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Robert J Bloch - One of the best experts on this subject based on the ideXlab platform.
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The FASEB Journal • Research Communication Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum
2013Co-Authors: Aikaterini Kontrogianni-konstantopoulos, Andrei B. Borisov, Mark W. Russell, Robert J Bloch, Dawn H. Catino, John C. Strong, Sarah Sutter, David W. Pumplin, R. Obscurin Modulates J. TheAbstract:ABSTRACT Obscurin (�800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca 2 � homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of Obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the �800 kDa form of Obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of Obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to Obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but �-actinin was not, suggesting that downregulation of Obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that Obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.
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electrostatic interactions mediate binding of Obscurin to small ankyrin 1 biochemical and molecular modeling studies
Journal of Molecular Biology, 2011Co-Authors: Ben Busby, Maegen A Ackermann, Aikaterini Kontrogiannikonstantopoulos, Taiji Oashi, Chris D Willis, Alexander D Mackerell, Robert J BlochAbstract:Abstract Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of Obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro , in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for Obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on Obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on Obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in Obscurin. We used molecular modeling to develop structural models of residues 6322–6339 of Obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for Obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of Obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on Obscurin and sAnk1 promote binding and complex formation.
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interaction of Obscurin a with small ankyrin 1
Biophysical Journal, 2011Co-Authors: Robert J Bloch, Maegen A Ackermann, Aikaterini Kontrogiannikonstantopoulos, Ben Busby, Taiji Oashi, Chris D Willis, Alexander D MackerellAbstract:We have studied the binding of a small product of the ankyrin 1 gene (sAnk1; Ank1.5), a ∼17.5 kDa integral protein of the sarcoplasmic reticulum, to the C-terminus of Obscurin A, a ∼720 kDa protein that envelopes sarcomeres at Z-disks and M-bands. Alanine scanning mutagenesis identified several lysines and arginines in the cytoplasmic sequence of sAnk1 that mediate binding to Obscurin, and several glutamates in the high affinity site of Obscurin that mediate binding to sAnk1. Complementary K- or R-to-E and E-to-K mutations identified specific pairs of residues involved in binding; mutagenesis of several of these eliminates binding. Molecular and Brownian dynamics simulation suggested several possible models for the docked complex but predicted only one in which D111 of sAnk1 and K6338 of Obscurin interact. We confirmed their interaction by complementary mutagenesis. We tested the model further by using similar approaches to examine the hydrophobic residues involved in binding, with results consistent with the predictions of a representative structure of the docked complex selected from cluster analysis of structures generated from molecular dynamics simulations. Our studies indicate that the Obscurin-binding region of sAnk1 is comprised of two ankyrin-like repeats, which establish specific electrostatic and hydrophobic contacts with the high affinity site on Obscurin, composed of an 18-residue α-helical polypeptide. We identify structures similar to this polypeptide in the binding regions of proteins that interact with other ankyrin repeat proteins. We propose that the 18-mer in the high affinity binding site on Obscurin for sAnk1 represents a prototypical ankyrin binding motif.Supported by grants to ADM from the NIH (CA120215; GM051501) and the University of Maryland Computer-Aided Drug Design Center, and from grants to AKK and RJB from the MDA and the NIH (RO1 AR052768 to AKK; RO1 AR056330 to RJB).
