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Siegfried Labeit - One of the best experts on this subject based on the ideXlab platform.
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a novel mouse model of Nebulin based nemaline myopathy
Biophysical Journal, 2013Co-Authors: Coen A C Ottenheijm, Siegfried Labeit, Danielle Buck, Josine M De Winter, Ger J M Stienen, Michael W Lawlor, Alan H Beggs, Henk GranzierAbstract:Nebulin - a giant sarcomeric protein - plays a pivotal role in skeletal muscle contractility by regulating thin filament length and function. Although mutations in the gene encoding Nebulin (NEB) are a frequent cause of nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which NEB exon 55 is deleted (NEBΔex55), a mutation known to frequently occur in NM patients.NEBΔex55 mice are born close to Mendelian ratio's, but show growth retardation after birth. Electronmicroscopy shows nemaline rods - a hallmark feature of NM - in muscle fibers from NEBΔex55 mice. Western blotting studies with Nebulin-specific antibodies reveal much reduced Nebulin levels in muscle from NEBΔex55 mice. Immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is reduced in muscle fibers from NEBΔex55 mice. In line with reduced thin filament length, the maximal force generating capacity of skinned muscle fibers is reduced in NEBΔex55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in NEBΔex55 mice the regulation of contraction is impaired, as evidenced by marked changes in cross bridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. This reduced calcium sensitivity was observed only at short sarcomere lengths, suggesting that Nebulin might play a role in the length dependence of activation.In conclusion, we have generated the first Nebulin-based NM model. Our data indicate that the phenotype of NEBΔex55 mice closely recapitulates that observed previously by us in patients harboring this particular mutation.
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the sarcomeric protein Nebulin another multifunctional giant in charge of muscle strength optimization
Frontiers in Physiology, 2012Co-Authors: Henk Granzier, Coen A C Ottenheijm, Siegfried LabeitAbstract:The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been successful in explaining many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and Nebulin. Whereas the role of titin rapidly progressed, Nebulin’s role in muscle structure and function remained long nebulous. An important feature of muscle structure and function that has remained relatively obscure concerns the mechanisms that are involved in regulating thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of Nebulin KO models, reveal that Nebulin plays an important role in the regulation of thin filament length, most likely by stabilizing F-actin assemblies. Another structural feature of skeletal muscle that has been incompletely understood concerns the mechanisms involved in maintaining Z-disk structure and the regular lateral alignment of adjacent sarcomeres during contraction. Recent studies indicate that Nebulin is part of a protein complex that mechanically links adjacent myofibrils. In addition to these structural roles in support of myofibrillar force generation, Nebulin has been also shown to regulate directly muscle contraction at the level of individual cross bridges: cycling kinetics and the calcium sensitivity of force producing cross-bridges is enhanced in the presence of Nebulin. Thus, these recent data all point to Nebulin being important for muscle force optimization. Consequently, muscle weakness as the lead symptom develops in the case of patients with nemaline myopathy that have mutations in the Nebulin gene. Here, we discuss these important novel insights into the role of Nebulin in skeletal muscle function.
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Nebulin a major player in muscle health and disease
The FASEB Journal, 2011Co-Authors: Siegfried Labeit, Coen A C Ottenheijm, Henk GranzierAbstract:Nebulin is a giant 600- to 900-kDa filamentous protein that is an integral component of the skeletal muscle thin filament. Its functions have remained largely nebulous because of its large size and the difficulty in extracting Nebulin in a native state from muscle. Recent improvements in the field, especially the development of knockout mouse models deficient in Nebulin (NEB-KO mice), indicate now that Nebulin performs a surprisingly wide range of functions. In addition to a major role in thin-filament length specification, Nebulin also functions in the regulation of muscle contraction, as indicated by the findings that muscle fibers deficient in Nebulin have a higher tension cost, and develop less force due to reduced myofilament calcium sensitivity and altered crossbridge cycling kinetics. In addition, the function of Nebulin extends to a role in calcium homeostasis. These novel functions indicate that Nebulin might have evolved in vertebrate skeletal muscles to develop high levels of muscle force effic...
