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Biochemistry Mar 2021Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein....
Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.
Topics: Dystrophin; Endopeptidase K; Exons; Humans; Protein Conformation; Protein Stability; Proteolysis
PubMed: 33656846
DOI: 10.1021/acs.biochem.0c00827 -
Journal of Neuropathology and... Oct 2021Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy...
Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.
Topics: Adolescent; Child; Child, Preschool; Dystrophin; Female; Gene Expression; High-Throughput Screening Assays; Humans; Male; Molecular Imaging; Muscular Dystrophy, Duchenne
PubMed: 34498054
DOI: 10.1093/jnen/nlab088 -
Proceedings of the National Academy of... Oct 1990Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in...
Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.
Topics: Animals; Antibodies; Chickens; Dystrophin; Fluorescent Antibody Technique; Gizzard, Avian; Immunoblotting; Microscopy, Electron; Muscle, Smooth
PubMed: 2236001
DOI: 10.1073/pnas.87.20.7851 -
Annals of Neurology Aug 2022Duchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute...
OBJECTIVES
Duchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute reliable outcome measures to assess efficacy of treatments in the mdx mouse lacking the muscle and neuronal full-length dystrophins. The present study aimed to evaluate whether these deficits could be alleviated by the restoration of brain dystrophin.
METHODS
We performed intracerebroventricular administration of a new potent tricyclo-DNA antisense oligonucleotide (tcDNA-ASO) containing a full phosphodiester backbone conjugated to a palmitic acid moiety (tcDNA-ASO), designed to skip the mutated exon 23 of mdx mice.
RESULTS
We first show that the tcDNA-ASO rescues expression of brain dystrophin to 10-30% of wild-type levels and significantly reduces the abnormal unconditioned fear responses in mdx mice in a dose-dependent manner, 5 weeks post-injection. Exon skipping efficiency, ASO biodistribution, protein restoration and effect on the fear response were optimal with a dose of 400 μg at 6-7 weeks post-injection, with synaptic-like expression in brain tissues such as the hippocampus and amygdala. Furthermore, this dose of tcDNA-ASO restored long-term memory retention of mdx mice in an object recognition task, but only had minor effects on fear conditioning.
INTERPRETATION
These results suggest for the first time that postnatal re-expression of brain dystrophin could reverse or at least alleviate some cognitive deficits associated with Duchenne muscular dystrophy. ANN NEUROL 2022;92:213-229.
Topics: Animals; Brain; Disease Models, Animal; Dystrophin; Exons; Mice; Mice, Inbred mdx; Muscular Dystrophy, Duchenne; Oligonucleotides; Oligonucleotides, Antisense; Tissue Distribution
PubMed: 35587226
DOI: 10.1002/ana.26409 -
Trends in Biochemical Sciences Jan 1996
Review
Topics: Amino Acid Sequence; Animals; Dystrophin; Molecular Sequence Data; Plants; Sequence Alignment; Zinc Fingers
PubMed: 8848831
DOI: No ID Found -
Journal of Molecular Graphics &... Sep 2020Dystrophin-β-dystroglycan interaction has gained a special attention during current years due to its association with the pathogenesis of muscular dystrophies....
Dystrophin-β-dystroglycan interaction has gained a special attention during current years due to its association with the pathogenesis of muscular dystrophies. Dystrophin is an important component of dystrophin associated protein complex that functions in the normal physiology and cell signaling in addition to membrane stabilization and provides integrity to skeletal muscle fibers. WW, EF-hand and ZZ domains of dystrophin are known to bind with extreme C-terminal region of beta-dystroglycan (β-DG) containing PPxY motif and this interaction is experimentally proven to be coordinated and regulated by two tyrosine (Tyr and Tyr) residues in the C-terminus of beta-dystroglycan. These tyrosine residues are phosphorylated in adhesion dependent manner that disrupts dystrophin-β-DG interaction. The failure of dystrophin to interact with β-DG causes muscular dystrophies. In this study, we have performed molecular docking analysis of dystrophin with phosphorylated and mutated variants of β-DG to pinpoint the actual nature of this interaction at molecular level. We have discovered significant structural and conformational changes in β-DG molecule caused by mutations and tyrosine phosphorylation that alter the nature and site of its interaction with dystrophin. Our results not only support the previous findings but also bring to attention previously unreported discoveries about the nature of this interaction and behavior of different β-DG variants with dystrophin WW, EF-hand and ZZ domains.
Topics: Amino Acid Sequence; Dystroglycans; Dystrophin; Molecular Docking Simulation; Phosphorylation; Protein Binding; Tyrosine
PubMed: 32348939
DOI: 10.1016/j.jmgm.2020.107623 -
Proceedings of the National Academy of... Nov 1997Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction....
Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins' homologous coiled-coil motifs that flank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.
Topics: Amino Acid Sequence; DNA, Complementary; Dimerization; Dystrophin; Dystrophin-Associated Proteins; Molecular Sequence Data; Neuropeptides; Protein Binding; Protein Conformation; Saccharomyces cerevisiae; Sequence Alignment
PubMed: 9356463
DOI: 10.1073/pnas.94.23.12413 -
Journal of Muscle Research and Cell... Dec 1994
Review
Topics: Amino Acid Sequence; Animals; Cytoskeletal Proteins; Dystrophin; Genes; Humans; Male; Membrane Proteins; Mice; Mice, Inbred mdx; Microscopy, Electron; Models, Molecular; Molecular Sequence Data; Multigene Family; Muscular Dystrophies; Muscular Dystrophy, Animal; Sequence Alignment; Utrophin
PubMed: 7706416
DOI: 10.1007/BF00121067 -
Molecular and Cellular Biochemistry Jan 1994Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle,...
Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.
Topics: Amino Acid Sequence; Animals; Antibody Specificity; Cell Membrane; Cross Reactions; Cytosol; Dystrophin; Glycoproteins; Heart Ventricles; Mice; Mice, Inbred mdx; Molecular Sequence Data; Muscles; Myocardium; Rabbits; Sarcolemma; Sarcomeres; Sarcoplasmic Reticulum; Swine
PubMed: 8190121
DOI: 10.1007/BF01084268 -
Neuromuscular Disorders : NMD Jun 2014
Topics: Biomarkers; Dystrophin; Humans; Male; Muscular Dystrophy, Duchenne
PubMed: 24767581
DOI: 10.1016/j.nmd.2014.03.007