Browsing by Subject "Muscular Dystrophy"
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Item A biochemical and biophysical study of dystrophin.(2011-07) Henderson, Davin MichaelThe 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.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 Calorimetric Determination of Dystrophin ABD1 Unfolding Energetics(2018-12) Coffman, ChristianMuscular 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.Item Development and Synthesis of Utrophin Actin Binding Domain 1 (ABD1)(2016) McMahon, Kate; Horn, Ben; Gauer, Jacob; Hinderliter, AnneDuchenne 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.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 Signals and Systems Tools for Advanced Nanoscale Investigation with Atomic Force Microscopy(2017-03) Ghosal, SayanThe atomic force microscope (AFM) is one of the major advancements in recent science that has enabled imaging of samples at the nanometer and sub-nanometer scale. Over the years, different techniques have been developed to improve the speed, resolution and accuracy of imaging using AFM. Further, the application spectrum of AFMs has extended beyond topography imaging, examples of which include material characterization, probe based data storage systems, and also single molecule force spectroscopy. In spite of the remarkable achievements by AFM technologies, many challenges exist. While majority of this thesis aims to address important challenges that exist with state of the art AFM methodologies using tools from signal processing and systems theory, it also reports some surprising new phenomena that are observed from AFM based mechanical characterization of protein molecules. The techniques developed in each chapter are extensively verified with simulation and experimental results. A key issue that remains largely unaddressed in the AFM literature is the assessment of fidelity of the measurement data. The first contribution of this thesis is to develop a quantitative measure for the fidelity of images obtained from a fast dynamic mode AFM technique. The developed paradigm facilitates user specific priority for either detection of sample features with high decision confidence or on not missing detection of true features. The fidelity measures developed in this thesis are suitable for real-time implementation. The second contribution of this thesis is to develop and compare the performance of different methods to characterize mechanical properties of materials utilizing the dynamic mode of AFM operation. The dynamic mode AFM is particularly suitable for investigating soft-matter. Here, an important enabler is the viewpoint of an equivalent cantilever. The parameters of the equivalent cantilever need to be estimated to derive material properties. In this thesis, we develop a new steady-state based estimation of equivalent parameters (SEEP) and compare it with the recursive estimation of equivalent parameters (REEP). We show that the SEEP is considerably simpler to implement, however, SEEP is a low bandwidth method when compared to REEP. Both methods yield material parameters that quantitatively agree in the domain of validity of the methods. This thesis also streamlines the process of material identification and outlines the key pitfalls that need to be avoided for quantitative estimation of material parameters. Extensive design of a system identification module is reported which implements the REEP algorithm on modern field programmable gate arrays (FPGA). The step by step design procedure of the module explained in this thesis is employable to the development of a wide variety of FPGA based signal processing systems. The third contribution of this thesis is a new system model detection technique called the innovations squared mismatch. Such detection of a model from a set of models that best describes the behavior of a system is of primary importance in many applications. Here, two discriminating signals are derived from measurements for a plant that switches between two model behaviors, where the transfer functions from inputs to the two signals are identical when one model is effective while they are negative of one another when the other model is effective. Further, we report sequence based detection approaches to extend the use of the signals for high bandwidth applications. In such applications the plant behavior can switch from one model to another at high rates and the transients from a previous behavior affect the current behavior causing inter-symbol interference (ISI). Methods developed are specialized for probe based data storage where experimental data demonstrates that they offer significant advantages over current methods. The fourth contribution of this thesis is the first ever characterization of mechanical properties of utrophin protein molecule and its different terminal fragments using AFM based force spectroscopy experiments. Utrophin and its homologue dystrophin are proteins which are believed to play vital roles in mechanically stabilizing the muscle cells during stretch and relax cycles. These proteins are also under active research for finding possible cure for the disease muscular dystrophy. In this thesis we report markedly different mechanical characteristics for the utrophin constructs where previous thermodynamic studies measured identical thermal denaturation profiles. Our findings signify the need for force spectroscopy based characterization of molecules that are believed to play important mechanical roles in human body.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.