Deciphering the mechanism of the human proteasome by mass photometry

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In every eukaryotic cell, the proteasome is a vital molecular machine responsible for degradation of polypeptides. It recycles proteins into reusable amino acids and prevents accumulation of peptides into toxic aggregates. For this, the proteasome recognizes proteins flagged by a ubiquitin tag and pulls them in its catalytic lumen for degradation. The proteasome is a central part of many regulatory pathways as it facilitates adjusting the relative concentrations of proteins. This renders it an ideal target for drugs addressing cells which suffer from miss-regulated protein degradation, e.g. caused by neurodegenerative diseases or cancer.For targeted drug design, a profound understanding of the interaction between ubiquitin and the proteasomal recognition sites as well as the proteasome’s catalytic action is essential. Although the human 26S proteasome is being studied for several decades, kinetic data is barely available and requires merging results of an immense amount of different techniques. Consequently, a comprehensive understanding of substrate recognition, binding, and targeted degradation remains elusive. This is due to the fact that mainly bulk experiments are available, which do not allow tracking the complex catalytic proceedings of the 26S proteasome.In the described research action, I will make use of a novel technique, which detects light scattered by single molecules without the need for introducing artificial labels: interferometric scattering microscopy (iSCAT). When combined with a suitable calibration, this technique –termed mass photometry (MP)– enables determining the mass of proteins at a precision of up to tens of kDa and within few seconds.Mass photometry will allow me to study the interaction between substrates and the 26S proteasome. I aim to analyze binding of substrates to the proteasome and the downstream processing steps of deubiquitylation as well as proteolysis in a single-molecule-based assay depending on the quality of the ubiquitin tag. First, I will apply MP to determine the average binding affinities and deubiquitylation/proteolysis rates by acquiring equilibrium mass distributions of ubiquitylated substrates and proteasomes. In a second set of experiments, the mass distribution of the protein complexes in solution will be measured as a function of time after rapid mixing. This will yield separate kinetic information for the processes after binding (deubiquitylation and proteolysis). Thirdly, I will immobilize the proteasome on an interface and supply substrates in the solution on top. This will allow following the complete catalytic cycle in real-time on a molecular level within a consistent experimental design.The described experiments will have an immediate impact on our understanding of proteasomal degradation processes: a detailed mechanistic and kinetic description of the whole catalytic cycle. This will stimulate novel concepts for the design of drugs in neurodegenerative and cancer research.