Browsing by Subject "Dystrophin"

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A biochemical and biophysical study of dystrophin.
(2011-07) Henderson, Davin Michael
The primary role of a muscle cell is to contract and produce force that moves an organism. A vast majority of a muscle is made of the proteins in contraction machinery and nearly all energy utilized by the cell is consumed in this process. However, an equally important but substantially smaller portion of the muscle cell is dedicated to the preservation of cell membrane integrity. The costamere is an elaborate matrix of cytoskeleton associated and transmembrane proteins that form a support lattice between the plasma membrane and contractile apparatus. The dystrophin glycoprotein complex (DGC) is a structurally important member of the costamere and has been shown to link microtubules, thin and intermediate filaments of the cytoskeleton with major components of the extracellular matrix. In the DGC, dystrophin is responsible for attachments with intracellular cytoskeletal components and the transmembrane protein dystroglycan. One of the most common diseases afflicting muscle is Duchenne muscular dystrophy (DMD), which is caused by mutations in the gene encoding the protein dystrophin. The focus of my thesis is to better understand the biochemical and biophysical properties of dystrophin. Specifically, I investigated the actin binding properties of dystrophin in the context of its functional domains as well as the consequences of disease causing missense mutations localized to actin-binding domain 1 (ABD1). Additionally, I characterized the biophysical properties of internally truncated dystrophin proteins under development for treatment of DMD. It has been twenty years since dystrophin was hypothesized to bind actin and even today we are learning more about this fascinating interaction. My thesis expands our understanding of the dystrophin-actin interaction in three ways. First, I showed that full-length dystrophin interacts with the actin isoforms expressed in muscle with equivalent affinities. Second, I showed that the thermally stable C-terminal domain of dystrophin is required for full actin binding activity. Third, in collaboration with the Thomas lab, we showed that dystrophin and utrophin uniquely alter the physical properties of actin filaments. Disease causing missense mutations in the dystrophin gene are scattered in many functional domains but we chose to study a cluster localized to ABD1 with hope that we would find amino acids important for actin binding. We hypothesized that mutations in ABD1 would disrupt actin binding and therefore lead to disease though loss of an essential interacting partner. However, no mutation dramatically disrupted actin binding but instead lead to loss of thermal stability and protein aggregation. My thesis work was the first to show evidence that protein stability and aggregation may play a role in the pathogenesis of dystrophinopathies. DMD currently has no effective treatment but many promising therapies are being pursued. Many laboratories are pursuing therapies for DMD and multiple techniques are being pursued including adeno-associated viral gene therapy, protein replacement therapy, exon skipping therapy and stop codon read though therapy. For gene therapy and protein therapy, the size of the dystrophin or utrophin coding sequence has been reduced by deletion of internal domains, which retains important N- and C-terminal ligand binding sites. I set out to test the stability of internally deleted therapy proteins to ensure that no unwanted structural perturbations were caused by internal deletion. Additionally, I tested a set of N-and C-terminal truncations of dystrophin and a dystrophin related protein, utrophin for comparison to internally deleted versions of these proteins used in therapy. I found that the thermal stability of utrophin was uniform from N- to C-terminus and that internal deletion did not affect protein stability. I also found that the N-terminal half of dystrophin had a lower thermal stability compared to the C-terminal half and, to our surprise, internally deleted dystrophin proteins showed marked thermal instability and aggregation.
A biochemical and molecular analysis of functional differences between dystrophin and utrophin
(2013-11) Belanto, Joseph John
The 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.
Biophysical and functional consequences of sequence changes on dystrophin and utrophin
(2017-04) McCourt, Jackie
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.
Biophysical, cellular, and animal models of dystrophin missense mutations
(2014-12) Talsness, Dana
The 427kDa protein dystrophin is expressed in skeletal muscle where it localizes to the costamere and physically links the interior of muscle fibers to the extracellular matrix. Mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a mild form known as Becker (BMD). Currently, there is no cure for DMD or BMD, but there are several therapies being investigated that target specific types of mutations found in the DMD gene. Nonsense mutations almost always lead to a complete lack of dystrophin protein, with stop codon read-through drugs being studied for personalized treatments. Out-of-frame deletions and insertions also cause nearly a complete lack of dystrophin, for which exon-skipping is currently being investigated. Missense mutations in dystrophin, however, cause a wide range of phenotypic severity in patients, the molecular and cellular consequences of such mutations are not well understood, and there are no therapies currently targeting this genotype. Here, we report on three separate model systems of missense mutations in dystrophin: an in vitro biochemical model, a myoblast cell culture model, and an in vivo animal model. Together, they provide evidence that different missense mutations cause variable degrees of thermal instability, which leads to proportionally decreased dystrophin expression, and subsequently causes dystrophic phenotypes. In addition, our initial studies of small molecule treatments show that it is possible to increase the levels of mutant dystrophin, and may lead to personalized therapeutics for patients with missense mutations.
