Exploring the Structure and Function of Proteasome Complexes

The proteasome is essential in all eukaryotes for the highly regulated ATP dependent proteolysis of ubiquitin-tagged proteins, including key cell regulators. In eukaryotes its proteolytic core is formed by four hetero-heptameric rings arranged as an α_1-7β_1-7β_1-7α_1-7 barrel shaped stack, the full activation of which requires the binding of regulators at the outer surfaces of the α‑subunits. The 19S regulatory particle is the proteasome activator that recruits fully folded ubiquitinated proteins for degradation, associates with the core to form the fully active 26S proteasome and comprises a remarkable variety of functions, including ubiquitin receptors, deubiquitinases, ATP dependent unfoldase and translocase, and scaffolding. These are accomplished by at least 18 distinct canonical subunits, together with additional transiently bound proteins that further modulate the proteasome activity. Recently there was substantial progress in determining the 26S proteasome structure. However, a complete characterisation of how the proteasome components orchestrate the individual steps of substrate processing, from their recognition to the release of small peptides, is still missing. This information is required to fully understand the proteasome fundamental role and in order to optimise specific ligands as therapeutic drugs. We are developing new approaches for the preparation of proteasome complexes, namely by exploring recombinant expression systems, in order to obtain samples highly suitable for a detailed in vitro functional characterisation. These samples are also highly suitable for structural analysis, for which we use high resolution cryo‑EM.