7526), offer superior lot-to-lot consistency and specificity for a single epitope . GeneTex has also developed a recombinant rabbit monoclonal UBE3A antibody [HL1660] (GTX637258) that has been validated by knockdown experiments .
Polyclonal antibodies, such as Abcam's ab10488 and Thermo Fisher's 10344-1-AP, recognize multiple epitopes on the UBE3A protein, potentially offering greater sensitivity but with possible variations between lots . These antibodies are generated by immunizing animals (typically rabbits) with synthetic peptides corresponding to specific regions of the UBE3A protein.
Specificity and Performance Evaluation
The study assessed these antibodies in two common assays: immunofluorescence and western blot. The research revealed that among the seven antibodies tested, three demonstrated substantial nonspecific immunoreactivity, making them less reliable for precise UBE3A detection .
Comparative Performance in Different Models
Table 2: Performance of UBE3A Antibodies in Different Experimental Models
| Antibody Performance | Mouse Brain Sections | Human Cerebral Organoids | Undifferentiated Human Pluripotent Stem Cells |
|---|---|---|---|
| Specific localization | 4 out of 7 antibodies | 4 out of 7 antibodies | Variable patterns |
| Background signal | Variable | Variable | Significant variation |
| Nonspecific reactivity | 3 out of 7 antibodies | 3 out of 7 antibodies | Not specified |
Dynamic Spatiotemporal Detection
The study also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models . This assessment is particularly valuable as it helps identify antibodies capable of tracking UBE3A expression changes during neural development and in response to various stimuli.
Applications of UBE3A Antibodies
UBE3A antibodies serve multiple purposes in biomedical research, from basic protein detection to more complex applications in developmental neuroscience.
Common Research Applications
Table 3: Common Applications of UBE3A Antibodies
UBE3A Detection in Experimental Models
UBE3A antibodies have been crucial in studying the protein's role in various experimental models. In mice with a yellow fluorescent protein (YFP) fused to the paternal allele of Ube3a, antibodies targeting UBE3A or GFP (to detect the fusion protein) have enabled researchers to monitor the expression of the paternal allele following various treatments .
In human cerebral organoids, UBE3A antibodies have facilitated the investigation of UBE3A's spatiotemporal expression patterns during early brain development, providing insights into its role in neurogenesis and synaptogenesis .
UBE3A Antibodies in Neurodevelopmental Disorder Research
UBE3A antibodies have been instrumental in advancing our understanding of neurodevelopmental disorders, particularly Angelman syndrome.
Angelman Syndrome Research
Angelman syndrome results from the loss of UBE3A function in neurons, typically due to mutations or deletions of the maternal UBE3A allele . UBE3A antibodies have enabled researchers to verify the loss of UBE3A protein in neurons derived from Angelman syndrome models and patients .
A recent study demonstrated that the compound (S)-PHA533533 can unsilence the paternal UBE3A allele in neurons. UBE3A antibodies were essential in confirming the increased UBE3A protein expression following treatment, highlighting their value in therapeutic development research .
UBE3A and Synaptic Function
UBE3A antibodies have helped elucidate the role of UBE3A in regulating synaptic function. Research has shown that UBE3A regulates the degradation of ARC, a protein involved in AMPA receptor internalization . Using UBE3A antibodies, researchers demonstrated that in Ube3A knockout mice, ARC protein levels are significantly elevated, leading to decreased AMPA receptor expression at excitatory synapses .
Co-immunoprecipitation experiments using mouse brain extracts, facilitated by UBE3A antibodies, confirmed that ARC and UBE3A interact in the intact brain . Mass spectrometry further revealed that UBE3A catalyzes the polyubiquitination of ARC on Lysine 268 and 269, marking it for degradation .
Future Perspectives in UBE3A Antibody Research
The field of UBE3A antibody research continues to evolve, with several promising directions for future development.
Emerging Technologies
Recent advances in antibody engineering, including the development of recombinant monoclonal antibodies, offer improved specificity and reproducibility for UBE3A detection . GeneTex's recombinant rabbit monoclonal UBE3A antibody represents this new generation of research tools that provide enhanced performance in various applications .
Therapeutic Applications
UBE3A antibodies are playing a critical role in developing and validating potential therapeutic approaches for Angelman syndrome. For instance, they have been instrumental in confirming the efficacy of compounds like (S)-PHA533533 in unsilencing the paternal UBE3A allele, potentially offering a treatment strategy for Angelman syndrome .
Challenges and Opportunities
Despite advances in UBE3A antibody development, challenges remain. The evaluation study of seven commercial antibodies highlighted significant variability in specificity and performance , suggesting the need for more rigorous validation protocols and potentially the development of new, more reliable antibodies.