Calorimetric Determination of Dystrophin ABD1 Unfolding Energetics
(2018-12) Coffman, Christian
Muscular Dystrophy (MD) is a disease that effects the structural integrity of muscle cells. Studies have linked the Dystrophin protein to MD as the most commonly altered gene in patients with MD. Using empirical models established to predict the change in heat capacity associated with protein unfolding, we we correlated the likelihood of a mutation as being disease causing with an associated change in the heat capacity at that amino acid position. These studies focused on the first Actin Binding Domain of Dystrophin (ABD1, 27kDa) using Differential Scanning Calorimetry (DSC), as it is the region with the highest density of disease causing mutations. ABD1 is comprised of two Calmodulin Homology domains (CH1 and CH2) connected by a short linker region and is predicted to be slightly disordered. [1] Analyzing the data acquired from DSC proved to be rather difficult as it was highly dependent on the baseline definition, which can be rather noisy. This thesis describes the evolution of our DSC analysis starting with an analysis published in the Biophysical Journal. This method suggested the a change in heat capacity (∆Cp of 5 ± 5 kcal/mol). However, the model showed some systematic deviation from the experimental data so the data was fit to a Gaussian and Hubbert distribution. Then a deconvolution approach revealed the presence of an appreciable occupancy (approximately 50%) of intermediate states that helps account for the deviations from a two-state model. Deconvoluting the transitions revealed at least one intermediate transition with ∆G(37◦C) of 2 ± 1kcal/mol and an unfolding free energy of 2.2 ± 0.6kcal/mol and a change in heat capacity that is smaller than predicted. This free energy is comparable to that which has been determined for actin binding thus implicating unfolding of ABD1 upon binding actin, possibly through separating the CH domains or some other mechanism.
Conformational changes in actinin-type actin binding domains: probing actin-induced structural dynamics in dystrophin and utrophin using EPR spectroscopy.
(2014-12) Crain, Jonathan
The 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.
Development and Synthesis of Utrophin Actin Binding Domain 1 (ABD1)
(2016) McMahon, Kate; Horn, Ben; Gauer, Jacob; Hinderliter, Anne
Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease containing point mutations in the muscle protein Dystrophin causing the protein to lose its function. Specifically, Dystrophin is critical for dissipating the mechanical stress placed on muscles during physical activity. Although Dystrophin is nonfunctional in DMD patients, its fetal homolog, Utrophin, is often present in higher amounts than common to adult cells. Because Utrophin and Dystrophin share 85% homology in their first actin binding domains (ABD1), the interrelatedness of structure and function validate Utrophin as a proposed therapeutic tool for combating DMD. To test this hypothesis, the thermodynamic character of Utrophin ABD1 and Dystrophin ABD1 will be compared. As Utrophin is not regularly studied, the gene for Utrophin ABD1 was designed, synthesized, and expressed in E.coli cells. Prokaryotic cells were utilized to express a eukaryotic protein because of rapid growth rate and the presence of an extra, self-replicating, circular DNA called a plasmid. A plasmid is evolutionarily advantageous because it can be passed quickly from prokaryotic cell to prokaryotic cell without the entire genome replicating, thus increasing variability. This unique attribute was utilized to express Utrophin ABD1 in E. coli cells. Although eukaryotic systems often have posttranslational modifications, this did not pose a threat for the prokaryotic cell amplification. The gene encoding the protein was designed using specific amino acid residues, not nucleotide sequences; the splicing of nucleotide sequences was irrelevant as posttranslational modification occurs before the amino acids are assembled into their primary structure. Specifically, Utrophin ABD1 was designed with BamHI and XhoI restriction enzymes flanking the 246 amino acid Utrophin ABD1 construct which was synthesized in a pUC57 E. coli plasmid. Using BamHI and XhoI, the amino acid sequence was restriction digested and subcloned into an expression vector containing components critical for nickel column chromatography like a histidine tag, TEV protease cut site, and maltose binding protein. The expression vector also contains a selective marker to find the correct ligated species such as the antibiotic Kanamycin. These plasmids were transformed into competent E. coli cells so the E. coli cells would replicate the inserted DNA the same way it replicates a plasmid. During the rapid growth, inclusion bodies, protein aggregates of overexpressed protein, are accounted for by the addition of the maltose binding protein which maintains solubility. The transformed cells were stored in a glycerol stock. Synthesis of this gene then allows growth and purification of the Utrophin ABD1 protein in a similar manner to those already classified for histidine tagged proteins. Purification is carried out at a pH of 8 so that the six histidines will be deprotonated and bind to the nickel column, thus washing out all other protein expect for the Utrophin bound to the column. Purification is important in that it insures pure protein by cleaving off the maltose binding protein using the tobacco etch virus (TEV) protease that recognizes a specific nucleotide sequence rarely found in the eukaryotic genome. Finally, thermodynamic analysis of this protein will give insight into the structure and function of Utrophin ABD1 and its potential capabilities as a therapeutic agent for patients with DMD.
