Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome

Trezza, Rossella Avagliano; Sonzogni, Monica; Bossuyt, Stijn N V; Zampeta, F. Isabella; Punt, A. Mattijs; Berg, Marlene van den; Rotaru, Diana C.; Koene, Linda M. C.; Munshi, Shashini T.; Stedehouder, Jeffrey; Kros, Johan M.; Williams, Mark; Heussler, Helen; Vrij, Femke M.S. de; Mientjes, Edwin; Woerden, Geeske M. van; Kushner, Steven A.; Distel, Ben; Elgersma, Ype

doi:10.1038/s41593-019-0425-0

Cited by 71 publications

(93 citation statements)

References 56 publications

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“…2c and 2d). Altogether, these experiments indicate direct binding between E6AP AZUL and hRpn10 305–377 , consistent with recent publications implicating AZUL and hRpn10 to E6AP interaction with the proteasome 43, 45 .…”

Section: Resultssupporting

confidence: 91%

“…As human E6AP isoform 3 localizes to the nucleus in an hRpn10- and AZUL-dependent manner 43 , it may be that the E6AP’s function in the nucleus is primarily related to its association with hRpn10. By contrast, the decreased affinity for E6AP when RAZUL is phosphorylated at Y326 may imply that certain cellular contexts require less E6AP associated with the proteasome.…”

Section: Discussionmentioning

confidence: 99%

“…How aberrant E6AP mechanistically contributes to these neurological diseases is an active area of investigation. It distributes in an isoform-dependent manner between the nucleus and cytosol of neurons 42, 43 and contains an N-terminal Zn-binding AZUL ( a mino-terminal z inc-binding domain of u biquitin E3a ligase) domain 44 that binds to Rpn10 in the proteasome 45 , and is required for E6AP nuclear localization 43 . An Angelman syndrome-associated missense mutation in the E6AP HECT domain interferes with E6AP nuclear localization 43 , although the connection between this mutation and the requirement for the AZUL domain is not known.…”

Section: Introductionmentioning

confidence: 99%

“…It distributes in an isoform-dependent manner between the nucleus and cytosol of neurons 42, 43 and contains an N-terminal Zn-binding AZUL ( a mino-terminal z inc-binding domain of u biquitin E3a ligase) domain 44 that binds to Rpn10 in the proteasome 45 , and is required for E6AP nuclear localization 43 . An Angelman syndrome-associated missense mutation in the E6AP HECT domain interferes with E6AP nuclear localization 43 , although the connection between this mutation and the requirement for the AZUL domain is not known. E6AP also stimulates Wnt/β-catenin signaling, a function that requires its ubiquitin ligase activity and interaction with the proteasome, and is disrupted by an autism-linked E6AP mutation 45, 46 .…”

Section: Introductionmentioning

confidence: 99%

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Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Buel

Chen

Chari

et al. 2019

Preprint

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Regulated proteolysis by the proteasome involves ~800 enzymes for substrate modification with ubiquitin, of which ~600 are E3 ligases. We report here that E6AP/UBE3A is distinguished from other ubiquitin E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, this domain in hRpn10 locks into a novel well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL domain, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at the proteasome, where associated ubiquitin is correspondingly reduced, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10. KeywordsRpn10/PSMD4/S5a; E6AP/UBE3A; proteasome; ubiquitin E3 ligase; AZUL domain 1 1 RAZUL ( Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 4c). Structure of the RAZUL:AZUL complexAt the molecular interface, a 4-helix bundle is formed by two pairs of helices from AZUL and RAZUL stacking against each other (Fig. 4b). RAZUL α1 is centered between the two AZUL helices by hydrophobic interactions involving F334, L335, V338 and L339 as well as L342 and V345 from the RAZUL α1/ α2 loop ( Fig. 5a and 5b). These residues interact with A29, I33, and Y37 from AZUL H1 and A69 and L73 from AZUL H2 ( Fig. 5a and 5b). From the 3 10 -helix, V328 and M329 form hydrophobic interactions with AZUL A29, L73 and Y76 (Fig. 5c), capping the hydrophobic contact surface formed by RAZUL α1. RAZUL α2 is more peripheral compared to α1, with A351, I352, A355, M356, and L359 interacting with A67, L70 and L73 from AZUL H2 (Fig. 5d).The RAZUL N-terminal end (E322-D327) is rich in acidic residues ( Supplementary Fig. 1a) and proximal to the positively charged AZUL N-terminal end, which includes K25 and R26 (Fig. 5e).These AZUL residues contribute three hydrogen bonds to the complex, engaging RAZUL E323, D324, and Y326 (Fig. 5c). Y326, which is phosphorylated in Jurkat cells 60 , forms a hydrogen bond with the AZUL R26 sidechain in 80% of calculated structures, as well as hydrophobic contacts with AZUL K25 and R26 (Fig. 5c). We tested whether adding a bulky phosphate group at this location could be deleterious for AZUL binding by synthesizing RAZUL peptides that span E322-D366 without and with Y326 phosphorylated and measuring affinity by ITC. This shorter wild-type peptide bound with an affinity within error of hRpn10 305-377 ( Fig. 5f and Supplementary samples were validated by mass spectrometry. 26S proteasome (human) was purchased (Enzo Life Sciences, Inc. BML-PW9310). Peptide synthesishRpn10 322-3...

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Buel

Chen

Chari

et al. 2019

Preprint

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“…Interestingly, only very recently, the critical importance of the nuclear isoform of UBE3A for the Angelman syndrome phenotype was characterized. Mice lacking the nuclear isoform but not mice lacking the cytosolic isoform displayed all major behavioural phenotypes and synaptic deficits also seen upon complete UBE3A knockout in the previous Angelman syndrome mouse model 54 . The hypothesis of ubiquitin-ligase dependent regulation of a putative nuclear substrate such as MEF2C by UBE3A is also supported by our genetic interaction findings.…”

Section: Genetic Interaction Between Ube3a and Mef2mentioning

confidence: 83%

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Straub

Gregor

Sauerer

et al. 2020

Sci Rep

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neurodevelopmental disorders (nDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities.

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Ubiquitin receptors play redundant roles in the proteasomal degradation of the p53 repressor MDM2

2022

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Much remains to be determined about the participation of ubiquitin receptors in proteasomal degradation and their potential as therapeutic targets. Suppression of the ubiquitin receptor S5A/PSMD4/hRpn10 alone stabilises p53/ TP53 but not the key p53 repressor MDM2. Here, we observed S5A and the ubiquitin receptors ADRM1/PSMD16/hRpn13 and RAD23A and B functionally overlap in MDM2 degradation. We provide further evidence that degradation of only a subset of ubiquitinated proteins is sensitive to S5A knockdown because ubiquitin receptor redundancy is commonplace. p53 can be upregulated by S5A modulation while degradation of substrates with redundant receptors is maintained. Our observations and analysis of Cancer Dependency Map (DepMap) screens show S5A depletion/loss substantially reduces cancer cell line viability. This and selective S5A dependency of proteasomal substrates make S5A a target of interest for cancer therapy.

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Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome

Cited by 71 publications

References 56 publications

Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Ubiquitin receptors play redundant roles in the proteasomal degradation of the p53 repressor MDM2

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