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surface exposed hydrophobic residues on small ankyrin 1 mediate binding to Obscurin
Biophysical Journal, 2010Co-Authors: Chris D Willis, Taiji Oashi, Ben Busby, Alexander D Mackerell, Robert J BlochAbstract:Small ankyrin-1 (sAnk1, Ank1.5) is a splice variant of the ANK1 gene that binds to the large modular protein, Obscurin A, with nanomolar affinity, a reaction that may help to organize the sarcoplasmic reticulum in striated muscle. A subset of lysine and arginine residues in the 2 ankyrin repeats of sAnk1 interact specifically with 4 glutamate residues in a stretch of 30 amino acids of Obscurin to mediate binding. Homology modeling and molecular dynamics simulations have revealed a “hot spot” of 4 hydrophobic residues exposed on the surface of the ankyrin repeat domain of sAnk1. We used site-directed mutagenesis of bacterially expressed fusion proteins, followed by blot overlays and surface plasmon resonance assays, to study the contribution of these 4 residues, V70, F71, I102 and I103, to binding to the 30-mer of Obscurin. Alanine mutations of each of these four residues inhibited binding to residues 6316-6345 of Obscurin (Obsc6316-6345). In contrast, V70A and I102A mutations had no effect on binding to a second sAnk1 binding site on Obscurin, located within residues 6231-6260 (Obsc6231-6260 ). Using the same methods, we mutated the 5 hydrophobic residues present in Obsc6316-6345 to alanine and identified V6328, I6332, and V6334 as critical for proper binding. Our results suggest that hydrophobic interactions as well as electrostatic interactions are important for the binding of sAnk1 to Obsc6316-6345, consistent with studies of the complexes formed by other ankyrin repeat proteins with their ligands. Hydrophobic interactions are likely to contribute to the difference in affinity of sAnk1 for Obsc6316-6345 and Obsc6231-6260, and for the dominant role played by the more C-terminal sequence in binding.Supported by grant R01- AR056330 from the NIH to RJB and training grants T32 GM08181 (to RJB) and T32 AR07592 (to M. Schneider).
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the rho guanine nucleotide exchange factor domain of Obscurin activates rhoa signaling in skeletal muscle
Molecular Biology of the Cell, 2009Co-Authors: Diana L Fordspeelman, Amber L Bowman, Joseph A Roche, Robert J BlochAbstract:Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of Obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of Obscurin binds selectively to rhoA, and that rhoA colocalizes with Obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of Obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of Obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of Obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of Obscurin's rhoGEF domain. Our results suggest that Obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
Aikaterini Kontrogiannikonstantopoulos - One of the best experts on this subject based on the ideXlab platform.
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deletion of Obscurin immunoglobulin domains ig58 59 leads to age dependent cardiac remodeling and arrhythmia
Basic Research in Cardiology, 2020Co-Authors: Alyssa Grogan, Andrew K Coleman, Humberto C Joca, Henk Granzier, Mark W Russel, Christopher W Ward, Aikaterini KontrogiannikonstantopoulosAbstract:Obscurin comprises a family of giant modular proteins that play key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of Obscurin mediate binding to essential modulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban (PLN). Importantly, missense mutations localized within the Obscurin-Ig58/59 region that affect binding to titins and/or PLN have been linked to the development of myopathy in humans. To elucidate the pathophysiological role of this region, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses Obscurin lacking Ig58/59, and determined the consequences of this manipulation on cardiac morphology and function under conditions of acute stress and through the physiological process of aging. Our studies show that young Obscn-ΔIg58/59 mice are susceptible to acute β-adrenergic stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop left ventricular hypertrophy that progresses to dilation, contractile impairment, atrial enlargement, and arrhythmia as a function of aging with males being more affected than females. Experiments in ventricular cardiomyocytes revealed altered Ca2+ cycling associated with changes in the expression and/or phosphorylation levels of major Ca2+ cycling proteins, including PLN, SERCA2, and RyR2. Taken together, our work demonstrates that Obscurin-Ig58/59 is an essential regulatory module in the heart and its deletion leads to age- and sex-dependent cardiac remodeling, ventricular dilation, and arrhythmia due to deregulated Ca2+ cycling.
