Characterization of Calsequestrin-1 mutations in patients affected by myopathies

Calsequestrin1 (CASQ1) is the main Ca2+ buffer protein localized in the lumen of the skeletal muscle sarcoplasmic reticulum. Two isoforms of CASQ, encoded by two different genes, have been identified: the skeletal isoform (CASQ1) expressed in fast-twich skeletal fibres and the cardiac isoform (CASQ2) expressed in cardiac muscle and in slow-twich skeletal fibres. Crystallographic studies have shown that the protein is made of three thioredoxin-like domains linked by short amino acidic sequences. The main characteristic of this molecule is its ability to polymerize in presence of Ca2+, binding more and more Ca2+ ions. Mutations in CASQ2 have been reported in an autosomal-recessive form of Catecholaminergic Polymorphic Ventricular Tachycardia, whereas recently a heterozygous missense mutation has been identified in the CASQ1 gene in patients with a vacuolar aggregate myopathy. This thesis is focused on the characterization of four missense variants in the CASQ1 gene, identified in patients with different myopathies. The identified mutations affect conserved amino acids of the CASQ1 protein. Turbidity measurements in presence of increasing Ca2+ concentrations showed that two of these mutations lead to a significant reduction in Ca2+-dependent aggregation. In agreement, limited trypsin proteolysis experiments showed that these amino acid substitutions enhanced the conformational flexibility of CASQ1, which become more susceptible to trypsin cleavage in comparison to wild type. On the contrary, the CASQ1 mutation associated with the vacuolar myopathy showed an increased Ca2+-dependent aggregation. These results support the hypothesis that distinct mutations in the CASQ1 gene may cause different phenotypes.