4-Phenylbutyrate restores localization and membrane repair to human dysferlin mutations.
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PROVIDER: S-EPMC8741482 | BioStudies |
REPOSITORIES: biostudies
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Project description:Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region.We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing.The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.
| S-EPMC3898210 | BioStudies
Project description:Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development.
| S-EPMC3188867 | BioStudies
Project description:Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is a transmembrane protein involved in several muscle functions like T-tubule maintenance and membrane repair. In 2009, a study showed the existence of fourteen dysferlin transcripts generated from alternative splicing. We were interested in dysferlin transcripts containing the exon 40a, and among them the transcript 11 which contains all the canonical exons and exon 40a. This alternative exon encodes a protein region that is cleaved by calpains during the muscle membrane repair mechanism. Firstly, we tested the impact of mutations in exon 40a on its cleavability by calpains. We showed that the peptide encoded by the exon 40a domain is resistant to mutations and that calpains cleaved dysferlin in the first part of DYSF exon 40a. To further explore the implication of this transcript in cell functions, we performed membrane repair, osmotic shock, and transferrin assay. Our results indicated that dysferlin transcript 11 is a key factor in the membrane repair process. Moreover, dysferlin transcript 11 participates in other cell functions such as membrane protection and vesicle trafficking. These results support the need to restore the dysferlin transcript containing the alternative exon 40a in patients affected with dysferlinopathy.
| S-EPMC8650162 | BioStudies
Project description:Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.
| S-EPMC3870721 | BioStudies
Project description:Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues.We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use of in vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients.
| S-EPMC5240545 | BioStudies
Project description:<h4>Introduction</h4>Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf<sup>-/-</sup> ) mice.<h4>Methods</h4>Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf<sup>-/-</sup> and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice.<h4>Results</h4>Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf<sup>-/-</sup> muscles.<h4>Discussion</h4>We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies.
| S-EPMC6771521 | BioStudies
Project description:Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations.
| S-EPMC3323038 | BioStudies
Project description:Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca<sup>2+</sup>-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysf<sup>tm1Kcam</sup>/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca<sup>2+</sup>-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca<sup>2+</sup>-dependent pathway known to be important for sarcolemmal membrane repair.
| S-EPMC5628796 | BioStudies
Project description:The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.
| S-EPMC3376115 | BioStudies
Project description:Deficits in membrane repair may contribute to disease progression in dysferlin-deficient muscular dystrophy. Dysferlin, a type-II transmembrane phospholipid-binding protein, is hypothesized to regulate fusion of repair vesicles with the sarcolemma to facilitate membrane repair, but the dysferlin-containing compartments involved in membrane repair and the mechanism by which these compartments contribute to resealing are unclear. A dysferlin-pHluorin [dysf-pH-sensitive green fluorescent protein (pHGFP)] muscle-specific transgenic mouse was developed to examine the dynamic behavior and subcellular localization of dysferlin during membrane repair in adult skeletal muscle fibers. Live-cell confocal microscopy of uninjured adult dysf-pHGFP muscle fibers revealed that dysferlin is highly enriched in the sarcolemma and transverse tubules. Laser-wounding induced rapid recruitment of ?30 ?m of local dysferlin-containing sarcolemma, leading to formation of stable dysferlin accumulations surrounding lesions, endocytosis of dysferlin, and formation of large cytoplasmic vesicles from distal regions of the fiber. Disruption of the actin cytoskeleton decreased recruitment of sarcolemma-derived dysferlin to lesions in dysf-pHGFP fibers without affecting endocytosis and impaired membrane resealing in wild-type fibers, similar to findings in dysferlin deficiency (a 2-fold increase in FM1-43 uptake). Our data support a new mechanism whereby recruitment of sarcolemma-derived dysferlin creates an active zone of high lipid-binding activity at wounds to interact with repair vesicles and facilitate membrane resealing in skeletal muscle.
| S-EPMC4101652 | BioStudies