Riassunto analitico
Congenital myasthenic syndrome 22 (CMS22, OMIM 616224) is a rare genetic disease caused by homozygous or compound heterozygous mutation in the PREPL gene (609557) on chromosome 2p21. The pathophysiological basis of PREPL deficiency and the physiological substrates of PREPL remain mostly unknown. PREPL (prolyl endopeptidase-like) is an enzyme composed of two domains: an amino-terminal seven-bladed β-propeller domain, featuring four antiparallel β-strands in each blade, and a carboxy-terminal catalytic domain adopting an α/β hydrolase fold. Despite its initial classification within the prolyl oligopeptidase subfamily based on its sequence and structural resemblance to PREP (prolyl endopeptidase), a recent work by our research group has unveiled that PREPL has a (thio)esterase rather than peptidase activity. In my thesis project, I characterized three mutants observed in CMS22 patients (Arg243Cys, Ile412Arg, Arg647Gln) with the aim of understanding at the molecular level how these mutants can lead to the disease. While mutant Ile412Arg was aggregated when purified in vitro, the other two behaved like the wt protein, showing similar dimensions as a monomer, overall fold and thermal stability as measured by circular dichroism and binding affinities to a PREPL inhibitor (1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]-pyridine-4-carbonitrile). Moreover, the cryo-electron microscopy structure of Arg243Cys PREPL at 4 Ǻ resolution showed a similar structure as the wt protein. Despite having the same overall conformation, Arg647Gln showed compromised binding to 2-Bromopalmitate, perhaps due to changes in the putative binding site. The results obtained for the Arg243Cys and Arg647Gln mutants allowed us to exclude that these mutations cause the disease due to changes on the protein stability or structure. Further studies are ongoing to elucidate whether these mutations affect PREPL thioesterase activity and other PREPL functions by disrupting other molecular activities like protein-protein interactions.
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