Cells continuously respond to physiological signals and potentially pathological perturbations. In response, protein synthesis and protein degradation, the latter of which is predominantly driven by the ubiquitin- proteasome system, reciprocally remodel the intracellular proteome. The dynamics of protein turnover determine the physiological response to a large diversity of signals or perturbations and have major ramifications on human physiology. Indeed, over four decades of work on the ubiquitin conjugating cascade and the 26S proteasome has elucidated essential roles for the ubiquitin-proteasome system in nearly every cellular process. The prevailing principles in protein turnover have been that ubiquitylation is necessary for substrate tagging and that the 26S proteasome is the only proteasome species that degrades ubiquitin-protein conjugates. Though 20S proteasomes form the core of the 26S complex, they remain largely understudied because of a prior lack of clear evidence for functional 20S particles in cells and no insight into 20S-specific substrate targeting. I recently discovered a new mechanism of ubiquitin-independent protein turnover through a highly specialized 20S proteasome that is tightly associated with neuronal plasma membranes. These neuronal membrane proteasomes (NMPs) directly associate with ribosomes to degrade ~250-500 nascent chain substrates independent of ubiquitylation. The NMP degrades substrates across the membrane, releasing resulting peptide fragments into the extracellular space that induce signaling in other neurons, and therefore represents a new mechanism of neuromodulation. Here, I propose studies that will lay the foundation necessary to understand this new paradigm in protein turnover. In my first aim, I will identify how NMPs associate with the plasma membrane and reveal the molecular components of this membrane complex. In the second aim, I will determine how the specificity of NMP-mediated degradation of nascent chains is achieved. In the final aim, I will gain insights into the biological processes that NMP-mediated degradation regulates. The proposed research is significant because it opens a new field of research into non-canonical protein turnover in neurons. This work will generate the tools and mechanistic insight necessary to understanding how NMP-mediated degradation is codified in and relevant to the vertebrate nervous system. This will not only shed light onto the new mechanism of neuromodulation through NMPs, but also provide a framework relevant to abnormalities in protein turnover that underlie multiple human neuropathologies.
Deficits in proteasome-mediated protein turnover, which was previously thought to be a monolithic process, have been shown to underlie many neurological disorders. The proposed research will yield fundamental knowledge about the mechanisms, role, and significance of a novel and unstudied process of protein turnover through neuronal-specific plasma membrane-bound proteasomes. Understanding this differentiated process of protein turnover will help reveal the causality of neurological disorders specific to degradation through these neuronal membrane proteasomes and provide the knowledge and tools required to target this system for therapeutic benefit.
