Ferlin Family Research Articles

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

A missense variant in MYOF is associated with ARVC and sudden cardiac death

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is rare autosomal dominant genetic disorder that leads to severe arrhythmia and sudden cardiac death. Although previous studies in clinical, pathological and genetics of ARVC established consensus diagnostic criteria and expanded the spectrum of pathogenic genes, there is still a proportion of patients with unclear causative factors.Here, whole-exome sequencing was employed to investigate the genetic etiology of a 15-year-old sudden cardiac death female caused by ARVC. A novel variant of MYOF (NM_013451.3: c.4723G > C: p.D1575H) was identified, which is a member of the Ferlin family of proteins is associated with cardiomyopathy. And the bioinformatics analysis predicted the pathogenicity of this variant. We report the first variant of MYOF in ARVC, which imply a vital role of MYOF in cardiomyopathy.

P97/VCP Promotes the Recycling of Endocytic Cargo.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

Spermiogenesis in Caenorhabditis elegans: An Excellent Model to Explore the Molecular Basis for Sperm Activation.

C. elegans spermiogenesis converts non-motile spermatids into motile, fertilization-competent spermatozoa. Two major events include the building of a pseudopod required for motility and fusion of membranous organelles (MOs)-intracellular secretory vesicles-with the spermatid plasma membrane required for the proper distribution of sperm molecules in mature spermatozoa. The mouse sperm acrosome reaction-a sperm activation event occurring during capacitation-is similar to MO fusion in terms of cytological features and biological significance. Moreover, C. elegans fer-1 and mouse Fer1l5, both encoding members of the ferlin family, are indispensable for MO fusion and acrosome reaction, respectively. Genetics-based studies have identified many C. elegans genes involved in spermiogenesis pathways; however, it is unclear whether mouse orthologs of these genes are involved in the acrosome reaction. One significant advantage of using C. elegans for studying sperm activation is the availability of in vitro spermiogenesis, which enables combining pharmacology and genetics for the assay. If certain drugs can activate both C. elegans and mouse spermatozoa, these drugs would be useful probes to explore the mechanism underlying sperm activation in these two species. By analyzing C. elegans mutants whose spermatids are insensitive to the drugs, genes functionally relevant to the drugs' effects can be identified.

A Novel Homozygous Abnormal Splice Variant in the Myoferlin Gene Leading to Floppy Infant Syndrome in a Saudi Family

Myoferlin (MYOF) (OMIM#604603) is a type II membrane protein that belongs to the ferlin family, which is expressed in cardiac and skeletal muscles. This protein has seven C2 domains that mediate calcium-dependent membrane fusion events and membrane trafficking, while MYOF dysfunction is associated with muscular disorder. We are reporting a case from Saudi Arabia of an 18-month-old male patient with generalized hypotonia, which might be a floppy infant syndrome. In this study, whole exome sequencing (WES) was done, and a novel homozygous abnormal splice variant c.4982+1G>T, p.Val1661fs was identified in the MYOF gene. The results of WES were further validated by using Sanger sequencing; the proband showed homozygous mutation while both parents were heterozygous at this position. Implementing WES improves the screening and detection of novel and causative genetic variants and comprehends patient management. The results of this study, therefore, will add to the literature on the role of MYOF gene and any pathogenic variants that might lead to muscular dysfunction. Furthermore, this will establish a disease database, providing a groundwork for understanding the critical genomic regions.

Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function.

Ferlins are complex, multi-domain proteins, involved in membrane trafficking, membrane repair, and exocytosis. The large size of ferlin proteins and the lack of consensus regarding domain boundaries have slowed progress in understanding molecular-level details of ferlin protein structure and function. However, in silico protein folding techniques have significantly enhanced our understanding of the complex ferlin family domain structure. We used RoseTTAFold to assemble full-length models for the six human ferlin proteins (dysferlin, myoferlin, otoferlin, Fer1L4, Fer1L5, and Fer1L6). Our full-length ferlin models were used to obtain objective domain boundaries, and these boundaries were supported by AlphaFold2 predictions. Despite the differences in amino acid sequence between the ferlin proteins, the domain ranges and distinct subdomains in the ferlin domains are remarkably consistent. Further, the RoseTTAFold/AlphaFold2 in silico boundary predictions allowed us to describe and characterize a previously unknown C2 domain, ubiquitous in all human ferlins, which we refer to as C2-FerA. At present, the ferlin domain-domain interactions implied by the full-length in silico models are predicted to have a low accuracy; however, the use of RoseTTAFold and AlphaFold2 as a domain finder has proven to be a powerful research tool for understanding ferlin structure.

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth.

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.

An Emerging Therapeutic Approach by Targeting Myoferlin (MYOF) for Malignant Tumors.