Furthermore, as research expands into more complex models such as human cerebral organoids and patient-derived neurons, there is an opportunity to develop and validate antibodies specifically optimized for these applications, enabling more translational research into UBE3A-related disorders.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Paternal UBE3A also partially colocalizes with SOX2, a marker of neural progenitors. This suggests that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. PMID: 27306933
- In vivo patch-clamp electrophysiology studies have measured the visually evoked responses to square-wave drifting gratings in L2/3 regular-spiking (RS) neurons in control mice, Ube3a-deficient mice (Angelman syndrome model), and mice in which Ube3a was conditionally reinstated in GABAergic neurons. These studies found that Ube3a-deficient mice exhibited enhanced pyramidal neuron excitability in vivo as well as weaker orientation tuning. PMID: 28468997
- Research indicates that neuronal overexpression of Ube3a isoform 2 causes phenotypes translatable to neurodevelopmental disorders. PMID: 29016856
- Findings suggest that aberrant function of Ube3a could influence the progression of Alzheimer's disease and restoring normal levels of Ube3a might be beneficial for this disease. PMID: 29016862
- Encephalomyocarditis virus (EMCV) 3C protease accumulates to higher levels in EMCV-infected E6AP knockdown cells compared to control cells. This indicates a role for E6AP in in vivo 3C protease concentration regulation. PMID: 29054411
- The expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15(INK4b), p16(INK4a), and p19(ARF)) was decreased in E6AP(-/-) embryo fibroblasts. PMID: 28074012
- Maternal loss of Ube3a affects nociception through a central, but not peripheral, mechanism. PMID: 28931574
- Studies suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. PMID: 27339004
- Mice with maternally-inherited deletions of Ube3a, which models Angelman syndrome, show an increased social preference/interaction. PMID: 28411125
- Increasing UBE3A in the nucleus downregulates Cbln1, a glutamatergic synapse organizer, which is needed for sociability in mice. PMID: 28297715
- Research suggests that imprinting of UBE3A does not function to reduce the dosage of UBE3A in neurons, but rather to regulate some other, as yet unknown, aspect of gene expression or protein function. PMID: 28515788
- The role of UBE3A in neurite contact guidance during neuronal development has been investigated. PMID: 26845073
- Results confirm that GABAergic Ube3a loss is the primary cause of circuit hyperexcitability in Angelman syndrome mice, providing insight into ictogenic mechanisms in AS. PMID: 27021170
- Loss of Ube3a from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behavior measured by optical self-stimulation. PMID: 26869263
- Results indicate that the phenotypes observed in Angelman syndrome mice may be influenced by factors independent of Ube3a genotype. PMID: 26028516
- UBE3A dampens ERK pathway signaling in HPV E6 transformed HeLa cells. PMID: 25815718
- The normal window of development in Angelman syndrome patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this critical epoch of early development. PMID: 25894543
- 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. PMID: 25660546
- This study demonstrated that abnormal sleep patterns arise from a deficit in accumulation of sleep drive, uncovering the Ube3a gene as a novel genetic regulator of sleep homeostasis. PMID: 26446213
- The deficiency of Ube3a in Huntington's disease (HD) mice brain also caused significant increase in global aggregates load, and these aggregates were less ubiquitinated when compared with age-matched HD mice. PMID: 25027318
- There are distinct neurodevelopmental windows when Ube3a restoration rescues Angelman-syndrome-like phenotypes. Motor deficits are rescued in adolescence. Anxiety, repetitive behavior, and epilepsy are rescued in early development. PMID: 25866966
- Mature oligodendrocytes express Ube3a in the cortex and white matter tracts during development. PMID: 24254964
- Findings provide novel insight into the regulation of Ube3a by synaptic activity and its potential role in kinase regulation. PMID: 24434871
- These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome. PMID: 24385930
- Activating UBE3A disrupts circadian oscillations in mouse embryonic fibroblasts and rhythms in endogenous mRNA and protein levels of BMAL1. PMID: 24728990
- Aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt. PMID: 24706802
- This study demonstrated that changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice. PMID: 24672001
- These results demonstrate that UBE3A plays a role in MC1R transcriptional regulation. PMID: 21733131
- E6AP may negatively control adipogenesis by inhibiting C/EBPalpha expression. PMID: 23762344
- MeCP2 and E6AP play a role in the transcriptional control of common target gene expression. PMID: 23791832
- This study described a novel phenotype of severely distended Golgi cisternae in the Angelman syndrome model mouse. PMID: 23447592
- The present study is the first to determine that the Ube3a protein ablation seen in the Angelman syndrome mouse model is also characteristic of Angelman syndrome patients. PMID: 22560727
- Findings showed that Ube3a-ATS is an atypical RNAPII transcript that functions to suppress paternal Ube3a expression. PMID: 22493002
- The loss of function of Ube3a might be associated with the synaptic abnormalities observed in Huntington's disease. PMID: 22787151
- A study reports that Ube3a regulates glucocorticoid receptor (GR) transactivation. The GR signaling pathway is disrupted in Ube3a-maternal-deficient mice brain, which eventually leads to increased susceptibility to stress and anxiety in these Angelman syndrome mice. PMID: 22215440
