Session: Parallel session 5 - New topics in OMICs

Mass spectrometry combined approaches highlight a defect in proteasome 20S assembly in patients with either immune or neurological disorders

The 20S proteasome is an enzyme made of 14 subunits α and β, successively assembled by chaperones. It represents the catalytic core of the ubiquitin-proteasome system, essential to maintain protein homeostasis. Lately, an increasing number of loss-of-function mutations have been identified in genes encoding for proteasome subunits in various patients. Intriguingly, such proteasome lesions induce two distinct phenotypes that are clinically classified as either autoinflammation or neurodevelopmental delay. Although these manifestations are not strictly mutually exclusive, they are considered to underlie two distinct diseases referred to as PRoteasome-Associated Autoinflammatory Syndrome (PRAAS) and Neurodevelopmental Disorders (NDD). Clinically, PRAAS and NDD have little in common.To date, the molecular basis for this dual phenotype is not understood. A peculiarity that is even harder to pinpoint, given that these two phenotypes cannot be mapped to specific proteasome genes.

To gain a better understanding of how those pathogenic variants of the same multi-subunit enzyme trigger such distinct diseases, we developed a strategy combining different mass spectrometry approaches. Our pipeline includes Bottom-Up and Top-Down analyses and allows, after immunopurification of 20S complexes, to investigate the interactome, composition, assembly, and PTMs of the proteasome. We applied this approach on T cells derived from patients and noticed a defect in 20S assembly in a NDD patient harboring a de novo mutation in PSMB3 gene and in a PRAAS patient mutation in PSMB4 gene. Indeed our data showed a striking increase in proteasome-associated chaperones revealing a stalling in proteasome assembly in these mutants. In this regard, we plan to dig deeper into this defect by producing mutated proteasomes via recombinant expression and to use structural MS to pinpoint more precisely at which step in assembly the mutations interfere and the effect they have on their structures.