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proteomic analysis of myocardia containing the Obscurin r4344q mutation linked to hypertrophic cardiomyopathy
Frontiers in Physiology, 2020Co-Authors: Aikaterini KontrogiannikonstantopoulosAbstract:Obscurin is a giant cytoskeletal protein with structural and regulatory roles encoded by the OBSCN gene. Recently, mutations in OBSCN were associated with the development of different forms of cardiomyopathies, including hypertrophic cardiomyopathy (HCM). We previously reported that homozygous mice carrying the HCM-linked R4344Q Obscurin mutation develop arrhythmia by 1-year of age under sedentary conditions characterized by increased heart rate, frequent incidents of premature ventricular contractions, and episodes of spontaneous ventricular tachycardia. In an effort to delineate the molecular mechanisms that contribute to the observed arrhythmic phenotype, we subjected protein lysates prepared from left ventricles of 1-year old R4344Q and wild-type mice to comparative proteomics analysis using tandem mass spectrometry; raw data are available via ProteomeXchange with identifier PXD017314. We found that the expression levels of proteins involved in cardiac function and disease, cytoskeletal organization, electropotential regulation, molecular transport and metabolism were significantly altered. Moreover, phospho-proteomic evaluation revealed changes in the phosphorylation profile of Ca2+ cycling proteins, including sAnk1.5, a major binding partner of Obscurin localized in the sarcoplasmic reticulum; notably, this is the first report indicating that sAnk1 undergoes phosphorylation. Taken together, our findings implicate Obscurin in diverse cellular processes within the myocardium, which is consistent with its multiple binding partners, localization in different subcellular compartments, and disease association.
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small Obscurins at the intercalated disc mediate structure cell size and adhesion
Biophysical Journal, 2016Co-Authors: Maegen A Ackermann, Nicole A Perry, Brendan King, Michael Rudloff, Nathan T Wright, Peter A Hecker, Christopher E Berndsen, Aikaterini KontrogiannikonstantopoulosAbstract:The intercalated disc (ID) of cardiac muscle embodies a highly ordered, multifunctional network, essential for the transmission of electrical stimuli and mechanical force resulting in the synchronous beating of the heart. Recently, a plethora of proteins have been identified as novel components of the ID. The challenge now lies in their characterization as it relates to the coupling of neighbouring cardiomyocytes and whole heart function. Here we focus on the functional characterization of two novel ID proteins, Obscurin-40 and Obscurin-80.Obscurins are a family of proteins expressed in striated muscle cells where they localize to distinct substructures, contributing to their assembly and integration with other sarcoplasmic elements. Our previous studies have shown that the Obscurin transcript, arising from the single OBSCN gene is extensively spliced, resulting in several Obscurin isoforms. Complex splicing at the 3’ end of OBSCN results in at least two novel products, obsc-40 and obsc-80, which preferentially concentrate at the ID. Consistent with their ID subcellular localization, biochemical assays demonstrated that obsc-40 and obsc-80 are in a complex with major ID proteins, including N-cadherin, connexin-43, vinculin, and ankyrin-G. In addition, the Obscurin pleckstrin homology (PH) domain present in both obsc-40 and obsc-80 binds specifically and directly to phosphatidylinositol 3,4 and 4,5 bisphosphates, likely targeting both proteins to the ID membrane. Overexpression of the Obscurin-PH domain in a cardiac derived cell line results in decreased activation of the PI3K/Akt and mTOR pathways. These molecular alterations lead to decreased cellular aggregation and overall cardiomyocyte size. Furthermore, both obsc-40 and obsc-80, but not giant Obscurins, exhibit reduced expression in myocardial infarction and transaortic constriction cardiac-stress models. Taken together these important findings implicate these novel small Obscurins in the maintenance of heart size and the prevention of hypertrophy.
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loss of giant Obscurins from breast epithelium promotes epithelial to mesenchymal transition tumorigenicity and metastasis
Oncogene, 2015Co-Authors: Marey Shriver, Kimberly M Stroka, Konstantinos Konstantopoulos, Michele Vitolo, Stuart S Martin, David L Huso, Aikaterini KontrogiannikonstantopoulosAbstract:Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. The OBSCN gene is highly mutated in different types of cancers. Loss of giant Obscurins from breast epithelial cells confers them with a survival and growth advantage, following exposure to DNA-damaging agents. Here we demonstrate that the expression levels and subcellular distribution of giant Obscurins are altered in human breast cancer biopsies compared with matched normal samples. Stable clones of non-tumorigenic MCF10A cells lacking giant Obscurins fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition and generate >100-μm mammospheres bearing markers of cancer-initiating cells. Obscurin-knockdown MCF10A cells display markedly increased motility as a sheet in 2-dimensional (2D) substrata and individually in confined spaces and invasion in 3D matrices. In line with these observations, actin filaments redistribute to extending filopodia where they exhibit increased dynamics. MCF10A cells that stably express the K-Ras oncogene and Obscurin short hairpin RNA (shRNA), but not scramble control shRNA, exhibit increased primary tumor formation and lung colonization after subcutaneous and tail vein injections, respectively. Collectively, our findings reveal that loss of giant Obscurins from breast epithelium results in disruption of the cell–cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced tumorigenesis, migration and invasiveness in vitro and in vivo.
