Human Filamin Research Articles

Interaction of the C-terminal immunoglobulin-like domains (Ig 22–24) of filamin C with human small heat shock proteins

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22–24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22–24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

Functional Validation of a Pathogenic Missense Variant in Human Filamin C Cardiomyopathy through Disruption of a Zebrafish Homologue Recapitulates Cardiac Disease

Purpose We recently performed whole-genome sequencing (WGS) of an East Asian proband and family members with diverse cardiac diseases, including arrhythmias, cardiomyopathies, premature heart failure requiring transplant, and identified a pathogenic missense variant (p.G2151S) in exon 39 of the filamin C gene, FLNC. In this study, we aim to demonstrate recapitulation of the pathogenic effects of the variant using a zebrafish (Danio rerio) model. Methods The proband, 3 other affected and 3 unaffected family members of a four-generation nonconsanguineous Hong Kong Chinese pedigree of 25 individuals were sequenced by WGS with 30X coverage. Single nucleotide variants and insertion-deletions that met our inclusion criteria were shortlisted, and bioinformatic analysis and variant prioritization were performed using Kaviar, gnomAD and dbNSFP databases to identify damaging variants in known cardiac-specific genes. The study was approved by the university/hospital's institutional review board. Sequence alignment of the pathogenic gene region with the zebrafish homologue was performed using the GENESTREAM network server, Institut de Genetique Humaine, Montpellier, France. Knockdown of the region was carried out using microinjection of morpholino oligonucleotides (MO), or control oligomers. Embryonic lethality, survival after birth, animal and cardiac morphology, heart rate, and histopathology were analyzed. Results Using a zebrafish model, we performed sequence alignment and identified the corresponding point mutation in flnca and flncb. Knockdown of the homologous sequences using antisense morpholino oligonucleotides resulted in stunted growth and development of the animal, cardiomegaly, pericardial edema, dose-dependent increase in embryonic lethality (survival rates of 95.8%, 46.6% and 9.7% at day 3 post-fertilization for control oligomer (n=24), 0.1 ng MO (n=88) and 0.5 ng MO (n=154), respectively), and reduced heart rate (134, 108 and 103 beats per minute, respectively; P Conclusion Damaging effects of the missense variant in human filamin C cardiomyopathy have been confirmed in the homologous region in this zebrafish model. The variable phenotypic manifestations within this East Asian family remain to be elucidated.

Non-syndromic Mitral Valve Dysplasia Mutation Changes the Force Resilience and Interaction of Human Filamin A

Filamin A (FLNa), expressed in endocardial endothelia during fetal valve morphogenesis, is key in cardiac development. Missense mutations in FLNa cause non-syndromic mitral valve dysplasia (FLNA-MVD). Here, we aimed to reveal the currently unknown underlying molecular mechanism behind FLNA-MVD caused by the FLNa P637Q mutation. The solved crystal structure of the FLNa3-5 P637Q revealed that this mutation causes only minor structural changes close to mutation site. These changes were observed to significantly affect FLNa's ability to transmit cellular force and to interact with its binding partner. The performed steered molecular dynamics simulations showed that significantly lower forces are needed to split domains 4 and 5 in FLNA-MVD than with wild-type FLNa. The P637Q mutation wasalso observed to interfere with FLNa's interactions with the protein tyrosine phosphatase PTPN12. Our results provide a crucial step toward understanding the molecular bases behind FLNA-MVD, which is critical for the development of drug-based therapeutics.

Structural basis of the filamin A actin-binding domain interaction with F-actin.

Actin-crosslinking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion and motility. Missense mutations in the tandem calponin homology (CH) domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-crosslinking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first CH domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD-F-actin binding.

Insilico characterization of Metal-Binding sites in human filamev protein

Human filamin are large actin-binding proteins and give mechanical force to cells by binding to actin filaments forming bundles or gel networks. There are three isoforms FLNA, FLNB and FLNC. Each isoforms consist of an N-terminal actin binding domain (ABB) and 24 repeated immunoglobulin Ig-like rod domains. Identifying metal binding sites is important for investigating a protein function and its interactions. In this study, metal binding sites were predicted for human filamin isoforms. The results suggest that filamin isoforms contain copper and cobalt ion as frequent occurrence as binding sites. This study will be helpful for further characterization of filamin protein and also for design of construct.

Skeletal Dysplasia Mutations Effect on Human Filamins\u2019 Structure and Mechanosensing

Cells’ ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered molecular dynamics simulations. The effects of skeletal dysplasia associated mutations of the structure and mechanosensing properties of Filamin were studied by combining various experimental and theoretical techniques. The results showed that Larsen syndrome associated mutations destabilize or even unfold domain 17. Interestingly, those Filamin functions that are mediated via domain 17 interactions with other proteins are not necessarily affected as strongly interacting peptide binding to mutated domain 17 induces at least partial domain folding. Mutation associated to Frontometaphyseal dysplasia, in turn, transforms 16–17 fragment from compact to an elongated form destroying the force-regulated domain pair.

