Function and regulation of the Cdc48-20S proteasome

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Proteasomes, the major ATP-dependent proteases in the eukaryotic and archaeal domains of life, are required for degradation of damaged or misfolded proteins during protein-quality control and for regulation of diverse cell-signalling pathways. To execute protein degradation, the 20S peptidase combines with the hexameric AAA+ enzymes of the 19S regulatory particle in eukarya or with the AAA+ ring of archaeal PAN.In pilot studies, I identified the Cdc48-20S complex as a novel proteasome in the archaeon Thermoplasma acidophilum. Cdc48 (alias p97/VCP/VAT) is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea and eukarya. The physiological role of archaeal Cdc48 has long been uncertain, but eukaryotic Cdc48 participates by largely unknown mechanism in a myriad of biological processes, including membrane fusion, cell-cycle regulation, and the ubiquitin-proteasome-system. Intriguingly, the sequence and structural features that allow archaeal Cdc48 and 20S to collaborate in protein degradation are also conserved in the eukaryotic enzymes. Proteolysis in collaboration with the 20S peptidase may therefore be an ancestral function of the Cdc48 family that has have been preserved over billions of years of evolution.Key questions about the modus operandi, physiological function, and cellular regulation of this novel proteasome remain to be answered. Solving a high-resolution cryo-EM structure of the archaeal Cdc48-20S proteasome will help understanding how both macromolecules interact. I will also explore how adapter proteins interact with Cdc48, affect its conformation, and allosterically regulate its function using biochemical interaction studies, mechanistic enzymology, and biophysical characterization. Finally, I will test the exciting possibility that an alternative Cdc48-20S proteasome also functions in the eukaryotic domain of life.I aim to understand how native and misfolded proteins are recognized, unfolded, translocated, and finally degraded by Cdc48-20S proteasome. My research will answer questions about the evolutionary origins of proteasomes and will provide insights into the operational principles of this ATP-dependent protease and related enzymes. My work will also help to clarify the role of eukaryotic Cdc48 during ubiquitin-dependent proteolysis, cell cycle regulation, and protein-misfolding diseases.