Insights into Proteasome Function

Synthesis and degradation of proteins in a cell must be carefully balanced to ensure organismal health. The 26S proteasome is a large protein complex that carries out most regulated protein degradation in eukaryotes. The assembly, structure, and function of proteasomes is highly conserved throughout the eukaryotic kingdom, and proteasomal proteolysis regulates nearly every biological process. Since degradation is irreversible, it is critical for proteasomes to function in a tightly regulated manner. Dysfunction of the proteasome is associated with multiple human diseases, including cancers and neurodegeneration, so it is imperative we gain insight into the requirements for proteasome activation and efficient turnover. The work described in this dissertation aims to uncover the mechanistic underpinnings of the proteasomal substrate processing toward the goal of therapeutically exploiting the proteasome for clinical intervention. The proteasome comprises over 70 individual subunits that must work in concert to selectively bind, unfold, and proteolyze substrates. Communication between subunits is a complex language dependent upon substrate-binding, ATP-binding, ATP-hydrolysis, conformational shifting, and more. Recent studies have contradicted what was long thought to be what triggers the proteasome to opens its central channel for substrate entry, so closer inspection was required. The first chapter of this dissertation sought to uncover how ATP-binding induced proteasome activation and opening of the central channel through which substrates are translocated to the peptidase active sites. Intersubunit communication is further explored in the second chapter with the investigation of how the smallest and sole intrinsically disordered subunit of the proteasome, Sem1, is responsible for enhancing substrate degradation despite making up less than 0.4% of the mass of the mature proteasome. Together, this work provides new insights into the mechanism of substrate degradation and furthers our understanding of how these processes may be compromised in proteostasis-imbalanced diseases.