Dynamic force sensing of filamin revealed in single-molecule experiments

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20-21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.

Normal X-inactivation mosaicism in corneas of heterozygous Flna Dilp2/+ female mice-a model of human Filamin A (FLNA) diseases

BackgroundSome abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance.ResultsX-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of FlnaDilp2/+ and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics.ConclusionsMosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.

Filamin's Force Sensing Mechanism Revealed by Optical Tweezers

Cell adhesion, spreading and migration, as it occurs during embryogenesis or wound healing and hemostasis, involve a complex interplay between the extracellular matrix, transmembrane adhesion receptors and the cytoskeleton. A prominent role plays human filamin A, since it is a widely expressed actin cross-linker which additionally binds a multitude of transmembrane receptors such as beta integrins or the von Willebrand Factor receptor GPIbα.Although it has been shown that beta integrins or GPIbα bind to the Ig-like domain 21 of filamin A, the molecular-level mechanism for filamin binding, especially its regulation, is still unclear. Filamin can auto-inhibit the interaction by hiding the binding site through intra-molecular interactions and it has been suggested that the binding site can be exposed by a force-induced conformational change.To address the question whether filamin's binding site in domain 21 was activated by force, we adapted a highly sensitive optical tweezers setup to pull at single domain pairs of human filamin A containing the receptor binding site and the auto-inhibiting region. We were able to apply and detect low forces in the physiological relevant regime around 3 pN and monitor the conformational change by measuring the length increase of the protein on a sub-millisecond timescale. This allowed us to distinguish in real-time between the inhibited closed conformation and the active open one. Additionally, we were able to detect the binding of different ligands added in solution and how the applied force increased the binding rate. Therefore, filamin A can be regarded as a force sensor.

Crystal structure of the dimerization domain of human filamin A

Crystal structure of the dimerization domain of human filamin A

Homology modeling and consensus protein disorder prediction of human filamin.

Filamins are dimeric actin-binding proteins participating in the organization of the actin-based cytoskeleton. Their modular domain organization is made up of an N-terminal actin-binding domain composed of two CH domains followed by flexible rod regions that consist of 24 Ig-like domains. Homology modeling was used to model human filamin using Modeller 9v5. The resulting model assessed by Verify 3D and PROCHECK showed that the final model is reliable. The conformational disorder prediction of human filamin residues were also mapped on the validated structure of human filamin. Prediction of protein disorder in filamin structures will help structural biologists to find suitable targets to be analyzed and for understanding protein function.

Structural Portrait of Filamin Interaction Mechanisms

We review the most recent findings on human filamin structure, with particular emphasis on the relationships between structure, function, and interaction. Filamin is a cytoskeletal actin-binding protein and it is therefore crucial in providing cells with the necessary mechanical and dynamical properties. Filamentous actin cross-linking by filamin is regulated by a number of other proteins and the molecular mechanisms of this complex interaction network can be understood by highlighting the structural features of isolated filamin moieties and of their complexes with several partners. Here we describe first the structure-function relationships of the isolated filamin, its flexibility, and its dimerization mechanism. Secondly, we illustrate the structural mechanism with which filamin can recognize its partners, both the actin filaments and the regulatory proteins.

Phosphorylation Facilitates the Integrin Binding of Filamin under Force

Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19–21 of filamin-A (IgFLNa-R19–R21). IgFLNa-R19–21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser2152, however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 β-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser2152 without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.

Identification and Characterization of Multiple Similar Ligand-binding Repeats in Filamin

The actin-binding protein filamin links membrane receptors to the underlying cytoskeleton. The cytoplasmic domains of these membrane receptors have been shown to bind to various filamin immunoglobulin repeats. Notably, among 24 human filamin repeats, repeat 17 was reported to specifically bind to platelet receptor glycoprotein Ibalpha and repeat 21 to integrins. However, a complete sequence alignment of all 24 human filamin repeats reveals that repeats 17 and 21 actually belong to a distinct filamin repeat subgroup (containing repeats 4, 9, 12, 17, 19, 21, and 23) that shares a conserved ligand-binding site. Using isothermal calorimetry and NMR analyses, we show that all repeats in this subgroup can actually bind glycoprotein Ibalpha, integrins, and a cytoskeleton regulator migfilin in similar manners. These data provide a new view on the ligand specificity of the filamin repeats. They also suggest a multiple ligand binding mechanism where similar repeats within a filamin monomer may promote receptor clustering or receptor cross-talking for regulation of the cytoskeleton organization and diverse filamin-mediated cellular activities.

Structure of the human filamin A actin-binding domain

Filamin A (FLNa) is a large dimeric protein that binds to actin filaments via its actin-binding domain (ABD). The crystal structure of this domain was solved at 3.2 A resolution. The domain adopts a closed conformation typical of other ABDs, but also forms a dimer both in crystallization conditions and in solution. The structure shows the localization of the residues mutated in patients with periventricular nodular heterotopia or otopalatodigital syndrome. Structural analysis predicts that mutations in both types of disorder may affect actin binding.