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Obscurins mechanistic involvement in signal transduction at the cardiac intercalated disc
Biophysical Journal, 2015Co-Authors: Maegen A Ackermann, Nicole A Perry, Aikaterini KontrogiannikonstantopoulosAbstract:The intercalated disc (ID) of cardiac muscle embodies a highly ordered, multifunctional network, which is essential for the transmission of electrical stimuli and mechanical force resulting in the synchronous contraction of the heart. Recently, a plethora of proteins have been identified as novel components of the ID. The challenge now lies in their characterization. Here we focus on the molecular and functional description of two novel members of the ID, Obscurin-80 and Obscurin-40.Obscurins are a family of proteins expressed in striated muscles where they localize to distinct subdomains. The members of the Obscurin family are multidomain proteins composed of adhesion modules and signaling domains, resulting from extensive alternative splicing of transcripts arising from the single OBSCN gene. Recent work from our laboratory has demonstrated that complex splicing at the 3’ end of the Obscurin transcript gives rise to at least two novel Obscurins, Obscurin-80 (obsc-80) and Obscurin-40 (obsc-40), named after their predicted molecular weights.Using immunofluorescence and immunoelectron microscopy, we show that obsc-80 and obsc-40 localize to the ID of developing and adult murine cardiomyocytes. Using biochemical assays we further demonstrate that both obsc-80 and obsc-40 exist in a complex with major ID proteins, including N-cadherin, connexin-43, vinculin, and ankyrin-G. The PH domain present in both obsc-80 and obsc-40 binds specifically and directly binds to phosphatidylinositol 3,4 and 4,5 bisphosphates, likely targeting both proteins to the ID membrane. Overexpression of the Obscurin PH domain results in decreased phosphorylation of Akt, therefore reducing Akt activation. This suggests a potential role for obsc-80 and obsc-40 in the regulation of cell growth and proliferation via the Akt pathway. Further experiments are underway to examine the functional activities of obsc-80 and obsc-40 at the ID and their regulation in health and disease.
Mathias Gautel - One of the best experts on this subject based on the ideXlab platform.
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when is an Obscurin variant pathogenic the impact of arg4344gln and arg4444trp variants on protein protein interactions and protein stability
Human Molecular Genetics, 2021Co-Authors: Atsushi Fukuzawa, Daniel Koch, Sarah Grover, Martin Rees, Mathias GautelAbstract:Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, Obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in Obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing Obscurin's Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between Obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/Obscurin interaction, the PLN/Obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these Obscurin variants as pathogenic.
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Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.
Structure (London England : 1993), 2016Co-Authors: Stefano Pernigo, Mathias Gautel, Atsushi Fukuzawa, Amy E. M. Beedle, Mark R. Holt, Adam Round, Alessandro Pandini, Sergi Garcia-manyes, Roberto A. SteinerAbstract:The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog Obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical Obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the Obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.
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binding partners of the kinase domains in drosophila Obscurin and their effect on the structure of the flight muscle
Journal of Cell Science, 2015Co-Authors: Anja Katzemich, Mathias Gautel, Atsushi Fukuzawa, John C Sparrow, Sean T Sweeney, Ryan J H West, Belinda BullardAbstract:Drosophila Obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike Obscurin in vertebrate skeletal muscle, Drosophila Obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of Obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.
