Cryo-EM studies of the proteasome: from function to drug design

Abstract

In recent years the field of biological structural electron microscopy has seen an enormous transformation. Cryo-EM is now recognised as a high resolution structural biology technique, with its own advantages and applications compared with other methods. We use cryo-EM as a tool to investigate the eukaryotic proteasome, a protease complex essential in all eukaryotes. The proteasome plays a key role in proteostasis and also in the highly regulated degradation of crucial proteins, the removal of which signals for fundamental processes such as cell cycle progression. The canonical proteasome substrates are proteins specifically tagged for degradation by K48 linked ubiquitin chains. While there has been significant progress towards the understanding of the proteasome architecture, a complete characterisation of how its 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’s fundamental role and in order to optimise specific inhibitors as therapeutic drugs: some such inhibitors are already in clinical use mainly against multiple myeloma. Our aim is to elucidate the functional mechanisms of the human proteasome, and for that we are improving our biochemical approaches to prepare and characterise relevant complexes, the structure of which we study by cryo-EM. We also investigate the use of cryo-EM and image processing for the study of inhibitor-bound proteasome complexes, revealing advantages of these methods for the overall study of protein‑ligand interactions. In this we have contributed to the validation of the Plasmodium falciparum proteasome as a therapeutic target and our high resolution cryo-EM structures are now being used to develop potential new antimalarials.