Description

Exon skipping is a therapy that seeks to correct or mitigate defects in proteins by masking of a specific exon during mRNA maturation. The first approved exon skipping compound in humans is eteplirsen, which targets exon 51 of dystrophin, the protein defective in the common and devastating... Show moreExon skipping is a therapy that seeks to correct or mitigate defects in proteins by masking of a specific exon during mRNA maturation. The first approved exon skipping compound in humans is eteplirsen, which targets exon 51 of dystrophin, the protein defective in the common and devastating genetic disease, Duchenne muscular dystrophy, DMD. DMD is most often caused by deletions that create a frame shift in the gene; this abolished protein expression so that the dystrophin protein is totally absent. Exon skipping therapy corrects the reading frame, restoring protein expression and hopefully mitigating this disease. However, this creates an edited protein, missing both the original defect as well as the therapeutically skipped region. Crucially, it is possible to correct many defects in alternative ways, typically by skipping and exon either before, or after the patient’s defect. This results in different, alternatively edited proteins, of possibly different properties and therapeutic consequences. Currently, there is only one exon skipping drug available, but many others are in late stage clinical trials and it is expected that in the near future for some patients there will be a choice of which exon to skip. Since at the edit site regions of the protein that are normally not in contact are juxtaposed, structural disturbances may result in instability and potentially loss of function. This has been the focus of our work. In this study we examined three such dystrophin exon skipped edits, comprising two pairs of alternative repairs of the same underlying DMD defect. We found that in both cases, one member of each alternative was clearly more stable than the other by a variety of thermodynamic and biochemical measures. We also examined the origin of these differences by molecular dynamics, MD, simulation. Here, we found that these stability differences manifested in different types of structural perturbations. In one case, the edit site was partially unfolded, and this resulted in additional perturbations in the protein domain in which it was localized. However, in the other reduced stability case, the edit site was well-structured, but unfolding was see distal to the edit site, at protein domain junctions which were severely unfolded resulting in excess molecular flexibility. This demonstrates that alternative exon skip repairs of the same underlying defect can have very different consequences at the level of protein structure and stability, and furthermore that these can arise either locally, or by more-subtle long-range perturbations.
M.S. in Molecular Biochemistry and Biophysics, May 2018 Show less