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differential splicing of the large sarcomeric protein Nebulin during skeletal muscle development
Journal of Structural Biology, 2010Co-Authors: Danielle Buck, Siegfried Labeit, Coen A C Ottenheijm, Bryan D Hudson, Henk GranzierAbstract:Abstract We studied differential splicing of Nebulin, a giant filamentous F-actin binding protein ( M r ∼700–800 kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that Nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied Nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse Nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS–agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of Nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that Nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of Nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.
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reduced myofibrillar connectivity and increased z disk width in Nebulin deficient skeletal muscle
Journal of Cell Science, 2010Co-Authors: Paola Tonino, Siegfried Labeit, Carol C. Gregorio, Christopher T. Pappas, Bryan D Hudson, Henk GranzierAbstract:A prominent feature of striated muscle is the regular lateral alignment of adjacent sarcomeres. An important intermyofibrillar linking protein is the intermediate filament protein desmin, and based on biochemical and structural studies in primary cultures of myocytes it has been proposed that desmin interacts with the sarcomeric protein Nebulin. Here we tested whether Nebulin is part of a novel biomechanical linker complex, by using a recently developed Nebulin knockout (KO) mouse model and measuring Z-disk displacement in adjacent myofibrils of both extensor digitorum longus (EDL) and soleus muscle. Z-disk displacement increased as sarcomere length (SL) was increased and the increase was significantly larger in KO fibers than in wild-type (WT) fibers; results in 3-day-old and 10-day-old mice were similar. Immunoelectron microscopy revealed reduced levels of desmin in intermyofibrillar spaces adjacent to Z-disks in KO fibers compared with WT fibers. We also performed siRNA knockdown of Nebulin and expressed modules within the Z-disk portion of Nebulin (M160-M170) in quail myotubes and found that this prevented the mature Z-disk localization of desmin filaments. Combined, these data suggest a model in which desmin attaches to the Z-disk through an interaction with Nebulin. Finally, because Nebulin has been proposed to play a role in specifying Z-disk width, we also measured Z-disk width in Nebulin KO mice. Results show that most Z-disks of KO mice were modestly increased in width (~80 nm in soleus and ~40 nm in EDL fibers) whereas a small subset had severely increased widths (up to ~1 μm) and resembled nemaline rod bodies. In summary, structural studies on a Nebulin KO mouse show that in the absence of Nebulin, Z-disks are significantly wider and that myofibrils are misaligned. Thus the functional roles of Nebulin extend beyond thin filament length regulation and include roles in maintaining physiological Z-disk widths and myofibrillar connectivity.
Carol C. Gregorio - One of the best experts on this subject based on the ideXlab platform.
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Nebulin a multi functional giant
The Journal of Experimental Biology, 2016Co-Authors: Carol C. Gregorio, Christopher T. PappasAbstract:Efficient muscle contraction in skeletal muscle is predicated on the regulation of actin filament lengths. In one long-standing model that was prominent for decades, the giant protein Nebulin was proposed to function as a ‘molecular ruler’ to specify the lengths of the thin filaments. This theory was questioned by many observations, including experiments in which the length of Nebulin was manipulated in skeletal myocytes; this approach revealed that Nebulin functions to stabilize filamentous actin, allowing thin filaments to reach mature lengths. In addition, more recent data, mostly from in vivo models and identification of new interacting partners, have provided evidence that Nebulin is not merely a structural protein. Nebulin plays a role in numerous cellular processes including regulation of muscle contraction, Z-disc formation, and myofibril organization and assembly.
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the desmin coil 1b mutation k190a impairs Nebulin z disc assembly and destabilizes actin thin filaments
Journal of Cell Science, 2011Co-Authors: Gloria M Conover, Carol C. GregorioAbstract:Desmin intermediate filaments intimately surround myofibrils in vertebrate muscle forming a mesh-like filament network. Desmin attaches to sarcomeres through its high-affinity association with Nebulin, a giant F-actin binding protein that co-extends along the length of actin thin filaments. Here, we further investigated the functional significance of the association of desmin and Nebulin in cultured primary myocytes to address the hypothesis that this association is key in integrating myofibrils to the intermediate filament network. Surprisingly, we identified eight peptides along the length of desmin that are capable of binding to C-terminal modules 160–170 in Nebulin. In this study, we identified a targeted mutation (K190A) in the desmin coil 1B region that results in its reduced binding with the Nebulin C-terminal modules. Using immunofluorescence microscopy and quantitative analysis, we demonstrate that expression of the mutant desmin K190A in primary myocytes results in a significant reduction in assembled endogenous Nebulin and desmin at the Z-disc. Non-uniform actin filaments were markedly prevalent in myocytes expressing GFP-tagged desmin K190A, suggesting that the near-crystalline organization of actin filaments in striated muscle depends on a stable interaction between desmin and Nebulin. All together, these data are consistent with a model in which Z-disc-associated Nebulin interacts with desmin through multiple sites to provide efficient stability to satisfy the dynamic contractile activity of myocytes.
