We are interested in the structural and biochemical underpinnings of neurotransmission and neurodegeneration, as well as the homeostasis of the proteins contributing to these processes. Primarily, we will be applying cryo-electron microscopy (cryo-EM) to reconstruct the biochemical processes underlying these problems. We firmly believe that the most informative mechanistic insights into biology come from a deep understanding of the biological machines that govern them.
The vast majority of synapses (~90%) in the central nervous system (CNS) are glutamatergic. Here, ionotropic glutamate receptors (iGluRs) in the post-synaptic membrane bind glutamate released from pre-synpatic neurons, which triggers cation influx into the post-synaptic neuron and depolarization. A subset of these iGluRs, α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) receptors, dictate fast excitatory neurotransmission and thus the initial depolarization of the post-synaptic neuron. AMPA receptor complexes at the post-synaptic membrane, and the dynamics of their recruitment to, and removal from it, have profound effects on synaptic plasticity – the process by which patterns of synaptic activity result in a strengthening or weakening of transmission. The altered neurotransmission affects high cognitive processes such as learning and memory, and its dysregulation leads to neurodegeneration. A wealth of processes regulates AMPA receptors, from assembly in the endoplasmic reticulum to their function within the post-synaptic membrane, and their eventual endocytosis and degradation. We aim to reconstitute these processes in vitro to characterize them biochemically, and to use cryo-EM and structural biology techniques to understand structure-function relationships. We expect for these studies to not only provide new insights into the basic mechanisms of neurotransmission and insights into neurodegeneration, but also lay new foundations for rational drug design.
Ubiquitin, AAA+ ATPases, and Protein Quality Control
Protein quality control and recycling are essential for cell viability and life. Ubiquitin is a post-translational modification that marks proteins for degradation by the proteasome so that they may be recycled. Generally, this is a critical process for cellular upkeep and protein quality control. However, proteins in tight complexes, or embedded within membranes, are not immediately accessible to the proteasome for degradation, even when tagged with ubiquitin. These proteins are prepared for degradation and made accessible for proteasomal degradation by first being extracted and unfolded by the AAA+ (ATPases Associated with diverse cellular Activities) p97. AAA+ ATPases, typically hexameric assemblies, convert energy from ATP hydrolysis to translocate and/or unfold proteins. We are particularly interested in how p97 and its co-factors alter neuronal protein homeostasis, with an emphasis on impacting iGluR homeostasis. p97 also relies on many co-factors for sub-cellular targeting of its function, and ablation of function often leads to neurodegeneration. For example, one such p97 co-factor, ataxin, forms fibrils in Machado-Joseph disease/SCA3, which would ultimately alter p97-dependent homeostasis. We aim to characterize these processes biochemically through in vitro reconstitution and reconstruct them structurally with cryo-EM. These data will provide new insights into neuronal protein homeostasis and lay foundations for rational drug design.