Myoferlin (MYOF), as a member of the ferlin family, is a type II transmembrane protein with a single transmembrane domain at the carbon terminus. Studies have shown that MYOF is involved in pivotal physiological functions related to numerous cell membranes, such as extracellular secretion, endocytosis cycle, vesicle trafficking, membrane repair, membrane receptor recycling, and secreted protein efflux. Recently, the studies have also revealed that MYOF is overexpressed in a variety of cancers such as colorectal cancer, pancreatic cancer, breast cancer, melanoma, gastric cancer, and non-small-cell lung cancer. High expression of MYOF is associated with the high invasion of tumors and poor clinical prognosis. MYOF medicates the expression, secretion, and distribution of proteins, which were closely related to cancers, as well as the energy utilization of cancer cells, lipid metabolism and other physiological activities by regulating the physiological processes of membrane transport. In this short article, we briefly summarize the latest progress related to MYOF, indicating that small molecule inhibitors targeting the MYOF-C2D domain can selectively inhibit the proliferation and migration of cancer cells, and MYOF may be a promising target for the treatment of malignant tumors.

Novel properties of myoferlin in glucose metabolism via pathways involving modulation of adipose functions.

While adipose tissue is required to maintain glucose metabolism, excessive calorie intake induces obesity via mechanisms including accelerated proliferation and differentiation of preadipocytes, leading to insulin resistance. Here, we investigated the role of myoferlin (MYOF), a ferlin family protein, in regulating glucose metabolism by mainly focusing on its unknown role in adipose tissue. Whereas young MYOF knockout (KO) mice on a normal diet showed aggravated glucose tolerance and insulin sensitivity, those on a high-fat diet (HFD) showed preserved glucose tolerance with an attenuated gain of body weight, reduced visceral fat deposits, and less severe fatty liver. The Adipose MYOF expression was reduced by aging but was restored by an HFD along with the retained expression of NFAT transcription factors. Loss-of-function of MYOF in preadipocytes suppressed proliferation and differentiation into mature adipocytes along with the decreased expression of genes involved in adipogenesis. The MYOF expression in preadipocytes was reduced with differentiation. Attenuated obesity in MYOF KO mice on an HFD was also accompanied with increased oxygen consumption by an unidentified mechanism and with reduced adipose inflammation due to less inflammatory macrophages. These insights suggest that the multifunctional roles of MYOF involve the regulation of preadipocyte function and affect glucose metabolism bidirectionally depending on consumed calories.

Myoferlin, a Membrane Protein with Emerging Oncogenic Roles.

Myoferlin (MYOF), initially identified in muscle cells, is a member of the Ferlin family involved in membrane fusion, membrane repair, and membrane trafficking. Dysfunction of this protein is associated with muscular dysfunction. Recently, a growing body of studies have identified MYOF as an oncogenic protein. It is overexpressed in a variety of human cancers and promotes tumorigenesis, tumor cell motility, proliferation, migration, epithelial to mesenchymal transition, angiogenesis as well as metastasis. Clinically, MYOF overexpression is associated with poor outcome in various cancers. It can serve as a prognostic marker of human malignant disease. MYOF drives the progression of cancer in various processes, including surface receptor transportation, endocytosis, exocytosis, intercellular communication, fit mitochondrial structure maintenance and cell metabolism. Depletion of MYOF demonstrates significant antitumor effects both in vitro and in vivo, suggesting that targeting MYOF may produce promising clinical benefits in the treatment of malignant disease. In the present article, we reviewed the physiological function of MYOF as well as its role in cancer, thus providing a general understanding for further exploration of this protein.

Myoferlin Regulates Wnt/β-Catenin Signaling-Mediated Skeletal Muscle Development by Stabilizing Dishevelled-2 Against Autophagy.

Myoferlin (MyoF), which is a calcium/phospholipid-binding protein expressed in cardiac and muscle tissues, belongs to the ferlin family. While MyoF promotes myoblast differentiation, the underlying mechanisms remain poorly understood. Here, we found that MyoF not only promotes C2C12 myoblast differentiation, but also inhibits muscle atrophy and autophagy. In the present study, we found that myoblasts fail to develop into mature myotubes due to defective differentiation in the absence of MyoF. Meanwhile, MyoF regulates the expression of atrophy-related genes (Atrogin-1 and MuRF1) to rescue muscle atrophy. Furthermore, MyoF interacts with Dishevelled-2 (Dvl-2) to activate canonical Wnt signaling. MyoF facilitates Dvl-2 ubiquitination resistance by reducing LC3-labeled Dvl-2 levels and antagonizing the autophagy system. In conclusion, we found that MyoF plays an important role in myoblast differentiation during skeletal muscle atrophy. At the molecular level, MyoF protects Dvl-2 against autophagy-mediated degradation, thus promoting activation of the Wnt/β-catenin signaling pathway. Together, our findings suggest that MyoF, through stabilizing Dvl-2 and preventing autophagy, regulates Wnt/β-catenin signaling-mediated skeletal muscle development.

