The third human FER-1-like protein is highly similar to dysferlin

Dysferlin, the protein product of the gene mutated in patients with an autosomal recessive limb-girdle muscular dystrophy type 2B (LGMD2B) and a distal muscular dystrophy, Miyoshi myopathy, is homologous to a Caenorhabditis elegans spermatogenesis factor, FER-1. Analysis of fer-1 mutants and of sequence predictions of the FER-1 and dysferlin ORFs has predicted a role in membrane fusion. Otoferlin, another human FER-1-like protein (ferlin), has recently been shown to be responsible for autosomal recessive nonsyndromic deafness (DFNB9). In this report we describe the third human ferlin gene, FER1L3, which maps to chromosome 10q23.3. Expression analysis of the orthologous mouse gene shows ubiquitous expression but predominant expression in the eye, esophagus, and salivary gland. All the ferlins are characterized by sequences corresponding to multiple C2 domains that share the highest level of homology with the C2A domain of rat synaptotagmin III. They are predicted to be Type II transmembrane proteins, with the majority of the protein facing the cytoplasm anchored by the C-terminal transmembrane domain. Sequence and predicted structural comparisons have highlighted the high degree of similarity of dysferlin and FER1L3, which have sequences corresponding to six C2 domains and which share more than 60% amino acid sequence identity.     

Proteomic investigation of the molecular pathophysiology of dysferlinopathy

Mutations in dysferlin gene cause several types of muscular dystrophy in humans, including the limb-girdle muscular dystrophy type 2B and the distal muscular dystrophy of Miyoshi. The dysferlin gene product is a membrane-associated protein belonging to the ferlins family of proteins. The function of the dysferlin protein and the cause of deterioration and regression of muscle fibres in its absence, are incompletely known. A functional clue may be the presence of six hydrophilic domains, C2, that bind calcium and mediate the interaction of proteins with cellular membranes. Dysferlin seems to be involved in the membrane fusion or repair. Molecular diagnosis of dysferlinopathies is now possible and the types of gene alterations that have been characterized so far include missense mutations, deletions and insertions.     

Dysferlin and the plasma membrane repair in muscular dystrophy

Muscular dystrophy covers a group of genetically determined disorders that cause progressive weakness and wasting of the skeletal muscles. Dysferlin was identified as a gene mutated in limb-girdle muscular dystrophy (type 2B) and Miyoshi myopathy. The discovery of dysferlin revealed a new family of proteins, known as the ferlin family, which includes four different genes. Recent work suggests the function of dysferlin in membrane repair and demonstrates that defective membrane repair is a novel mechanism of muscle degeneration. These findings reveal the importance of a basic cellular function in skeletal muscle and a new class of muscular dystrophy where the defect lies in the maintenance, not the structure, of the plasma membrane. Here, we discuss the current knowledge of dysferlin function in the repair of the plasma membrane of the skeletal muscle cells.     

Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

Ferlins are an ancient family of C2 domain-containing proteins, with emerging roles in vesicular trafficking and human disease. Dysferlin mutations cause inherited muscular dystrophy, and dysferlin also shows abnormal plasma membrane expression in other forms of muscular dystrophy. We establish dysferlin as a short-lived (protein half-life ∼4–6 h) and transitory transmembrane protein (plasma membrane half-life ∼3 h), with a propensity for rapid endocytosis when mutated, and an association with a syntaxin-4 endocytic route. Dysferlin plasma membrane expression and endocytic rate is regulated by the C2B-FerI-C2C motif, with a critical role identified for C2C. Disruption of C2C dramatically reduces plasma membrane dysferlin (by 2.5-fold), due largely to accelerated endocytosis (by 2.5-fold). These properties of reduced efficiency of plasma membrane expression due to accelerated endocytosis are also a feature of patient missense mutant L344P (within FerI, adjacent to C2C). Importantly, dysferlin mutants that demonstrate accelerated endocytosis also display increased protein lability via endosomal proteolysis, implicating endosomal-mediated proteolytic degradation as a novel basis for dysferlin-deficiency in patients with single missense mutations. Vesicular labeling studies establish that dysferlin mutants rapidly transit from EEA1-positive early endosomes through to dextran-positive lysosomes, co-labeled by syntaxin-4 at multiple stages of endosomal transit. In summary, our studies define a transient biology for dysferlin, relevant to emerging patient therapeutics targeting dysferlin replacement. We introduce accelerated endosomal-directed degradation as a basis for lability of dysferlin missense mutants in dysferlinopathy, and show that dysferlin and syntaxin-4 similarly transit a common endosomal pathway in skeletal muscle cells.     