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Deciphering the Functional Properties of Nebulin: It is a Stabilizer!
Biophysical Journal, 2011Co-Authors: Chris T. Pappas, Paul A Krieg, Carol C. GregorioAbstract:Striated muscle cells display one of the most extreme examples of molecular organization found in nature. Efficient muscle contraction requires precise regulation of actin (thin) filament lengths. In one highly cited model, the giant protein Nebulin (∼750-900 kDa) has been proposed to function as a “molecular ruler” specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of Nebulin (mini-Nebulin). When endogenous Nebulin was replaced with mini-Nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-Nebulin; an observation that is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-Nebulin were remarkably maintained at lengths either matching or longer than mini-Nebulin. This indicates that mini-Nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of Nebulin also resulted in more dynamic populations of thin filament components, while expression of mini-Nebulin decreased the dynamics at both ends of the filament (i.e., efficiently recovered loss of endogenous full-length Nebulin). Taken together, our data reveals that Nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.
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The Nebulin family: an actin support group
Trends in Cell Biology, 2010Co-Authors: Christopher T. Pappas, Katherine T. Bliss, Anke Zieseniss, Carol C. GregorioAbstract:Nebulin, a giant, actin-binding protein, is the largest member of a family of proteins (including N-RAP, nebulette, lasp-1 and lasp-2) that are assembled in a variety of cytoskeletal structures, and expressed in different tissues. For decades, Nebulin has been thought to act as a molecular ruler, specifying the precise length of actin filaments in skeletal muscle. However, emerging evidence suggests that Nebulin should not be viewed as a ruler but as an actin filament stabilizer required for length maintenance. Nebulin has also been implicated recently in an array of regulatory functions independent of its role in actin filament length regulation. In this review, we discuss the current evolutionary, biochemical, and functional data for the Nebulin family of proteins — a family whose members, both large and small, function as cytoskeletal scaffolds and stabilizers.
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Nebulin regulates actin filament lengths by a stabilization mechanism
Journal of Cell Biology, 2010Co-Authors: Christopher T. Pappas, Paul A Krieg, Carol C. GregorioAbstract:Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein Nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of Nebulin (mini-Nebulin). When endogenous Nebulin was replaced with mini-Nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-Nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-Nebulin were remarkably maintained at lengths either matching or longer than mini-Nebulin. This indicates that mini-Nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of Nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-Nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous Nebulin). Thus, Nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.
Henk Granzier - One of the best experts on this subject based on the ideXlab platform.
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in vivo characterization of skeletal muscle function in Nebulin deficient mice
Muscle & Nerve, 2020Co-Authors: Charlotte Gineste, Henk Granzier, Augustin C Ogier, Isabelle Varlet, Zaynab Hourani, Monique Bernard, David Bendahan, Julien GondinAbstract:INTRODUCTION: The conditional Nebulin knockout mouse is a new model mimicking nemaline myopathy, a rare disease characterized by muscle weakness and rods within muscle fibers. We investigated the impact of Nebulin (NEB) deficiency on muscle function in vivo. METHODS: Conditional Nebulin knockout mice and control littermates were studied at 10 to 12 months. Muscle function (force and fatigue) and anatomy (muscles volume and fat content) were measured in vivo. Myosin heavy chain (MHC) composition and Nebulin (NEB) protein expression were assessed by protein electrophoresis. RESULTS: Conditional Nebulin knockout mice displayed a lower NEB level (-90%) leading to a 40% and 45% reduction in specific maximal force production and muscles volume, respectively. Nebulin deficiency was also associated with higher resistance to fatigue and increased MHC I content. DISCUSSION: Adult Nebulin-deficient mice displayed severe muscle atrophy and weakness in vivo related to a low NEB content but an improved fatigue resistance due to a slower contractile phenotype.
