Session: Parallel session 9 - Top-down and Structural analysis

Understanding proteasome regulations with structural mass spectrometry

Angelique DAFUN¹, Amélie BOSC-ROSATI¹, Dušan ZIVKOVI?¹, Odile BURLET-SCHILTZ¹, Marie-Pierre BOUSQUET¹, Julien MARCOUX¹

¹Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III - Paul Sabatier (UPS), Toulouse, France
²University of Oxford, Department of Biology, Oxford, United Kingdom

Objective

The 20S proteasome is crucial for cellular protein degradation and its dysregulation has been implicated in various diseases, offering a promising therapeutic target. To fully understand its regulation, significant work has been put into analyzing its structure and function. However, understanding its dynamics remains a challenge due to its complexity and heterogeneity. To contribute to this effort, this project was to investigate the interplay of structure-centered proteasome regulations (subunit composition variations, partnering with activators, and post-translational modifications or PTMs) using structural mass spectrometry (MS) approaches.

Methodology

An efficient workflow combining Top-down and Bottom-up MS techniques was optimized for characterizing and quantifying proteoforms of the 20S proteasome associated with inflammation. The binding effects of PA28 activators and proteasome inhibitors like Bortezomib and ONX-0914 on the structure and dynamics of the 20S proteasome were then examined using mass photometry, native-, Top-down-, and HDX-MS.

Results

The study identified various proteoforms of the 20S proteasome associated with inflammation, including post-translational modifications, induction of immunoproteasomes, and genetic variations affecting assembly. Differences in structural dynamics induced by different PA28 activators were observed, with insights into the differential effects on immunoproteasome versus constitutive proteasome, particularly in inflammation-related conditions. Investigation into the binding of proteasome inhibitors revealed distinct effects on the structural dynamics of the 20S proteasome, confirming binding sites and preferences while identifying potential allosteric effects.

Conclusion

These findings underscore the complexity of proteasome regulation, dependent on its subunit composition and associated activators. This deeper understanding holds promise for developing targeted therapies tailored to specific disease conditions.