- This study demonstrated that Ube3a-deficient mice produce an excitatory/inhibitory imbalance through neuron type-specific synaptic in visual cortex. PMID: 22681684
- Research implicates E6AP as a significant regulator of the cellular response to stress, particularly through the regulation of replicative and oncogene-induced senescence. PMID: 21927031
- Results suggest that Ube3a gene dosage may contribute to the autism traits of individuals with maternal 15q11-13 duplication and support the idea that increased E3A ubiquitin ligase gene dosage results in reduced excitatory synaptic transmission. PMID: 21974935
- In a mouse model of Angelman syndrome, hippocampal and Purkinje cells have the highest expression of UBE3A. Therefore, these cells would have the greatest sensitivity to the UBE3A m-/p+ maternal/paternal genotype. PMID: 19563863
- HERC2 acts as a regulator of E6AP. PMID: 21493713
- The levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. PMID: 20826237
- Data show RNA interference that targets Ube3a in P19 cells caused downregulation of Mc1r and Nr4a2, whereas overexpression of Ube3a results in the upregulation of Mc1r and Nr4a2. PMID: 20571502
- Ube3a exhibits brain cell type-specific imprinting, with monoallelic expression from the maternal allele in neurons, but biallelic expression in glial cells. The antisense Ube3a transcript is reciprocally imprinted only in neurons. PMID: 12668607
- Ube3a expression is primarily neuronal in all brain regions and present in GABAergic interneurons as well as principal neurons. PMID: 20423730
- E6-associated protein is required for human papillomavirus type 16 E6 to cause cervical cancer. PMID: 20530688
- Polycomb protein Ring1B regulation by self-ubiquitination or by E6-AP may have implications for the pathogenesis of Angelman syndrome. PMID: 20351251
- As demonstrated by optical imaging, rapid ocular dominance plasticity after brief monocular deprivation was severely impaired during the critical period in the visual cortex of Ube3a maternal-deficient (m-/p+) mice. PMID: 20212164
- Disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant deregulation in AMPA receptors at synapses, which may contribute to the cognitive dysfunction that occurs in Angelman Syndrome. PMID: 20211139
- The imprinted Ube3a antisense transcript is regulated by the U exons rather than Snurf/Snrpn exon 1 (Ube3a antisense RNA). PMID: 15226413
- Loss of E6AP catalytic activity and improper regulation of E6AP substrate are important in the development of Angelman syndrome. PMID: 15263005
Q&A
Basic Research Questions
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What is UBE3A and what is its primary function in neuronal cells?
UBE3A is a HECT domain E3 ubiquitin ligase that targets substrate proteins, including itself, for proteasomal degradation . It has a dual role as both an E3 ligase in the ubiquitin proteasome pathway and as a transcriptional coactivator where it co-activates steroid hormone receptors such as progesterone and estrogen . In neurons, UBE3A is expressed exclusively from the maternal allele due to tissue-specific paternal imprinting (silencing) . This protein plays a critical role in neuronal development and synaptic function, as evidenced by the severe neurodevelopmental consequences (Angelman Syndrome) that result from maternal UBE3A deficiency .
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How does UBE3A expression differ between neurons and glial cells?
UBE3A exhibits a distinct expression pattern between neurons and glial cells due to genomic imprinting. In neurons, UBE3A is expressed exclusively from the maternal allele because the paternal allele is silenced by a long non-coding RNA called UBE3A-ATS . In contrast, glial cells express UBE3A biallelically (from both maternal and paternal alleles) . This cell-type specific expression pattern is critical for understanding UBE3A's role in neurodevelopmental disorders and for designing therapeutic strategies. Immunohistochemical studies using specific antibodies have confirmed this differential expression by showing that in Ube3a maternal deficient mice (Ube3a m-/p+), neurons lack UBE3A immunoreactivity while glial cells maintain it .
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What are the known substrates of UBE3A and their relevance to neuronal function?
Several substrates of UBE3A have been identified through proteomic and biochemical studies:
Disruption of UBE3A function leads to increased Arc expression and decreased AMPA receptors at excitatory synapses, potentially contributing to cognitive dysfunction in Angelman Syndrome and ASDs .
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How does neuronal activity regulate UBE3A expression?
Experience-driven neuronal activity dynamically regulates UBE3A expression through multiple mechanisms:
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Membrane depolarization or glutamate receptor activation significantly increases UBE3A mRNA expression in cultured neurons
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Conversely, blocking neuronal activity with NMDA receptor inhibitors (APV), sodium channel blockers (TTX), or AMPA receptor inhibitors (NBQX) results in decreased UBE3A mRNA expression
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UBE3A protein levels are higher in neurons activated by membrane depolarization and lower in neurons whose activity has been blocked
This activity-dependent regulation of UBE3A suggests that it plays a role in experience-dependent synaptic plasticity, providing insight into how UBE3A dysfunction may contribute to cognitive impairments in Angelman Syndrome and autism spectrum disorders.
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