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In vivo characterization of skeletal muscle function in Nebulin‐deficient mice
Muscle & Nerve, 2020Co-Authors: Charlotte Gineste, Henk Granzier, Augustin C Ogier, Isabelle Varlet, Zaynab Hourani, Monique Bernard, David Bendahan, Julien GondinAbstract:INTRODUCTION: The conditional Nebulin knockout mouse is a new model mimicking nemaline myopathy, a rare disease characterized by muscle weakness and rods within muscle fibers. We investigated the impact of Nebulin (NEB) deficiency on muscle function in vivo. METHODS: Conditional Nebulin knockout mice and control littermates were studied at 10 to 12 months. Muscle function (force and fatigue) and anatomy (muscles volume and fat content) were measured in vivo. Myosin heavy chain (MHC) composition and Nebulin (NEB) protein expression were assessed by protein electrophoresis. RESULTS: Conditional Nebulin knockout mice displayed a lower NEB level (-90%) leading to a 40% and 45% reduction in specific maximal force production and muscles volume, respectively. Nebulin deficiency was also associated with higher resistance to fatigue and increased MHC I content. DISCUSSION: Adult Nebulin-deficient mice displayed severe muscle atrophy and weakness in vivo related to a low NEB content but an improved fatigue resistance due to a slower contractile phenotype.
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Nebulin stiffens the thin filament and augments cross bridge interaction in skeletal muscle
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Balazs Kiss, Paola Tonino, Frank Li, Srboljub M Mijailovich, Thomas C Irving, Henk GranzierAbstract:Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in Nebulin cause muscle weakness in nemaline myopathy patients, suggesting that Nebulin plays important roles in force generation, yet little is known about Nebulin’s influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in Nebulin (using a conditional Nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 A) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when Nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 A) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that Nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.
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the sarcomeric protein Nebulin another multifunctional giant in charge of muscle strength optimization
Frontiers in Physiology, 2012Co-Authors: Henk Granzier, Coen A C Ottenheijm, Siegfried LabeitAbstract:The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been successful in explaining many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and Nebulin. Whereas the role of titin rapidly progressed, Nebulin’s role in muscle structure and function remained long nebulous. An important feature of muscle structure and function that has remained relatively obscure concerns the mechanisms that are involved in regulating thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of Nebulin KO models, reveal that Nebulin plays an important role in the regulation of thin filament length, most likely by stabilizing F-actin assemblies. Another structural feature of skeletal muscle that has been incompletely understood concerns the mechanisms involved in maintaining Z-disk structure and the regular lateral alignment of adjacent sarcomeres during contraction. Recent studies indicate that Nebulin is part of a protein complex that mechanically links adjacent myofibrils. In addition to these structural roles in support of myofibrillar force generation, Nebulin has been also shown to regulate directly muscle contraction at the level of individual cross bridges: cycling kinetics and the calcium sensitivity of force producing cross-bridges is enhanced in the presence of Nebulin. Thus, these recent data all point to Nebulin being important for muscle force optimization. Consequently, muscle weakness as the lead symptom develops in the case of patients with nemaline myopathy that have mutations in the Nebulin gene. Here, we discuss these important novel insights into the role of Nebulin in skeletal muscle function.
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Nebulin a major player in muscle health and disease
The FASEB Journal, 2011Co-Authors: Siegfried Labeit, Coen A C Ottenheijm, Henk GranzierAbstract:Nebulin is a giant 600- to 900-kDa filamentous protein that is an integral component of the skeletal muscle thin filament. Its functions have remained largely nebulous because of its large size and the difficulty in extracting Nebulin in a native state from muscle. Recent improvements in the field, especially the development of knockout mouse models deficient in Nebulin (NEB-KO mice), indicate now that Nebulin performs a surprisingly wide range of functions. In addition to a major role in thin-filament length specification, Nebulin also functions in the regulation of muscle contraction, as indicated by the findings that muscle fibers deficient in Nebulin have a higher tension cost, and develop less force due to reduced myofilament calcium sensitivity and altered crossbridge cycling kinetics. In addition, the function of Nebulin extends to a role in calcium homeostasis. These novel functions indicate that Nebulin might have evolved in vertebrate skeletal muscles to develop high levels of muscle force effic...
