Dysfunction of the UBE3A gene encoding the HECT-type ubiquitin ligase (E3) UBE3A/E6AP is implicated in autism spectrum disorder (ASD). Increased UBE3A dosage due to duplication of chromosomal region 15q11-13.1 is a critical determinant of penetrance for autism, while the loss of the maternally inherited UBE3A allele causes Angelman syndrome (AS). Numerous experimental studies suggest that UBE3A is a dosage-sensitive gene and that the expression level of UBE3A in neurons must be regulated tightly within a physiologically tolerated range during brain development. Despite the prominent association of UBE3A with ASD, the mechanisms by which deregulated UBE3A expression leads to neurodevelopmental disorders are unknown. In particular, information about physiological substrates of UBE3A-mediated ubiquitination is limited, leaving the downstream pathways poorly understood. This gap in knowledge is mostly due to the technical challenges of identifying direct ubiquitination substrates of E3s. To identify previously undefined substrates of UBE3A and other E3s, we recently developed a proteomic screening method called Orthogonal Ubiquitin Transfer (OUT). OUT is based on engineering of ubiquitination enzymes (E1-E2-E3) to generate a mutant enzyme cascade (designated as xE1-xE2-xE3) orthogonal to its native cascade, which enables tracking of proteins conjugated with an affinity-tagged mutant ubiquitin (xUB) in cells. Our recent OUT screens for UBE3A substrates identified over 100 potential targets in HEK293 cells, including known substrates, e.g., HHR23A (RAD23A), HHR23B (RAD23B), PSMD4/S5a, RNF2, ADRM1, and -catenin. The successful development and application of the xUba1(xE1)-xUbcH7(xE2)-xUBE3A(xE3) OUT cascade now prompts us to identify neuron-specific UBE3A substrates and reveal their roles in the pathobiology of ASD and AS. In this proposal we will engineer induced pluripotent stem cells (iPSCs) for physiologically controlled expression of the UBE3A OUT cascade and conduct screens for substrates in iPSC-derived neural progenitor cells (NPCs) and forebrain cortical neurons.
|Effective start/end date||12/1/20 → 11/30/22|
- Simons Foundation (645756)