Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.
ABSTRACT: Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.
Project description:Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a-specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.
Project description:Dysferlin has been implicated in acute membrane repair processes, whereas myoferlin's activity is maximal during the myoblast fusion stage of early skeletal muscle cell development. Both proteins are similar in size and domain structure; however, despite the overall similarity, myoferlin's known physiological functions do not overlap with those of dysferlin. Here we present for the first time the X-ray crystal structure of human myoferlin C2A to 1.9 Å resolution bound to two divalent cations, and compare its three-dimensional structure and membrane binding activities to that of dysferlin C2A. We find that while dysferlin C2A binds membranes in a Ca2+-dependent manner, Ca2+ binding was the rate-limiting kinetic step for this interaction. Myoferlin C2A, on the other hand, binds two calcium ions with an affinity 3-fold lower than that of dysferlin C2A; and, surprisingly, myoferlin C2A binds only marginally to phospholipid mixtures with a high fraction of phosphatidylserine.
Project description:Ferlin proteins participate in such diverse biological events as vesicle fusion in C. elegans, fusion of myoblast membranes to form myotubes, Ca2+-sensing during exocytosis in the hair cells of the inner ear, and Ca2+-dependent membrane repair in skeletal muscle cells. Ferlins are Ca2+-dependent, phospholipid-binding, multi-C2 domain-containing proteins with a single transmembrane helix that spans a vesicle membrane. The overall domain composition of the ferlins resembles the proteins involved in exocytosis; therefore, it is thought that they participate in membrane fusion at some level. But if ferlins do fuse membranes, then they are distinct from other known fusion proteins. Here we show that the central FerA domain from dysferlin, myoferlin, and otoferlin is a novel four-helix bundle fold with its own Ca2+-dependent phospholipid-binding activity. Small-angle X-ray scattering (SAXS), spectroscopic, and thermodynamic analysis of the dysferlin, myoferlin, and otoferlin FerA domains, in addition to clinically-defined dysferlin FerA mutations, suggests that the FerA domain interacts with the membrane and that this interaction is enhanced by the presence of Ca2+.
Project description:Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.
Project description:Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
Project description:Ferlins are multiple-C2-domain proteins involved in Ca2+-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development.
Project description:Ferlins are large multi-C2 domain membrane proteins involved in membrane fusion and fission events. In this study, we investigate the effects of binding of the C2 domains of otoferlin, dysferlin, and myoferlin on the structure of lipid bilayers. Fluorescence measurements indicate that multi-C2 domain constructs of myoferlin, dysferlin, and otoferlin change the lipid packing of both small unilamellar vesicles and giant plasma membrane vesicles. The activities of these proteins were enhanced in the presence of calcium and required negatively charged lipids like phosphatidylserine or phosphatidylglycerol for activity. Experiments with individual domains uncovered functional differences between the C2A domain of otoferlin and those of dysferlin and myoferlin, and truncation studies suggest that the effects of each subsequent C2 domain on lipid ordering appear to be additive. Finally, we demonstrate that the activities of these proteins on membranes are insensitive to high salt concentrations, suggesting a nonelectrostatic component to the interaction between ferlin C2 domains and lipid bilayers. Together, the data indicate that dysferlin, otoferlin, and myoferlin do not merely passively adsorb to membranes but actively sculpt lipid bilayers, which would result in highly curved or distorted membrane regions that could facilitate membrane fusion, membrane fission, or recruitment of other membrane-trafficking proteins.
Project description:The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.
Project description:Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.
Project description:Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC.EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing.Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.