Kuan Wang - One of the best experts on this subject based on the ideXlab platform.
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Elasticity of Intrinsically Disordered Nebulin Modules
Biophysical Journal, 2011Co-Authors: Jeffrey G. Forbes, Richard J. Wittebort, Alexander Grishaev, Wanxia L. Tsai, Kuan WangAbstract:The elasticity of native full length Nebulin, demonstrated recently via atomic force microscopy with site-specific antibody pairs as force handles (Langmuir, 2009, 25, 7496), suggests that in the thin filaments, Nebulin is stretched to cope with the actin length and imposes significant force and influences the functions of the underlying actins. This pre-stressed mechanical state of thin filaments may have important implications for the role of Nebulin as a length ruler and as a regulator of actomyosin interaction. The structural basis of Nebulin elasticity remains open. We report here the structural characterization of modules from the super-repeat and single repeat regions by a combination of circular dichroism (CD), NMR, SAXS, AFM, structural predictions and steered molecular dynamics simulations. In aqueous solutions of common buffers, these modules are intrinsically disordered, but are poised to form alpha-helices, especially in the presence of trifluoroethanol. SAXS analysis of a four-module construct indicates an elongated structure with a radius of gyration of 3.6 nm and, as modeled with DAMMIN, shows a contour length of ∼15 nm. Interestingly, this extended structure is also evident in a small population of the structural models as predicted by ROSETTA++. AFM images of the modules on an inert surface are predominantly compact with an average height of ∼ 2.5 nm, consistent with the bulk of the ROSETTA predictions. These structural ensembles of compact and extended structures are significantly shorter than what it would take for Nebulin modules to wrap around the perimeter of actin filaments (∼6 nm per module). We propose that Nebulin modules’ disorder-order transition of alpha helices, contributes to its elasticity and how Nebulin juxtapositions itself onto the actin to form a pre-stressed thin filaments in the muscle sarcomere.
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interaction of Nebulin sh3 domain with titin pevk and myopalladin implications for the signaling and assembly role of titin and Nebulin
FEBS Letters, 2002Co-Authors: Kuan WangAbstract:Skeletal muscle Nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of Nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to Nebulin SH3 with micromolar affinity (∼31 and 3.4 μM, respectively), whereas the other four peptides bind weakly (>100 μM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating Nebulin to the Z-line during sarcomere assembly.
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each actin subunit has three Nebulin binding sites implications for steric blocking
Current Biology, 2002Co-Authors: Natalya Lukoyanova, Kuan Wang, Margaret S Vanloock, Albina Orlova, Vitold E Galkin, Edward H EgelmanAbstract:Nebulin is a giant protein that spans most of the muscle thin filament [1, 2]. Mutations in Nebulin result in myopathies and dystrophies [3, 4]. Nebulin contains ∼200 copies of ∼35 residue modules, each believed to contain an actin binding site, organized into seven-module superrepeats [5, 6]. The strong correlation between the number of Nebulin modules and the length of skeletal muscle thin filaments in different species suggests that Nebulin determines thin filament length [2, 7, 8]. Little information exists about the interactions between intact Nebulin and F-actin. More insight has come from working with fragments of Nebulin, containing from one to hundreds of actin binding modules. However, the observed stoichiometry of binding between these fragments and actin has ranged from 0.4 to 13 modules per actin subunit [9–12]. We have used electron microscopy and a novel method of helical image analysis to characterize complexes of F-actin with a Nebulin fragment. The fragment binds as an extended structure spanning three actin subunits and binding to different sites on each actin. Muscle regulation involves tropomyosin movement on the surface of actin, with binding in three states. Our results suggest the intriguing possibility that intact Nebulin may also be able to occupy three different sites on F-actin.
