Browsing by Subject "Utrophin"
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Item A biochemical and molecular analysis of functional differences between dystrophin and utrophin(2013-11) Belanto, Joseph JohnThe DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.Item Biophysical and functional consequences of sequence changes on dystrophin and utrophin(2017-04) McCourt, JackieMutations 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.Item Conformational changes in actinin-type actin binding domains: probing actin-induced structural dynamics in dystrophin and utrophin using EPR spectroscopy.(2014-12) Crain, JonathanThe underlying cause of Duchenne and Becker muscular dystrophies is a lack of functional dystrophin, a large multidomain protein. Dystrophin is normally expressed in muscle, where it links the extracellular matrix to the cortical actin cytoskeleton via a complex of associated proteins. Dystrophin, and its autosomal homologue utrophin, connect with the actin cytoskeleton through two F-actin binding domains, including an N-terminal "actinin-type" actin binding domain (ABD).In addition to dystrophin and utrophin, actinin-type ABDs are found in a large number of proteins. Nonetheless, the actin binding mechanism remains poorly understood: x-ray crystallography and electron microscopy have produced conflicting models. Electron paramagnetic resonance (EPR) spectroscopy, especially double electron-electron resonance (DEER), can be used to distinguish between these models or to build new models. In this thesis, I present data from DEER experiments which suggest that actinin-type ABDs of dystrophin and utrophin adopt unexpected conformations in solution.Item From structure and dynamics to novel therapeutic development for muscular dystrophy.(2012-07) Lin, Ava YunDystrophin is defective in Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are debilitating X-linked diseases that currently have no cure. Dystrophin links the actin cytoskeleton at its N-terminus and a glycoprotein complex (DGC) embedded in the sarcolemma at its C-terminus, apparently providing mechanical stability to the muscle during contraction. Due to the large size (427 kD) and filamentous nature of dystrophin, studies of its function and attempts to develop effective therapeutics have developed slowly, despite intensive efforts. Utrophin (395 kD) is a homolog of dystrophin that has shown therapeutic promise in mdx mice, which lack dystrophin. Utrophin is endogenously expressed in the cytoskeleton of fetal and developing muscle but is replaced by dystrophin as the muscle matures (8-10). Both dystrophin and utrophin belong to the spectrin superfamily of actin-binding proteins, which carry out diverse functions in the cytoskeleton of most cells. Of the many proteins included in this superfamily, dystrophin and utrophin are among the least studied in terms of structural dynamics, limiting the understanding of their function at the sarcolemma. In order to target the root of dystrophin malfunction in muscular dystrophy, we need to better understand the native functions of dystrophin and utrophin. Lack of structural information about dystrophin and its interactions adds to the complexity of tying clinical presentations to the diverse disease-causing mutations, and hinders therapeutic advancement in gene or drug therapy. There are numerous mouse-model studies, but there are varied results across several parameters tested, and no construct or drug has been found that restores normal muscle force in the mdx mouse. Exon-skipping morpholinos are expensive to produce with variable delivery and efficacy to muscle groups and require a customized oligo design for each mutation, making it difficult to test them individually in mouse models. In order to (a) understand disease mechanisms and (b) design better therapies rationally, we need more fundamental information about the structures and interactions of specific regions of dystrophin and utrophin. That is the goal of this project.Item A Substitute for Dystrophin: Why Utrophin Fails(2017) Ostgaard, CyrinaEach year 1 in 3500 males in the United States are born with Muscular Dystrophy (MD). In serious cases this disease is marked by heavily atrophied musculature, mental impairment, cardiomyopathy, and a shortened lifespan (~20 years). Currently, there is no cure for this debilitating disease and treatment options remain abysmal. Knowing how utrophin fails to replace dystrophin has potential for introducing factors that could fix this failure. That is part of the future of where this project is headed in terms of drug design and possible implications that could be had for disease treatment. This is one of the first projects that looks into the thermodynamics and molecular dynamics of these proteins to find how they function as little is still known about the way in which dystrophin transduces force and what role utrophin plays.