Dysferlin forms a dimer mediated by the C2 domains and the transmembrane domain in vitro and in living cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ～460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.     

Dysferlin homozygous mutation G1418D causes limb-girdle type 2B in a Mexican family

Dysferlin protein (DYSF) is a ferlin family member found in sarcolemma and is involved in membrane repair, muscle differentiation, membrane fusion, etc. The deficiency of DYSF due to mutations is associated with different pathologic phenotypes including the autosomal recessive limb-girdle type 2B phenotype (LGMD2B), a distal anterior compartment myopathy (DMAT), and the Miyoshi myopathy (MM). In this study, we determined a missense mutation c.4253G>A on the DYSF gene in a Mexican family from an endogamic population. This mutation was assumed to be the cause of dystrophy because only homozygous individuals of the family manifest a clinical phenotype. Structural implications caused by G/D substitution at amino acid position 1418 are discussed in terms of potential importance of the dysferlin neighboring sequence.     

Dysferlin deficiency confers increased susceptibility to coxsackievirus‐induced cardiomyopathy

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin‐deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin‐deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus‐induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy.     

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles.Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.     

Proteasomal inhibition restores biological function of mis-sense mutated dysferlin in patient-derived muscle cells

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.     

Caveolin regulates endocytosis of the muscle repair protein, dysferlin

Dysferlin and Caveolin-3 are plasma membrane proteins associated with muscular dystrophy. Patients with mutations in the CAV3 gene show dysferlin mislocalization in muscle cells. By utilizing caveolin-null cells, expression of caveolin mutants, and different mutants of dysferlin, we have dissected the site of action of caveolin with respect to dysferlin trafficking pathways. We now show that Caveolin-1 or -3 can facilitate exit of a dysferlin mutant that accumulates in the Golgi complex of Cav1(-/-) cells. In contrast, wild type dysferlin reaches the plasma membrane but is rapidly endocytosed in Cav1(-/-) cells. We demonstrate that the primary effect of caveolin is to cause surface retention of dysferlin. Caveolin-1 or Caveolin-3, but not specific caveolin mutants, inhibit endocytosis of dysferlin through a clathrin-independent pathway colocalizing with internalized glycosylphosphatidylinositol-anchored proteins. Our results provide new insights into the role of this endocytic pathway in surface remodeling of specific surface components. In addition, they highlight a novel mechanism of action of caveolins relevant to the pathogenic mechanisms underlying caveolin-associated disease.     

Absence of dysferlin alters myogenin expression and delays human muscle differentiation "in vitro"

Mutations in dysferlin cause a type of muscular dystrophy known as dysferlinopathy. Dysferlin may be involved in muscle repair and differentiation. We compared normal human skeletal muscle cultures expressing dysferlin with muscle cultures from dysferlinopathy patients. We quantified the fusion index of myoblasts as a measure of muscle development and conducted optic and electronic microscopy, immunofluorescence, Western blot, flow cytometry, and real-time PCR at different developmental stages. Short interference RNA was used to corroborate the results obtained in dysferlin-deficient cultures. A luciferase reporter assay was performed to study myogenin activity in dysferlin-deficient cultures. Myoblasts fusion was consistently delayed as compared with controls whereas the proliferation rate did not change. Electron microscopy showed that control cultured cells at 10 days were fusiform, whereas dysferlin-deficient cells were star-shaped and large. After 15 days the normal multinucleated appearance and structured myofibrils were not present in dysferlin-deficient cells. Strikingly, myogenin was not detected in myotubes from dysferlin-deficient cultures using Western blot, and mRNA analysis showed low levels (p < 0.05) compared with controls. Flow cytometry and immunofluorescence also showed reduced levels of myogenin in dysferlin-deficient cultures. When the dysferlin gene was knocked down ( approximately 80%), myogenin mRNA leveled down to approximately 70%. MyoD and desmin mRNA levels in controls and dysferlin-deficient cultures were similar. The reporter luciferase assay demonstrated a low myogenin activity in dysferlin-deficient cultures. These results point to a functional link between dysferlin and myogenin, and both proteins may share a new signaling pathway involved in differentiation of skeletal muscle in vitro.     

Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains

Mutations in dysferlin, a novel membrane protein of unknown function, lead to muscular dystrophy. Myoferlin is highly homologous to dysferlin and like dysferlin is a plasma membrane protein with six C2 domains highly expressed in muscle. C2 domains are found in a variety of membrane-associated proteins where they have been implicated in calcium, phospholipid, and protein-binding. We investigated the pattern of dysferlin and myoferlin expression in a cell culture model of muscle development and found that dysferlin is expressed in mature myotubes. In contrast, myoferlin is highly expressed in elongated "prefusion" myoblasts and is decreased in mature myotubes where dysferlin expression is greatest. We tested ferlin C2 domains for their ability to bind phospholipid in a calcium-sensitive manner. We found that C2A, the first C2 domain of dysferlin and myoferlin, bound 50% phosphatidylserine and that phospholipid binding was regulated by calcium concentration. A dysferlin point mutation responsible for muscular dystrophy was engineered into the dysferlin C2A domain and demonstrated reduced calcium-sensitive phospholipid binding. Based on these data, we propose a mechanism for muscular dystrophy in which calcium-regulated phospholipid binding is abnormal, leading to defective maintenance and repair of muscle membranes.     

Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle

Dysferlin is a type II transmembrane protein implicated in surface membrane repair in muscle. Mutations in dysferlin lead to limb girdle muscular dystrophy 2B, Miyoshi Myopathy and distal anterior compartment myopathy. Dysferlin''s mode of action is not well understood and only a few protein binding partners have thus far been identified. Using affinity purification followed by liquid chromatography/mass spectrometry, we identified alpha-tubulin as a novel binding partner for dysferlin. The association between dysferlin and alpha-tubulin, as well as between dysferlin and microtubules, was confirmed in vitro by glutathione S-transferase pulldown and microtubule binding assays. These interactions were confirmed in vivo by co-immunoprecipitation. Confocal microscopy revealed that dysferlin and alpha-tubulin co-localized in the perinuclear region and in vesicular structures in myoblasts, and along thin longitudinal structures reminiscent of microtubules in myotubes. We mapped dysferlin''s alpha-tubulin-binding region to its C2A and C2B domains. Modulation of calcium levels did not affect dysferlin binding to alpha-tubulin, suggesting that this interaction is calcium-independent. Our studies identified a new binding partner for dysferlin and suggest a role for microtubules in dysferlin trafficking to the sarcolemma.     

Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity

Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.     

Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.     

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.     

Translational research and therapeutic perspectives in dysferlinopathies

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.     

Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy

Dysferlin deficiency leads to a peculiar form of muscular dystrophy due to a defect in sarcolemma repair and currently lacks a therapy. We developed a cell therapy protocol with wild-type adult murine mesoangioblasts. These cells differentiate with high efficiency into skeletal muscle in vitro but differ from satellite cells because they do not express Pax7. After intramuscular or intra-arterial administration to SCID/BlAJ mice, a novel model of dysferlinopathy, wild-type mesoangioblasts efficiently colonized dystrophic muscles and partially restored dysferlin expression. Nevertheless, functional assays performed on isolated single fibers from transplanted muscles showed a normal repairing ability of the membrane after laser-induced lesions; this result, which reflects gene correction of an enzymatic rather than a structural deficit, suggests that this myopathy may be easier to treat with cell or gene therapy than other forms of muscular dystrophies.     

Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf-/- mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.     

Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing

Mutations in the dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes.