Autism spectrum disorders (ASDs) are neurodevelopmental syndromes associated with diverse genetic alterations. One of the chromosomal abnormalities observed most frequently in ASD is duplication of chromosomal region 15q11.2-13.1. Genetic loci within this region include the UBE3A gene, which encodes the E3 ubiquitin ligase UBE3A/E6AP. Increased gene dosage of UBE3A is likely to be a key driver for autistic traits, as mice with triple Ube3a gene dosage exhibit decreased social interaction, impaired communication and increased repetitive behaviors. Furthermore, loss of the maternally inherited UBE3A allele causes Angelman syndrome, recapitulating the importance of UBE3A in neurodevelopment.
Despite the prominent association of UBE3A with ASD, the mechanisms by which deregulated UBE3A expression leads to neurodevelopmental conditions are unknown. In particular, information about physiological substrates of UBE3A-mediated ubiquitination is limited, leaving the downstream pathways poorly defined. This gap in knowledge is mostly due to technical challenges associated with identifying ubiquitination substrates of E3 ligases.
To overcome these challenges, Hiroaki Kiyokawa and his colleagues at Northwestern University, in collaboration with Jun Yin’s laboratory at Georgia State University, developed a proteomic screening method called ‘orthogonal ubiquitin transfer’ (OUT)1. OUT is based on the engineering of ubiquitination enzymes (E1-E2-E3) in such a way so as to generate a mutant enzyme cascade (designated as xE1-xE2-xE3) orthogonal to its native cascade, which enables the tracking of proteins conjugated with an affinity-tagged mutant ubiquitin (xUB) in cells. Their recent OUT screens for UBE3A substrates in vitro (using Human Embryonic Kidney 293 cells) identified over 100 potential substates, including β-catenin (a protein encoded by CTNNB1, which is also a high-confidence risk gene for ASD) and several other known targets1. The successful development of the xUBE3A OUT cascade prompted Kiyokawa and his team to identify neuronal-specific UBE3A substrates and reveal their roles in the pathobiology of ASD.
In the current project, Kiyokawa’s team plans to engineer induced pluripotent stem cells (iPSCs) from healthy individuals for physiologically controlled expression of the xUBE3A OUT cascade. They will then conduct screens for substrates in iPSC-derived neural progenitor cells and cortical neurons. Bioinformatic analyses of substrates identified by the screens and subsequent biochemical and cellular examinations will reveal ASD-relevant roles of UBE3A-mediated ubiquitination.
This project is expected to provide a novel high-resolution perspective about neuronal-specific pathways controlled by UBE3A-mediated ubiquitination and identify potential therapeutic targets for ASD2.