Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes

Martínez-Noël, Gustavo; Luck, Katja; Kühnle, Simone; Desbuleux, Alice; Szajner, Patricia; Galligan, Jeffrey T.; Zheng, Léon; Boyland, Kathleen; Leclere, Flavian; Zhong, Quan; Hill, David E.; Vidal, Marc; Howley, Peter M.

doi:10.1016/j.jmb.2018.01.021

Cited by 36 publications

(31 citation statements)

References 188 publications

(237 reference statements)

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“…The 26S proteasome is one of the major cellular complexes interacting with UBE3A (21)(22)(23)(24)(25)(26)(27)(28)(29). Although immunoprecipitation and MS cannot distinguish which specific protein subunit within the 26S proteasome directly binds UBE3A, PSMD4 was the only proteasomal subunit identified as a direct UBE3Abinding partner in a yeast two-hybrid screen we recently reported, suggesting that PSMD4 likely bridges the interaction of UBE3A with the 26S proteasome (24).…”

Section: The 26s Proteasome Binds To the N Terminus Of Ube3amentioning

confidence: 85%

“…We recently identified PSMD4 as a UBE3A-interacting protein in a yeast two-hybrid screen, indicating that PSMD4 is likely a direct interaction partner of UBE3A (24). To examine this possibility further, we tested binding of the isolated UBE3A AZUL domain to PSMD4 with recombinant proteins expressed in bacteria.…”

Section: Characterizing the Interaction Of Ube3a/e6ap With Psmd4mentioning

confidence: 99%

“…Although the proteins of the lid complex are responsible for the recruitment of ubiquitylated proteins, the barrel and its intrinsic proteolytic activities mediate the breakdown of proteins (20). UBE3A associates with the proteasome (21)(22)(23)(24)(25)(26)(27)(28)(29). In that context, the UBE3A-interacting protein PMSD4 (also known as Rpn10/S5a) is part of the regulatory lid complex and known to bind to polyubiquitin chains of ubiquitylated proteins via its ubiquitin-interacting motif (UIM), resulting in their recruitment to the proteasome (30 -32).…”

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confidence: 99%

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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

Kühnle¹,

Martínez-Noël²,

Leclere³

et al. 2018

Journal of Biological Chemistry

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Edited by George N. DeMartino 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 Zn 2؉-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.

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Section: The 26s Proteasome Binds To the N Terminus Of Ube3amentioning

confidence: 85%

Section: Characterizing the Interaction Of Ube3a/e6ap With Psmd4mentioning

confidence: 99%

mentioning

confidence: 99%

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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

Kühnle¹,

Martínez-Noël²,

Leclere³

et al. 2018

Journal of Biological Chemistry

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“…E6AP is known to ubiquitinate and degrade wide range of proteins that include p53, PML, C/EBPα, MNT, G‐CSFR and others involved in various cellular functions . Dysregulation of E6AP expression and function has been associated with numerous diseases such as cervical carcinogenesis, Angelman syndrome and others . In a recent study E6AP was shown to act as a tumor suppressor in non‐small cell lung cancer by maintaining INK4/ARF expression levels .…”

Section: Introductionmentioning

confidence: 99%

Nano‐LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells

et al. 2019

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E6AP (E6 associated protein) is a HECT domain containing protein having dual E3 ligase and ERα coactivation activity in breast cancer cells. Although E6AP is known to possess antitumorigenic activity, the underlying molecular mechanism is poorly understood. In the present study, we applied nano-LC based proteomics approach to identify E6AP-interacting proteins where we performed GST-pull down using GST-E6AP from whole cell extracts of MCF7 cells, resolved the differentially interacting proteins on 1D-SDS-PAGE, excised the gel bands that were trypsin digested followed by fractionation and spotting on MALDI-TOF/TOF plate through Nano-LC MALDI spotter. Subsequently, fractionated and spotted peptides were identified using MALDI-TOF/TOF. We identified several E6AP interacting proteins including previously reported such as HSP70 and new ones such as Enolase-1. We further confirmed that E6AP and Enolase1 interacted and colocalized more in the cytoplasmic periphery in breast cancer cells and further demonstrated that E6AP also targeted ENO1 for ubiquitin-mediated degradation in these cells. K E Y W O R D Sbreast cancer, E6AP, Enolase-1, mass spectrometry, Nano-LC-MALDI spotter Abbreviations: Co-IP, Co-immunoprecipitation; E6AP, E6-associated protein; ENO1, Enolase1; WCEs, whole cell extracts.