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JCB Article Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly
2013Co-Authors: Paul Young, Elisabeth Ehler, Mathias GautelAbstract:Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, Obscurin, encoded on chromosome 1q42. Obscurin is �800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of Obscurin reveals a modular architecture, consisting of �67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of ob-scurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of Obscurin interacts via two specific Ig-like domains with the NH2-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all Obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that Obscurin is involved in Ca 2 � /calmodulin, as well as G protein–coupled signal transduction in the sarcomere
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Structural insight into M-band assembly and mechanics from the titin-Obscurin-like-1 complex
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Stefano Pernigo, Atsushi Fukuzawa, Mark R. Holt, Roberto A. Steiner, Morten Bertz, Matthias Rief, Mathias GautelAbstract:In the sarcomeric M-band, the giant ruler proteins titin and Obscurin, its small homologue Obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of Obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands—Obscurin and obsl1—in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/Obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to Obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/Obscurin-titin links has severe consequences for normal muscle functions.
Mark W. Russell - One of the best experts on this subject based on the ideXlab platform.
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Dev Genes Evol (2004) 214: 352–359 DOI 10.1007/s00427-004-0413-5 SEQUENCE CORNER
2016Co-Authors: Sarah B. Sutter, Andrei B. Borisov, Maide O Raeker, Mark W. RussellAbstract:Abstract Myosin light chain kinases (MLCK) are a family of signaling proteins that are required for cyto-skeletal remodeling in myocytes. Recently, two novel MLCK proteins, SPEG and Obscurin-MLCK, were identified with the unique feature of two tandemly-arranged MLCK domains. In this study, the evolutionary origins of this MLCK subfamily were traced to a probable orthologue of Obscurin-MLCK in Drosophila melanogas-ter, Drosophila Unc-89, and the MLCK kinase domains of zebrafish SPEG, zebrafish Obscurin-MLCK, and human SPEG were characterized. Phylogenetic analysis of the MLCK domains indicates that the carboxy terminal kinase domains of Obscurin-MLCK, SPEG and Unc-89 are more closely related to each other than to the amino terminal kinase domains or to other MLCKs, supporting the assertion that Obscurin-MLCK is the vertebrate orthologue of Caenorhabditis elegans Unc-89, a giant multidomain protein that is required for normal myofibril assembly. The apparent lack of an invertebrate orthologue of SPEG and the conserved exon structure of the kinase domains between SPEG and Obscurin-MLCK suggests that SPEG arose from Obscurin-MLCK by a gene duplication event. The length of the primary amino acid sequence between the immunoglobulin (Ig) domains associated with the MLCK motifs is conserved in Obscurin-MLCK, SPEG and C. elegans Unc-89, suggesting that these putative protein interaction domains may target the kinases to highly conserved intracellular sites. The conserved arrangement of the tandem MLCK domains and their relatively restricted expression in striated muscle indicates that further characterization of this novel MLCK subfamily may yield important insights into cardiac and skeletal muscle physiology
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The FASEB Journal • Research Communication Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum
2013Co-Authors: Aikaterini Kontrogianni-konstantopoulos, Andrei B. Borisov, Mark W. Russell, Robert J Bloch, Dawn H. Catino, John C. Strong, Sarah Sutter, David W. Pumplin, R. Obscurin Modulates J. TheAbstract:ABSTRACT Obscurin (�800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca 2 � homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of Obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the �800 kDa form of Obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of Obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to Obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but �-actinin was not, suggesting that downregulation of Obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that Obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.
