a-scalable-cell-based-method-for-functional-assessment-ube3a

A Scalable, Cell-Based Method for the Functional Assessment of <em>Ube3a</em> Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

Washington University School of Medicine

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 β-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 suggest that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

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

The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

Article	Year
The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome. The Journal of biological chemistry\| PubMed ID: 28559284	2017
Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis. Nature communications\| PubMed ID: 34815418	2021

Jason J. Yi

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