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.
The fibulocalcaneus (peroneocalcaneus) internus (PCI) muscle is a rare variant leg muscle with a low prevalence of <1%‐3% in cadaveric dissection and imaging studies. The PCI arises from the medial aspect of the distal third of the fibula, descends posterior and lateral to the flexor hallucis longus (FHL) muscle, traverses the tarsal tunnel inferior to the sustentaculum tali of the calcaneus, and inserts into the plantar surface of the calcaneus. However, controversy exists concerning the exact location of the insertion site of the PCI muscle. The insertion of the PCI muscle was recently described as being located distal to the coronoid fossa, a small depression between the anterior tuberosity and the anterior apex of the sustentaculum tali of the calcaneus. However, historical descriptions described the PCI as inserting into either the sustentaculum tali itself or a small tubercle on the medical surface of the calcaneus distal to the sustentaculum tali. During routine dissection of a 53‐year‐old Caucasian male, a PCI muscle was identified, and it inserted into distal to the coronoid fossa on the plantar surface of the calcaneus. More specifically, it inserted into the plantar surface of the calcaneus in the space between the origin of the plantar talonavicular (spring) ligament and the plantar calcaneocuboid ligament. Knowledge of the PCI muscle is important due its involvement with various ankle pathologies, including predisposing individuals to tarsal tunnel syndrome, FHL tenosynovitis, and posterior ankle impingement and pain.
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