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targeted deletion of the zebrafish Obscurin a rhogef domain affects heart skeletal muscle and brain development
Developmental Biology, 2010Co-Authors: Maide O Raeker, Ashley N Bieniek, Alison S Ryan, Huaijen Tsai, Katelin M Zahn, Mark W. RussellAbstract:Obscurin is a giant structural and signaling protein that participates in the assembly and structural integrity of striated myofibrils. Previous work has examined the physical interactions between Obscurin and other cytoskeletal elements but its in vivo role in cell signaling, including the functions of its RhoGTPase Exchange Factor (RhoGEF) domain have not been characterized. In this study, morpholino antisense oligonucleotides were used to create an in-frame deletion of the active site of the Obscurin A RhoGEF domain in order to examine its functions in zebrafish development. Cardiac myocytes in the morphant embryos lacked the intercalated disks that were present in controls by 72 and, in the more severely affected embryos, the contractile filaments were not organized into mature sarcomeres. Neural abnormalities included delay or loss of retinal lamination. Rescue of the phenotype with co-injection of mini-Obscurin A expression constructs demonstrated that the observed effects were due to the loss of small GTPase activation by Obscurin A. The immature phenotype of the cardiac myocytes and the retinal neuroblasts observed in the morphant embryos suggests that Obscurin A-mediated small GTPase signaling promotes tissue-specific cellular differentiation. This is the first demonstration of the importance of the Obscurin A-mediated RhoGEF signaling in vertebrate organogenesis and highlights the central role of Obscurin A in striated muscle and neural development.
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developmental expression and differential cellular localization of Obscurin and Obscurin associated kinase in cardiac muscle cells
Journal of Cellular Biochemistry, 2008Co-Authors: Andrei B. Borisov, Maide O Raeker, Mark W. RussellAbstract:Obscurin and Obscurin-associated kinase are two products of the Obscurin transcriptional unit that encodes a recently identified giant muscle-specific protein Obscurin. In this study, we characterized the developmental expression and cellular localization of Obscurin and Obscurin-associated kinase in cardiac muscle cells. We cloned murine Obscurin-associated kinase and found that it is abundantly expressed in the heart as two isotypes encoded by 2.2 and 4.9 kb sequences. The 2.2 kb isotype of the kinase was more prominently expressed than the 4.9 kb isotype. Both Obscurin and the kinase-like domains were progressively upregulated since the early stages of cardiac development. Obscurin-associated kinase was expressed at higher levels than Obscurin at early stages of cardiomyogenesis. Increasing intensity of Obscurin expression in the developing heart positively correlated with progressive cell differentiation and was higher in the ventricles compared to the atria. These data were supported by the results of experiments with primary cardiac cell cultures. Obscurin localization changed from a weakly immunopositive diffuse pattern in poorly differentiated cells to an intensely immunolabeled cross-striated distribution at the level of mid-A-bands and Z-disks during the assembly of the myofibrillar contractile apparatus. In dividing myocytes, unlike the interphase cells, Obscurin translocated from disassembling myofibrils into a diffuse granulated pattern segregated separately from a-actinin- immunopositive aggregates. Obscurin-associated kinase was localized mainly to cell nuclei with increasing incorporation into the Z-disks during differentiation. Our results suggest that these two novel proteins are involved in the progression of cardiac myogenesis during the transition to advanced stages of heart development.
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Early incorporation of Obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis
Histochemistry and Cell Biology, 2008Co-Authors: Andrei B. Borisov, Marina G. Martynova, Mark W. RussellAbstract:Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of Obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of Obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that Obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of Obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of Obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of Obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that Obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of Obscurin and titin localization in these areas showed that Obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with Obscurin, assembled around each new sarcomere. These results suggest that Obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.
Amber L Bowman - One of the best experts on this subject based on the ideXlab platform.
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the rho guanine nucleotide exchange factor domain of Obscurin activates rhoa signaling in skeletal muscle
Molecular Biology of the Cell, 2009Co-Authors: Diana L Fordspeelman, Amber L Bowman, Joseph A Roche, Robert J BlochAbstract:Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of Obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of Obscurin binds selectively to rhoA, and that rhoA colocalizes with Obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of Obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of Obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of Obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of Obscurin's rhoGEF domain. Our results suggest that Obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
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Obscurin interacts with a novel isoform of mybp c slow at the periphery of the sarcomeric m band and regulates thick filament assembly
Molecular Biology of the Cell, 2009Co-Authors: Maegen A Ackermann, Robert J Bloch, Amber L Bowman, Aikaterini KontrogiannikonstantopoulosAbstract:Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of Obscurin (Ig2) in developing myotubes inhibits the assembly of A- and M-bands, but not Z-disks or I-bands. This effect is mediated by the direct interaction of the Ig2 domain of Obscurin with a novel isoform of myosin binding protein-C slow (MyBP-C slow), corresponding to variant-1. Variant-1 contains all the structural motifs present in the known forms of MyBP-C slow, but it has a unique COOH terminus. Quantitative reverse transcription-polymerase chain reaction indicated that MyBP-C slow variant-1 is expressed in skeletal muscles both during development and at maturity. Immunolabeling of skeletal myofibers with antibodies to the unique COOH terminus of variant-1 demonstrated that, unlike other forms of MyBP-C slow that reside in the C-zones of A-bands, variant-1 preferentially concentrates around M-bands, where it codistributes with Obscurin. Overexpression of the Ig2 domain of Obscurin or reduction of expression of Obscurin inhibited the integration of variant-1 into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of Obscurin at the M-band, MyBP-C slow variant-1 and suggest that their interaction contributes to the assembly of M- and A-bands.