Myoferlin, a multifunctional protein in normal cells, has novel and key roles in various cancers.

Myoferlin, a protein of the ferlin family, has seven C2 domains and exhibits activity in some cells, including myoblasts and endothelial cells. Recently, myoferlin was identified as a promising target and biomarker in non‐small‐cell lung cancer, breast cancer, pancreatic adenocarcinoma, hepatocellular carcinoma, colon cancer, melanoma, oropharyngeal squamous cell carcinoma, head and neck squamous cell carcinoma, clear cell renal cell carcinoma and endometrioid carcinoma. This evidence indicated that myoferlin was involved in the proliferation, invasion and migration of tumour cells, the mechanism of which mainly included promoting angiogenesis, vasculogenic mimicry, energy metabolism reprogramming, epithelial‐mesenchymal transition and modulating exosomes. The roles of myoferlin in both normal cells and cancer cells are of great significance to provide novel and efficient methods of tumour treatment. In this review, we summarize recent studies and findings of myoferlin and suggest that myoferlin is a novel potential candidate for clinical diagnosis and targeted cancer therapy.

Human colon cancer cells highly express myoferlin to maintain a fit mitochondrial network and escape p53-driven apoptosis

Colon adenocarcinoma is the third most commonly diagnosed cancer and the second deadliest one. Metabolic reprogramming, described as an emerging hallmark of malignant cells, includes the predominant use of glycolysis to produce energy. Recent studies demonstrated that mitochondrial electron transport chain inhibitor reduced colon cancer tumour growth. Accumulating evidence show that myoferlin, a member of the ferlin family, is highly expressed in several cancer types, where it acts as a tumour promoter and participates in the metabolic rewiring towards oxidative metabolism. In this study, we showed that myoferlin expression in colon cancer lesions is associated with low patient survival and is higher than in non-tumoural adjacent tissue. Human colon cancer cells silenced for myoferlin exhibit a reduced oxidative phosphorylation activity associated with mitochondrial fission leading, ROS accumulation, decreased cell growth, and increased apoptosis. We observed the triggering of a DNA damage response culminating to a cell cycle arrest in wild-type p53 cells. The use of a p53 null cell line or a compound able to restore p53 activity (Prima-1) reverted the effects induced by myoferlin silencing, confirming the involvement of p53. The recent identification of a compound interacting with a myoferlin C2 domain and bearing anticancer potency identifies, together with our demonstration, this protein as a suitable new therapeutic target in colon cancer.

Fer1l6 is essential for the development of vertebrate muscle tissue in zebrafish.

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein–based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.

A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion.

Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+ Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite's rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite's infection cycle.

FerA is a Membrane-Associating Four-Helix Bundle Domain in the Ferlin Family of Membrane-Fusion Proteins

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+.

Myoferlin controls mitochondrial structure and activity in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.

Emerging Functional Differences between the Synaptotagmin and Ferlin Calcium Sensor Families.

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

A muscle-specific protein 'myoferlin' modulates IL-6/STAT3 signaling by chaperoning activated STAT3 to nucleus.

Myoferlin, a member of ferlin family of proteins, was first discovered as a candidate gene for muscular dystrophy and cardiomyopathy. Recently myoferlin was shown to be also expressed in endothelial and cancer cells where it was shown to modulate VEGFR-2 and EGFR signaling by enhancing their stability and recycling. Based on these reports, we hypothesized that myoferlin might be regulating IL-6 signaling by modulating IL-6R stabilization and recycling. However, in our immunoprecipitation (IP) experiments, we did not observe myoferlin binding with IL-6R. Instead, we made a novel discovery that in resting cells myoferlin was bound to EHD2 protein and when cells were treated with IL-6, myoferlin dissociated from EHD2 and binds to activated STAT3. Interestingly, myoferlin depletion did not affect STAT3 phosphorylation, but completely blocked STAT3 translocation to nucleus. In addition, inhibition of STAT3 phosphorylation by phosphorylation defective STAT3 mutants or JAK inhibitor blocked STAT3 binding to myoferlin and nuclear translocation. Myoferlin knockdown significantly decreased IL-6-mediated tumor cell migration, tumorsphere formation and ALDH positive cancer stem cell population, in vitro. Furthermore, myoferlin knockdown significantly decreased IL-6-meditated tumor growth and tumor metastasis. Based on these results, we have proposed a novel model for the role of myoferlin in chaperoning phosphorylated STAT3 to the nucleus.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

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.