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The Histochemical Journal Jul 1999Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin... (Comparative Study)
Comparative Study
Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane. In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400 kDa dystrophins in Purkinje fibres.
Topics: Animals; Blotting, Western; Cattle; Cytoskeletal Proteins; Dystroglycans; Dystrophin; Dystrophin-Associated Proteins; Membrane Glycoproteins; Membrane Proteins; Muscle Proteins; Organ Specificity; Purkinje Fibers; Sarcoglycans; Utrophin
PubMed: 10475570
DOI: 10.1023/a:1003805905456 -
The Journal of Biological Chemistry Mar 1995Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V.,...
Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.
Topics: Animals; Cell Differentiation; Cell Line; Cell Membrane; Chickens; Chromatography, Affinity; Chromatography, Gel; Cysteine; Cytoskeletal Proteins; Dystrophin; Electrophoresis, Polyacrylamide Gel; Gizzard, Avian; Membrane Proteins; Methionine; Mice; Molecular Weight; Muscle, Skeletal; Muscle, Smooth; Phosphoglucomutase; Protein Binding; Sulfur Radioisotopes; Utrophin
PubMed: 7890770
DOI: 10.1074/jbc.270.11.6328 -
Journal of Molecular Biology Feb 2012Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the...
Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.
Topics: Actins; Animals; Dystrophin; Models, Molecular; Muscle Fibers, Skeletal; Protein Binding; Protein Conformation
PubMed: 22226838
DOI: 10.1016/j.jmb.2011.12.040 -
Experimental Gerontology Jan 2014Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the...
Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.
Topics: Aging; Animals; Animals, Genetically Modified; Cardiomyopathy, Dilated; Drosophila; Dystroglycans; Dystrophin; Genetic Therapy; Mutation; Myocytes, Cardiac; Nitric Oxide Synthase; Signal Transduction
PubMed: 24231130
DOI: 10.1016/j.exger.2013.10.015 -
Journal of Biochemistry Nov 1990We found six groups of proteins, A0-A5, besides dystrophin itself in a dystrophin preparation obtained by the reported method [Campbell, K.P. & Kahl, S.D.(1989) Nature...
We found six groups of proteins, A0-A5, besides dystrophin itself in a dystrophin preparation obtained by the reported method [Campbell, K.P. & Kahl, S.D.(1989) Nature 338, 259-262] with some modifications. Their molecular weights were 94, 62, 52, 43, 36, and 24 kDa, respectively. Their molar ratios to dystrophin were 0.14, 2.2, 0.88, 0.90, 1.7, and 0.34, respectively. Each of A1, A3, and A4 was split into several bands. But each group of bands except A3 seemed to behave like the same kind of protein. The doublet of A3 was subdivided into A3a and A3b in the decreasing order of molecular weight. All the A-proteins except A2 were cross-linked with dystrophin molecule by a cross-linker, bis(sulfosuccinimidyl)suberate, suggesting them to be dystrophin-associated proteins. When dystrophin preparation was treated with KI, which is known to break membrane cytoskeletal interactions, as described by Campbell and Kahl, A2, A3, and A4 were absorbed by wheat germ lectin (WGL) Sepharose, but the dystrophin molecule and A1 were not absorbed. On the other hand, A2 and A3b reacted with biotinyl WGL but A3a and A4 did not in blotting analysis. This apparent discrepancy can be explained if we postulate that A3a and/or A4 would associate with A2 and/or A3b. On the basis of these results including stoichiometric considerations, we are of the opinion that the complex of A2.A4 among various possible ones is the most important to anchor dystrophin to sarcolemma. In this A2.A4 complex, A4 but not A2 is directly associated with dystrophin.
Topics: Animals; Cross-Linking Reagents; Densitometry; Dystrophin; Glycoproteins; Molecular Weight; Muscles; Rabbits; Sarcolemma; Succinimides; Wheat Germ Agglutinins
PubMed: 2081733
DOI: 10.1093/oxfordjournals.jbchem.a123276 -
Muscle & Nerve Jan 1999Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two...
Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C2C12 myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427.
Topics: Actins; Animals; Blotting, Western; Cell Line; Cells, Cultured; Dystrophin; Fetus; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mice; Microscopy, Fluorescence; Muscle, Skeletal; Protein Isoforms; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Subcellular Fractions; Transfection
PubMed: 9883853
DOI: 10.1002/(sici)1097-4598(199901)22:1<16::aid-mus5>3.0.co;2-r -
PloS One 2020Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in...
Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in other tissues like retina and brain. Non-progressing cognitive dysfunction occurs in 20 to 50% of DMD patients. Furthermore, loss of expression of the Dp427 dystrophin isoform in the brain of mdx mice, the most used animal model of DMD, leads to behavioral deficits thought to be linked to insufficiencies in synaptogenesis and channel clustering at synapses. Mdx mice where the locomotor phenotype is mild also display a high and maladaptive response to stress. Recently, we generated Dmdmdx rats carrying an out-of frame mutation in exon 23 of the DMD gene and exhibiting a skeletal and cardiac muscle phenotype similar to DMD patients. In order to evaluate the impact of dystrophin loss on behavior, we explored locomotion parameters as well as anhedonia, anxiety and response to stress, in Dmdmdx rats aged from 1.5 to 7 months, in comparison to wild-type (WT) littermates. Pattern of dystrophin expression in the brain of WT and Dmdmdx rats was characterized by western-blot analyses and immunohistochemistry. We showed that dystrophin-deficient Dmdmdx rats displayed motor deficits in the beam test, without association with depressive or anxiety-like phenotype. However, Dmdmdx rats exhibited a strong response to restraint-induced stress, with a large increase in freezings frequency and duration, suggesting an alteration in a functional circuit including the amygdala. In brain, large dystrophin isoform Dp427 was not expressed in mutant animals. Dmdmdx rat is therefore a good animal model for preclinical evaluations of new treatments for DMD but care must be taken with their responses to mild stress.
Topics: Animals; Anxiety; Brain; Central Nervous System; Dystrophin; Locomotion; Maze Learning; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Animal; Myocardium; Protein Isoforms; Rats; Rats, Transgenic; Stress, Psychological
PubMed: 32160266
DOI: 10.1371/journal.pone.0230083 -
Journal of the Neurological Sciences Jul 1992Two 5-month-old male Domestic Shorthair littermates showed general skeletal muscle hypertrophy, multifocal submucosal lingual calcification with lingual enlargement, and...
Two 5-month-old male Domestic Shorthair littermates showed general skeletal muscle hypertrophy, multifocal submucosal lingual calcification with lingual enlargement, and excessive salivation. Both cats had a reduced level of activity, walked with a stiff gait, and tended to "bunny hop" when they ran. These clinical features were similar to those of previously reported dystrophin-deficient cats. Using multiple dystrophin antibodies, we found that the cats described in this report also showed marked dystrophin deficiency. The histopathology was remarkable for hypertrophy and splitting of fibers, and progressive accumulation of calcium deposits within the muscle. There was little or no endomysial fibrosis at 2 years of age. The natural history of dystrophin-deficiency in cats has not been described: both previous cats had been euthanized at 2 years of age prior to experiencing any life-threatening problems. At 6 months of age, one of the new cats developed megaesophagus because of severe progressive hypertrophy of the diaphragmatic muscles. The diaphragm completely occluded the esophagus, and the cat was euthanized for humane reasons. The second cat remained in good condition until age 18 months when it developed acute renal failure attributed to severe prolonged dehydration and hyperosmolality. The cat recovered after receiving supportive treatment but was unable to maintain fluid homeostasis. The insufficient water intake was attributed to glossal hypertrophy and dysfunction. At age 2 years, the cat received regular subcutaneous injections of low-sodium fluids to maintain proper hydration. The clinical consequence of dystrophin deficiency in cats is lethal muscle hypertrophy. We have called the feline disease "hypertrophic feline muscular dystrophy" (HFMD).
Topics: Animals; Cat Diseases; Cats; Dystrophin; Hypertrophy; Immunoblotting; Male; Muscles; Muscular Diseases
PubMed: 1506854
DOI: 10.1016/0022-510x(92)90022-d -
Biochemical and Biophysical Research... Nov 2022The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the...
The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/β importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.
Topics: Active Transport, Cell Nucleus; Animals; Dystrophin; Intracellular Space; Karyopherins; PC12 Cells; Protein Isoforms; RNA, Messenger; Rats; beta Karyopherins
PubMed: 36155058
DOI: 10.1016/j.bbrc.2022.09.035 -
American Journal of Medical Genetics Oct 1992Becker muscular dystrophy is usually caused by intragenic dystrophin gene deletions that result in production of an internally deleted protein. Previous studies have...
Becker muscular dystrophy is usually caused by intragenic dystrophin gene deletions that result in production of an internally deleted protein. Previous studies have detected what appears to be a unique dystrophin degradation product that appears only in muscle biopsies from patients with Becker muscular dystrophy. This dystrophin fragment is always seen in addition to the "full-size" dystrophin of the expected size for a given gene deletion. It is only found in biopsies from patients with mutations in the deletion-prone region encompassing exons 45-53, but it does not appear to correlate with any observable phenotype at the clinical level. By correlating the size and locations of dystrophin gene deletions with the size of this degradation product, together with use of region-specific dystrophin antisera, we find that proteolytic cleavage may occur at the deletion breakpoints, perhaps due to alterations of the secondary and/or tertiary structures of the protein. This cleavage results in loss of the carboxy-terminal domains that are thought to be important for interactions between dystrophin and other membrane-bound proteins.
Topics: Blotting, Western; Dystrophin; Gene Deletion; Humans; Molecular Weight; Muscles; Muscular Dystrophies
PubMed: 1488990
DOI: 10.1002/ajmg.1320440322