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mapping protein interfaces with a fluorogenic cross linker and mass spectrometry application to Nebulin calmodulin complexes
Biochemistry, 2001Co-Authors: Andrea Sinz, Kuan WangAbstract:: Nebulin is a giant multifunctional protein that is thought to serve as both a length-regulating protein ruler and calcium/CaM-mediated regulatory protein on the thin filaments of the skeletal muscle sarcomere. To define molecular interfaces between Nebulin and CaM, we thiolated lysines of CaM and ND66, a four-module cloned fragment from the C-terminus of Nebulin, with 2-iminothiolane and cross-linked the complex with dibromobimane, which alkylates thiol pairs within approximately 6 A of each other to form a fluorescent adduct. Such a two-stage cross-linking generated mainly 1:1 complexes of ND66 and CaM, with a limited extent of intramolecular cross-linking. In-gel chymotryptic digestion of the dibromobimane-cross-linked complexes yielded peptides that were first screened by HPLC with fluorescence detection and then scored for cross-linking with mass spectrometry. Several inter- and intramolecular sites were identified and confirmed further by ESI-MS/MS experiments, defining molecular interfaces and patterns of protein folding. In particular, five intermolecular cross-linking products of sequences within the region of amino acids 83-99 (YKENMGKGTPLPVTPEM) in ND66 and several sequences of CaM indicate that the Nebulin-CaM interface is close to, and may overlap with, the Nebulin-actin interface. This proximity suggests a potential competition between CaM and actin for this Nebulin interface. Intramolecular cross-linking of amino acids 13-16 (KEAF) and 13-18 (KEAFSL) with amino acids 145-148 (MTAK) and 146-148 (TAK) in CaM suggests the interaction of two lobes across the central helix. The cross-linking of amino acids 1-6 (MKTPEM) with amino acids 114-129 (YKENVGKATATPVTPE) and 115-129 (KENVGKATATPVTPE) in ND66 hints at an association of noncontiguous Nebulin modules in solution.
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high affinity actin binding Nebulin fragments influence the actos1 complex
Biochemistry, 2001Co-Authors: Douglas D Root, Kuan WangAbstract:Human Nebulin fragments, NA3 and NA4, corresponding to individual superrepeats display high-affinity interactions with individual actin protomers in cosedimentation and solid-phase binding assays. Stoichiometric analysis of Nebulin fragment-induced actin polymerization and inhibition of actin-activated S1 ATPase indicate that one superrepeat influences multiple actin molecules along the F-actin filament, consistent with a combination of strong and weak interactions of Nebulin over the length of the actin filament. The mechanisms by which human Nebulin fragments affect the interaction between actin and myosin S1 are studied by fluorescence quenching, polarization, and resonance energy transfer. We show that, under strong binding conditions, premixing actin with the NA3 prior to adding myosin subfragment 1 (S1) inhibits the rate of actoS1 association. The Nebulin fragments, NA3 and NA4, caused little effect on the extent of actoS1 binding at equilibrium but did alter the nature of the complex as evidenced b...
K Pelin - One of the best experts on this subject based on the ideXlab platform.
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a Nebulin super repeat panel reveals stronger actin binding toward the ends of the super repeat region
Muscle & Nerve, 2019Co-Authors: J Laitila, Johanna Lehtonen, Vilmalotta Lehtokari, L Sagath, Carina Wallgrenpettersson, Mikaela Gronholm, K PelinAbstract:INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of Nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the Nebulin super-repeat region into a super-repeat panel, and studied Nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different Nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between Nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The Nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating Nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.
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two alternatively spliced human Nebulin isoforms with either exon 143 or exon 144 and their developmental regulation
Scientific Reports, 2018Co-Authors: Ian Holt, K Pelin, J Laitila, Carina Wallgrenpettersson, Mubashir Hanif, C Sewry, Glenn E MorrisAbstract:Nebulin is a very large protein required for assembly of the contractile machinery in muscle. Mutations in the Nebulin gene NEB are a common cause of nemaline myopathy. Nebulin mRNA is alternatively-spliced so that each mRNA contains either exon 143 or exon 144. We have produced monoclonal antibodies specific for the regions of Nebulin encoded by these two exons, enabling analysis of expression of isoforms at the protein level for the first time. All antibodies recognized a protein of the expected size (600–900 kD) and stained cross-striations of sarcomeres in muscle sections. Expression of exon 143 is developmentally-regulated since newly-formed myotubes in cell culture expressed Nebulin with exon 144 only; this was confirmed at the mRNA level by qPCR. In fetal muscle, Nebulin with exon 143 was expressed in some myotubes by 12-weeks of gestation and strongly-expressed in most myotubes by 17-weeks. In mature human muscle, the exon 144 antibody stained all fibres, but the exon 143 antibody staining varied from very strong in some fibres to almost-undetectable in other fibres. The results show that Nebulin containing exon 144 is the default isoform early in myogenesis, while regulated expression of Nebulin containing exon 143 occurs at later stages of muscle development.
