Résumé :
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Tropomyosins together with the troponin complex regulate the binding of actin to myosin during muscle contraction. In humans tropomyosins are encoded by at least four different genes, TPM1-4. Mutations in the beta-tropomyosin (TPM2) gene have been reported in patients with nemaline myopathy (NM), cap myopathy and distal arthrogryposis. The aim of the study is to shed light on the pathogenetic pathways leading from six mutations identified in TPM2 to the structural abnormalities seen in the patients’ muscle fibres and to clinical muscle weakness. Here, we study the aberrant protein products and their effects on the binding of beta-tropomyosin to actin and on the ability of the tropomyosin molecules to form dimers. To confirm our preliminary results, based on E.coli- produced beta-tropomyosin, suggesting altered affinity, we are now expressing WT and aberrant variants of beta-tropomyosin proteins in a baculovirus expression system. This ensures acetylation of the NH2 terminus, which is important for the affinity of beta-tropomyosin to actin, and for the dimerisation of tropomyosin molecules. The mutations are introduced into mouse TPM2-cDNA clones with Stratagenes Quick Change® Site Directed Mutagenesis Kit. Actin binding is monitored by an in vitro cosedimentation assay, where G-actin is polymerised to F-actin and beta-tropomyosin binding to actin is analysed with SDS-PAGE and Coomassie blue staining. The dimerisation assay utilises baculovirus system-expressed alfa-tropomyosin also. The formation of homo- or heterodimers is evaluated using Western blot analysis. The study is ongoing.
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