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“…Recent research has illuminated diverse roles for UBE3A in neuronal and cellular functions. Proteomic analyses have suggested that UBE3A and its interacting proteins integrate several cellular processes including translation, intracellular trafficking, and cytoskeleton regulation, which are all necessary for neuronal function (8,9). In line with its role in neurodevelopmental disease, UBE3A has also been shown to be critical in synaptic formation and maintenance (10–12).…”

Section: Introductionmentioning

confidence: 99%

Imprinting effects of UBE3A loss on synaptic gene networks and Wnt signaling pathways

Lopez

Laufer

Beitnere

et al. 2019

Preprint

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17 The genomically imprinted UBE3A gene encodes a E3 ubiquitin ligase whose loss from 18 the maternal allele leads to the neurodevelopmental disorder Angelman syndrome.19 However, the mechanisms by which loss of maternal UBE3A contribute to severe 20 neurodevelopmental phenotypes are poorly understood. Previous studies of UBE3A 21 function have focused on mouse models or single targets, but these approaches do not 22 accurately reflect the complexity of imprinted gene networks in the brain nor the 23 systems-level cognitive dysfunctions in Angelman syndrome. We therefore utilized a 24 systems biology approach to better elucidate how UBE3A loss impacts the early 25 postnatal brain in a novel CRISPR/Cas9 engineered rat Angelman model of a complete 26 Ube3a deletion. Strand-specific transcriptome analysis of offspring derived from 27 maternally or paternally inherited Ube3a deletions revealed the expected parental 28 expression patterns of Ube3a sense and antisense transcripts by postnatal day 2 (P2) 29 in hypothalamus and day 9 (P9) in cortex, when compared to wild-type sex-matched 30 littermates. The dependency of genome-wide effects on parent-of-origin, Ube3a 31 genotype, and time (P2, P9) was investigated through transcriptome (RNA-seq of cortex 32 and hypothalamus) and methylome (whole genome bisulfite sequencing of 33 hypothalamus). Weighted gene co-expression and co-methylation network analyses 34 identified co-regulated networks in maternally inherited Ube3a deletion offspring 35 correlated with postnatal age that were enriched in developmental processes including 36 Wnt signaling, synaptic regulation, neuronal and glial functions, epigenetic regulation, 37 ubiquitin, circadian entrainment, and splicing. Furthermore, using this novel rat model, 38 we showed that loss of the paternally expressed Ube3a antisense transcript resulted in 3 39 both unique and overlapping dysregulated gene pathways, predominantly at the level of 40 differential methylation, when compared to loss of maternal Ube3a. Together, these 41 results provide the most holistic examination to date of the molecular impacts of UBE3A 42 loss in brain, supporting the existence of interactive epigenetic networks between 43 maternal and paternal transcripts at the Ube3a locus. 4445 Author Summary 46 The neurodevelopmental disorder Angelman syndrome is caused by loss of UBE3A 47 from the maternal chromosome. UBE3A is a genomically imprinted gene, which results 48 in parent-of-origin specific expression of a protein from the mother and a noncoding 49 RNA from the father. While mouse models have been useful in investigating diverse 50 roles for UBE3A, their partial mutations are of limited utility for investigating parental 51 imprinting effects or identifying a complete list of downstream differences in gene 52 pathways relevant to developing therapies for Angelman syndrome. To address this 53 limitation, we utilized a novel rat model with a CRISPR/Cas9 engineered full UBE3A 54 deletion and systems biology approaches to better understand how UBE3A los...

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Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes

Cited by 36 publications

References 188 publications

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

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

Nano‐LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells

Imprinting effects of UBE3A loss on synaptic gene networks and Wnt signaling pathways

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