From structure and dynamics to novel therapeutic development for muscular dystrophy.
(2012-07) Lin, Ava Yun
Dystrophin 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.
Microscopic Properties Determine Macroscopic Characteristics of Proteins
(2016-07) Horn, Benjamin
Macroscopic characteristics of a peptide or protein are dependent on microscopic properties. One microscopic property that has an effect on the macroscopic characteristics is the oxidation of methionine. Here we show that oxidation strengthens the methionine-aromatic interaction by 0.5-1.4 kcal/mol and that oxidation plays a role in the structure and biological function of calmodulin and lymphotoxin-α/TNFR1 binding. Another microscopic property is the sequence of amino acids. We have also shown that the intrinsically disordered linker region of synaptotagmin I plays a role in modulating the binding of Ca2+ and the interaction with membrane. The final portion of this work determined the microscopic properties, in this case hydrophobic and electrostatic interactions within the dystrophin ABD1 to shed light on the underlying mechanism by which structural changes occur under physiological conditions. This work consisted of using thermodynamic and structural approaches and computational techniques, namely database searching and molecular dynamics simulations.
Skeletal Muscle Microtubule Organization and Stability is Regulated by the Dystrophin-Glycoprotein Complex and Cortical Actin
(2020-08) Nelson, D'anna
Duchenne muscular dystrophy (DMD) is a fatal X-linked myopathy caused by the loss of dystrophin in striated muscle. DMD is frequently studied in the mdx mouse which also lacks dystrophin. It has been observed by multiple independent research groups that mdx skeletal muscle presents with a disorganized cortical microtubule lattice that primarily lacks transverse microtubules as compared to the orthogonal microtubule lattice of wildtype mouse skeletal muscle. While transgenic expression of dystrophin in mdx skeletal muscle does restore microtubule organization, it is not understood how dystrophin regulates microtubule organization in vivo. This thesis implicates two regions of the dystrophin rod domain as regulators of microtubule organization and stability. Singular absence of dystrophin spectrin like repeats R4-15 or R20-24 does not impact basal microtubule organization. However, removal of both R4-15 and R20-23 from micro-dystrophin constructs results in a miniaturized dystrophin that is incapable of fully restoring microtubule organization when transgenically expressed in mdx muscle. In addition to the intermediate microtubule organization by micro-dystrophins lacking R4-15 and R20-23, we have characterized a novel microtubule pathology where transverse microtubules are lost upon eccentric contraction in the absence of either R4-15 or R20-24. Transverse microtubule loss is specific to eccentric contractions and occurs rapidly via a ROS mediated mechanism. Multiple sources of ROS appear to be involved including NOX2 but not nNOS. Moreover, loss of γ-cytoplasmic actin, β-cytoplasmic actin, or the dystrophin-glycoprotein complex (DGC) member α-dystrobrevin all cause a highly similar microtubule phenotype where transverse microtubules are lost post eccentric contraction. While both the intermediate microtubule organization and microtubule susceptibility to eccentric contraction exhibited by micro-dystrophin rescued muscle may have implications for micro-dystrophin gene therapy, the work presented in this thesis has also widened our understanding of skeletal muscle microtubule regulation to include cytoplasmic actins and DGC stability.
A Substitute for Dystrophin: Why Utrophin Fails
(2017) Ostgaard, Cyrina
Each 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.

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