Résumé :
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Duchenne and Becker muscular dystrophy (DMD and BMD respectively) are caused by mutations of the dystrophin's gene coding for a skeletal muscles protein included in the dystrophin-glycoprotein sarcolemmal complex. Whereas complete deficit in dystrophin leads to the severe DMD, expression of truncated or mutated form leads mainly to the milder BMD phenotype. In this case, great majority of mutations consists of "in frame" exons deletions leading to lack of part of the central rod domain's spectrin-like repeats. A high number of these deletions are clustered in exons 45 to 53 coding for spectrin-like repeats 17 to 22.In order to investigate the relationship between the proteins expressed in patients and the great variability of BMD phenotypes, we aimed to compare the biochemical properties (such as stability, conformation, interactions) of recombinant proteins derived from deletion mutants. Two approaches were chosen: first, the so-called "microdystrophin" R4-23 and R2-21+H3 were expressed in baculovirus infected insect's cells ( R4-23 is reversing the phenotype in mdx mouse when R2-21+H3 is not). A second approach was focused on the deletion hot spot: a collection of rod domain fragments from repeat 16 to 21 (R16-21) or repeat 16 to 23 (R16-23) were produced in bacteria with truncation corresponding to exons deletions (eg R16-21 45-46, R16-23 45-55).It appears that lots of these truncated proteins were very unstable compared to full length analogs (eg R16-21 45-51 compared to R16-21): they were produced in inclusion bodies of bacteria or in insoluble material of insect's cells. When solubilized by detergent, some of them formed aggregates with contaminants and could not have been purified (R16-21 45-46, R16-21 45-48). Among proteins obtained with sufficient purity (above 90%), alpha-helix levels determined by circular dichroism were between 35 and 50 %, compared to 60% for full length R16-21.What emerge from all these results is that truncated proteins in our study were for a large part unfolded and susceptible to aggregation. It is then likely that although expressed in BMD patients, these proteins may not be able to fulfill their physiological functions. Due to a large heterogeneity of these proteins' properties, it remains important to further study them in vitro and to accumulate biochemical data to provide clinicians with arguments for designing gene replacement or exon skipping therapy.
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