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

Abstract

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.

Stem cell models of Angelman syndrome

“…There is a wide range of reported adverse mutations in the maternal copy of the UBE3A gene that cause AS (AS Mut), most of which are nonsense mutations that lead to frameshifts and premature stop codons ( Sadikovic et al, 2014 ). Besides mutations that result in the truncation of the maternal UBE3A transcript, loss of function of UBE3A may also result from missense mutations affecting active domains and protein stability ( Beasley et al, 2020 ), intracellular localization ( Bossuyt et al, 2021 ), and even gain of function ( Weston et al, 2021 ), although the latter is often associated with Dup15q syndrome and not AS ( Copping et al, 2017 ; Xing et al, 2023 ). AS individuals carrying UBE3A mutations often present the mildest phenotypes out of the four (epi)genetic causes, with less pronounced development delay ( Gentile et al, 2010 ; Yang et al, 2021 ), as seen by clinical scales of development in infancy ( Keute et al, 2021 ).…”

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases

“…Destabilization of the trimer formation also seems to be a feature of a specific loss-of-function Angelman syndrome mutation [ 33 ]. An AS-associated transversion mutation found in the HECT domain of UBE3A I827K destabilizes protein folding and causes C-lobe aggregation, which hinders UBE3A enzymatic activity [ 34 ]. A specific c-Abl-mediated tyrosine residue phosphorylation in UBE3A HECT domain has also been linked to its oligomerization and activity [ 35 ].…”