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Obscurin interacts with a novel isoform of myosin binding protein c slow to regulate the assembly of thick filaments
Biophysical Journal, 2009Co-Authors: Maegen A Borzok, Amber L Bowman, Robert J Bloch, John Strong, Aikaterini KontrogiannikonstantopoulosAbstract:Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. We used adenoviral-mediated gene transfer to overexpress its extreme NH2-terminus in developing myofibers, followed by immunofluorescence and ultrastructural methods to study its effects in sarcomerogenesis. We found that overexpression of Obscurin's second immunoglobulin domain (Ig2) inhibits the assembly of A- and M-bands, but not Z-disks and I-bands. This effect is mediated by the direct interaction of Obscurin's Ig2 with a novel isoform of the thick filament associated protein, Myosin Binding Protein-C Slow (MyBP-C slow), that corresponds to transcript variant-1. Variant-1 contains all the structural motifs known to be present in MyBP-C slow (variant-3), but has a unique COOH-terminus consisting of twenty-six amino acids and a new termination codon. RT-PCR showed that variant-1 is abundantly expressed in skeletal muscles during development and at maturity. Quantitative RT-PCR further demonstrated that transcripts containing the novel COOH-terminus are expressed in higher amounts than those lacking it. Three different antibodies to the unique COOH-terminus of variant-1 labeled M-bands and flanking regions in both developing and adult myofibers, suggesting that unlike other forms of MyBP-C that reside in C-zones, variant-1 preferentially concentrates in the middle of the A band. Adenoviral overexpression of Obscurin's Ig2 domain and reduction of Obscurin via siRNA inhibited the integration of variant 1 of MyBP-C slow into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of Obscurin at the M-band, MyBP-C slow variant-1, and suggest that its interaction with Obscurin contributes to the assembly and maintenance of M- and A-bands.
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the rho guanine nucleotide exchange factor domain of Obscurin regulates assembly of titin at the z disk through interactions with ran binding protein 9
Molecular Biology of the Cell, 2008Co-Authors: Amber L Bowman, Aikaterini Kontrogiannikonstantopoulos, Dawn H. Catino, John Strong, William R Randall, Robert J BlochAbstract:Obscurin is an ∼800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of Obscurin to understand i...
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different Obscurin isoforms localize to distinct sites at sarcomeres
FEBS Letters, 2007Co-Authors: Amber L Bowman, Mark W. Russell, Aikaterini Kontrogiannikonstantopoulos, Sarah B Geisler, Sara S Hirsch, Hugo Gonzalezserratos, Robert J BlochAbstract:We used four antibodies to regions of Obscurin isoforms A and B, encoded by the Obscurin gene, to investigate the location of these proteins in skeletal myofibers at resting and stretched lengths. Obscurin A ( approximately 800 kDa) which was recognized by antibodies generated to the N-terminal, Rho-GEF, and the non-modular C-terminal domain that lacks the kinase-like domains, localizes at the level of the M-band. Obscurin B ( approximately 900 kDa) which has the N-terminal, Rho-GEF, and the C-terminal kinase-like domains, localizes at the level of the A/I junction. Additional isoforms, which lack one or more of these epitopes, are present at the Z-disk and Z/I junction.