Single and multiple CH (calponin homology) domain containing multidomain proteins in Dictyostelium discoideum: an inventory

We present an inventory of single or multiple calponin homology (CH) domain containing proteins of Dictyostelium discoideum. A multiple alignment and a phylogenetic tree of all 60 CH domains found in 36 proteins showed that most CH domains can be assigned to one of 6 types. We have then distributed the proteins into several classes according to the type and arrangement of the CH domains. Most proteins belong to the class of ABD (actin-binding domain)-forming CH tandems (CH1-CH2) of the alpha-actinin and fimbrin families or to the class of CH3 domain-bearing proteins. There are a few examples of proteins with a single CH1 or CH2 domain, one with a CH1-CH1 doublet and a single representative of the CHe class of microtubule-binding proteins. A comparison with CH domain proteins in Homo sapiens suggests that while the individual domains are available in both species, the existence of identical multidomain proteins in toto is rare. Fimbrin 1, alpha-actinin and EB1 appear as perfect orthologs in both species, whereas filamin and interaptin may represent ancestral forms of human filamin and nesprins. In four more cases (NAV/Unc-53-, smoothelin-, transgelin- and Gas2-related proteins) functional data are needed in order to establish a potential relationship with a human counterpart. Although extensive data exist for a few of the D. discoideum CH proteins, most remain to be characterized and our analysis may help predicting some of their properties.

The Regulation Mechanism for the Auto-Inhibition of Binding of Human Filamin A to Integrin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Human filamins are large actin cross-linking proteins that connect integrins to the cytoskeleton. Filamin binding to the cytoplasmic tail of β integrins has been shown to prevent integrin activation in cells, which is important for controlling cell adhesion and migration. The molecular-level mechanism for filamin binding to integrin has been unclear, however, as it was recently demonstrated that filamin undergoes intramolecular auto-inhibition of integrin binding. In this study, using steered molecular dynamics simulations, we found that mechanical force applied to filamin can expose cryptic integrin binding sites. The forces required for this are considerably lower than those for filamin immunoglobulin domain unfolding. The mechanical-force-induced unfolding of filamin and exposure of integrin binding sites occur through stable intermediates where integrin binding is possible. Accordingly, our results support filamin's role as a mechanotransducer, since force-induced conformational changes allow binding of integrin and other transmembrane and intracellular proteins. This observed force-induced conformational change can also be one of possible mechanisms involved in the regulation of integrin activation.

Disease-associated Substitutions in the Filamin B Actin Binding Domain Confer Enhanced Actin Binding Affinity in the Absence of Major Structural Disturbance: Insights from the Crystal Structures of Filamin B Actin Binding Domains

Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F–actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6–7 °C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 μM (W148R) and 0.56 μM (M202V), compared to the wild type ABD Kd of 7.0 μM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein.

Mechanics of Biophysical Networks with Flexible Cross-links

Various mechanical properties and functions of euchariotic cells largely originate from the cytoskeleton. The predominant cytoskeletal constituent is the biopolymer filamentous actin (F-actin). In the presence of various cross-linking proteins, F-actins can comprise two rather different structures: isotropic orthogonal networks or bundled fibers. Actin bundles are formed mostly by short and stiff cross-linking proteins (like α-actinin and scruin), while large and flexible cross-linkers, such as filamin, lead to an orthogonal network. Orthogonal networks can also be formed at lower concentrations of short cross-linking proteins, but rheological experiments of in vitro F-actin networks showed that the mechanical response of such networks is different from that of networks cross-linked with filamin. Moreover, atomic force microscope stretching experiments on filamin demonstrated the possibility of force-induced domain unfolding, characterized by a sawtooth-like pattern in the force-displacement curve.Here we present a 3D discrete model of F-actin networks that extends our previous, rigidly cross-linked network model by incorporating a flexible cross-linking model for human filamin A (hsFLNa). The implemented hsFLNa element has a highly nonlinear response to stretching, incorporating the transition to a softer response that characterizes filamin domain unfolding. Simple shear simulations of F-actin/hsFLNa networks show that the response of such networks is dominated by the behavior of the hsFLNa cross-linkers, while F-actin behaves almost rigid. We observe that force-induced unfolding of the hsFLNa relaxes the stresses in actin filaments, thus allowing for large network strains. By contrast, the shearing of F-actin networks with rigid cross-links leads to a large number of actin filaments stressed well beyond their breaking force. An increase in actin concentration increases the initial shear modulus, while the maximum network stiffening depends on the hsFLNa axial stiffness. The calculated initial modulus of F-actin/hsFLNa networks is found to be comparable with experimental measurements.

Crystal structure of the dimerization domain of human filamin A

Crystal structure of the dimerization domain of human filamin A Min-Duk Seo,1y Seung-Hyeon Seok,1y Hookang Im, Ae-Ran Kwon, Sang Jae Lee, Hyung-Ryong Kim, Yongcheol Cho, Dongeun Park, and Bong-Jin Lee* 1 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea 2 Promeditech Ltd., Seoul 151-010, Korea 3Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk 570-749, Korea 4 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea