- Awarded: 2011
- Award Type: Explorer
- Award #: 221724
Autism is commonly seen in people with the genetic disorder tuberous sclerosis complex (TSC). TSC involves two genes, TSC1 and TSC2, encoding the proteins hamartin and tuberin, respectively. TSC proteins regulate an important signaling pathway known as the mTOR pathway, which plays an essential role in interconnectivity and communication between neurons in the brain.
A brain-specific protein known as PAM plays a critical role in communication between neurons. Vijaya Ramesh and her team’s earlier studies documented that PAM interacts with the TSC protein complex and regulates mTOR signaling1,2. One of the roles of PAM in neurons is as an E3 ubiquitin ligase, whereby it targets a selected set of proteins for ubiquitin-mediated degradation. This helps to maintain a delicate balance between protein synthesis and protein degradation at synapses.
Ramesh and her group previously reported that some, but not all, of the neurodevelopmental defects observed in mice deficient in PHR1 (the PAM homolog in mice) are due to dysregulation of mTOR signaling3. This suggests that PAM targets additional synaptic proteins that are responsible for the additional neurodevelopmental defects in PHR1-deficit mice that are independent of mTOR signaling.
As part of the current study, Ramesh and her team proposed to identify proteins that are targeted for degradation by PAM, which will have direct relevance for autism and other complex brain disorders. The researchers crossed hemagglutinin-tagged ubiquitin (HA-Ub) mice with PHR1-Magellan transgenic mice (Phr1Mag), which express a stable C-terminal truncated version of PHR1 that lacks E3 ubiquitin ligase activity. Transmission of the PHR1Mag transgene is autosomal, however, transmission of the HA-Ub transgene follows X-linked inheritance (XHA-Ub): Males can be wildtype or heterozygous and females can be wildtype, heterozygous, or homozygous for HA-Ub. First-generation (F1) double-heterozygous mice are viable and fertile. In addition, the group bred F1 mice to obtain double-homozygous F2 mice (XHA-UbXHA-Ub/PHR1Mag/Mag or XHA-UbY/PHR1Mag/Mag) and respective littermate controls.
In parallel, the researchers are undertaking complementary in vitro studies, which involve utilizing proteomic approaches to identify PAM-associated proteins in cultured HEK293T cells. Due to the large size of PAM, the researchers have cloned five overlapping fragments spanning the entire coding region of 4,640 amino acids for expression in cells, which is in progress.