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Nebulin interactions with actin and tropomyosin are altered by disease causing mutations
Skeletal Muscle, 2014Co-Authors: Minttu Marttila, J Laitila, Carina Wallgrenpettersson, Mikaela Gronholm, Mubashir Hanif, Elina Lemola, Kristen J Nowak, K PelinAbstract:Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of Nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site. We produced four wild-type (WT) Nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the Nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT Nebulin super repeats for WT α- and β–tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro. WT Nebulin was shown to interact with actin and tropomyosin. Both the Nebulin super repeats containing the p.Glu2431Lys mutation and Nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for Nebulin (super repeat 18). We demonstrate for the first time the existence of direct tropomyosin-Nebulin interactions in vitro, and show that Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in Nebulin and tropomyosin.
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p 9 3 repair of mutant Nebulin transcripts by exon exchange
Neuromuscular Disorders, 2013Co-Authors: J Laitila, James J Dowling, K PelinAbstract:The Nebulin gene (NEB) has 183 exons encoding transcripts up to 26 kb in length. Mutations found in NEB are dispersed throughout the gene. Mutations cause autosomal recessive nemaline myopathy, distal myopathy and core-rod myopathy, for which no therapy is available. The size of NEB limits the options of gene therapy development. Thus, our research has focused on methods correcting the mutation carrying transcripts. The exon exchange method can be developed to exchange a mutated exon by spliceosome mediated RNA trans-splicing occurring between the target pre-mRNA and a carefully designed therapy molecule, PTM (Pre-Trans-splicing Molecule), carrying the wild type exon. We have previously obtained successful results from our 3′ and internal exon exchange experiments using a minigene target in cell cultures. In our latest experiment we targeted the last exons of the Nebulin transcript produced by C2C12 mouse myoblasts. This is the first time the endogenous Nebulin has been used as a target for exon exchange. Our preliminary results indicate successful, but weak trans-splicing at the RNA level. By combining the knowledge gathered from the internal exon exchange experiments and the modification of the endogenous transcript, we are now planning a new experiment in which we target an internal exon of endogenous mutated Nebulin transcripts in vivo using a zebrafish model for nemaline myopathy. The fish has a splice site mutation causing abnormal splicing of Nebulin exon 43. Zebrafish Nebulin has very small introns which make selecting PTM binding sites in the mutated area challenging. We have now designed five different PTM constructs targeted to the mutated region of zebrafish Nebulin. The PTMs will first be tested in cell cultures targeting zebrafish Nebulin minigene transcripts and the successful PTMs will be used in the in vivo experiments. This will be the first attempt to correct a mutated Nebulin pre-mRNA in vivo.
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nemaline myopathy caused by mutations in the Nebulin gene may present as a distal myopathy
Neuromuscular Disorders, 2011Co-Authors: Vilmalotta Lehtokari, K Pelin, Agnes Herczegfalvi, Veronika Karcagi, J Pouget, Jerome Franques, Jean Francois Pellissier, Dominique Figarellabranger, Maja Von Der Hagen, Angela HuebnerAbstract:Mutations in the Nebulin gene are the main cause of autosomal recessive nemaline myopathy, with clinical presentations ranging from mild to severe disease. We have previously reported a nonspecific distal myopathy caused by homozygous missense mutations in the Nebulin gene in six Finnish patients from four different families. Here we describe three non-Finnish patients in two unrelated families with distal nemaline myopathy caused by four different compound heterozygous Nebulin mutations, only one of which is a missense mutation. One of the mutations has previously been identified in one family with the severe form of nemaline myopathy. We conclude that nemaline myopathy and distal myopathy caused by Nebulin mutations form a clinical and histological continuum. Nemaline myopathy should be considered as a differential diagnosis in patients presenting with an early-onset predominantly distal myopathy.
