Mutations in the DMD gene result in Duchenne (DMD) and Becker (BMD) muscular dystrophies. The DMD gene encodes the protein, dystrophin that is predominantly expressed in skeletal muscle. Dystrophin is part of a larger protein complex known as the dystrophin-glycoprotein complex (DGC) and, as part of the DGC, provides a mechanical link between the sub-sarcolemmal cytoskeleton and the extracellular matrix. BMD is typically caused by mutations that maintain the reading frame and most often produce variable levels of internally truncated, partially functional dystrophin wheras DMD is most frequently characterized by a complete loss of dystrophin protein or disruption of key ligand binding domains. Utrophin has a highly similar overall structure to dystrophin and is part of the homologous utrophin-glycoprotein complex (UGC) present during fetal development and is localized to neuromuscular and myotendinous junctions in adult muscle. Our lab has previously demonstrated that dystrophin protein in vitro thermal stability is sensitive to disease-causing missense mutations and internal deletions. In contrast, utrophin displays uniform stability upon internal deletion or terminal truncation. Several therapeutic strategies to treat DMD utilize internally deleted dystrophins, including the recently FDA approved exon-skipping drug, eteplirsen, as well as adeno-associated virus (AAV) mediated delivery of therapeutic micro-dystrophins. Here, we characterized therapeutically relevant, internally truncated dystrophin constructs that have been proposed by leading scientists in the field. We show that, as a group, gene therapy micro-dystrophins are significantly less stable in vitro than full-length dystrophin whereas exon-skipped dystrophins have stability profiles congruent with full-length dystrophin. To address the consequences of dystrophin instability in vivo, we generated two novel transgenic mouse models expressing missense mutant dystrophins reported in human DMD (L54R) and BMD (L172H) patients. The L54R and L172H missense mutants were previously evaluated in cultured myoblasts and shown to have missense-mutant dystrophin levels that were inversely proportional to in vitro stability and disease severity of the corresponding patients. Analysis of the L54R and L172H mouse lines as well as a homozygous L172H mouse reveal that disease severity inversely correlates with expression levels of dystrophin protein. Based on the increase of mutant dystrophin upon proteasome inhibition in cultured myoblasts, our hypothesis is that missense dystrophin proteins are being targeted to the proteasome for degradation through the ubiquitin-proteasome pathway. To determine the specific ligases involved in targeting missense dystrophins to the proteasome, we screened an siRNA library of over 500 ubiquitin-conjugating enzymes in cultured myoblasts and identified five putative dystrophin-specific E3 ligases. Two of the identified ligases, Amn1 and FBXO33, were observed in our transgenic mouse lines, with Amn1 protein levels showing significant increases correlating with the amount of missense dystrophin present. Our future studies will continue to evaluate the impact of Amn1 and FBXO33 activity on dystrophin protein levels in order to identify potential therapeutic targets. In addition to the characterization of dystrophin and utrophin stability, we have begun to interrogate a long-hypothesized but understudied function of dystrophin and utrophin, namely, their roles as molecular springs to mechanically stabilize the muscle membrane during muscle contraction. Using atomic force microscopy (AFM), we show here the first mechanical characterization of utrophin and functionally relevant utrophin fragments. Our data reveal striking differences in the mechanical properties of N- and C-terminal halves of utrophin despite having nearly identical thermal stabilities and high structural homology. The high unfolding forces observed in utrophin and the evidence of stiffening spring behavior suggest that utrophin may be acting as a stiff elastic element in series with the giant muscle protein, titin, at the myotendinous junction. Future studies will include evaluation of myotendinous defects in utrophin-deficient mice as well as mechanical characterization of full-length dystrophin.
University of Minnesota Ph.D. dissertation. April 2017. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: James Ervasti. 1 computer file (PDF); x, 139 pages.
Biophysical and functional consequences of sequence changes on dystrophin and utrophin.
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.