Proteasome puzzles: Exploring Proteasome Mutations in Neurodevelopmental Disorders

The 20S proteasome is an enzyme made of 14 highly conserved subunits α and β, that are successively assembled by chaperones. It represents the catalytic core of the ubiquitin-proteasome system, essential to maintain protein homeostasis and clearance. Over the past ten years, an increasing number of loss-of-function mutations have been identified in genes encoding for proteasome subunits in various patients that are clinically classified with a neurodevelopmental delay. This disease is referred to as Neurodevelopmental Disorders (NDD). To date, the molecular basis of this phenotype is not understood as it cannot be associated with specific proteasome genes.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. 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.