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Peter W Gunning - One of the best experts on this subject based on the ideXlab platform.
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Visualizing the in vitro assembly of Tropomyosin/actin filaments using TIRF microscopy
Biophysical Reviews, 2020Co-Authors: Miro Janco, Nicole S Bryce, Edna C Hardeman, Irina Dedova, Peter W GunningAbstract:Tropomyosins are elongated alpha-helical proteins that form co-polymers with most actin filaments within a cell and play important roles in the structural and functional diversification of the actin cytoskeleton. How the assembly of Tropomyosins along an actin filament is regulated and the kinetics of Tropomyosin association with an actin filament is yet to be fully determined. A recent series of publications have used total internal reflection fluorescence (TIRF) microscopy in combination with advanced surface and protein chemistry to visualise the molecular assembly of actin/Tropomyosin filaments in vitro. Here, we review the use of the in vitro TIRF assay in the determination of kinetic data on Tropomyosin filament assembly. This sophisticated approach has enabled generation of real-time single-molecule data to fill the gap between in vitro bulk assays and in vivo assays of Tropomyosin function. The in vitro TIRF assays provide a new foundation for future studies involving multiple actin-binding proteins that will more accurately reflect the physiological protein-protein interactions in cells.
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Actin–Tropomyosin distribution in non-muscle cells
Journal of Muscle Research and Cell Motility, 2020Co-Authors: Dietmar J Manstein, J. C. M. Meiring, E. C. Hardeman, Peter W GunningAbstract:The interactions of cytoskeletal actin filaments with myosin family motors are essential for the integrity and function of eukaryotic cells. They support a wide range of force-dependent functions. These include mechano-transduction, directed transcellular transport processes, barrier functions, cytokinesis, and cell migration. Despite the indispensable role of Tropomyosins in the generation and maintenance of discrete actomyosin-based structures, the contribution of individual cytoskeletal Tropomyosin isoforms to the structural and functional diversification of the actin cytoskeleton remains a work in progress. Here, we review processes that contribute to the dynamic sorting and targeted distribution of Tropomyosin isoforms in the formation of discrete actomyosin-based structures in animal cells and their effects on actin-based motility and contractility.
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colocation of tpm3 1 and myosin iia heads defines a discrete subdomain in stress fibres
Journal of Cell Science, 2019Co-Authors: Joyce C M Meiring, Edna C Hardeman, Nicole S Bryce, Maria Lastra Cagigas, Ales Benda, Renee Whan, Nicholas Ariotti, Robert G Parton, Jeffrey H Stear, Peter W GunningAbstract:Co-polymers of Tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform specific manner. Previous work has implicated specific roles for at least 5 different Tropomyosin isoforms in stress fibres, as depletion of any of these 5 isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude Tropomyosins. In this study we investigate Tropomyosin organisation in stress fibres using super resolution light microscopy and electron microscopy with genetically tagged, endogenous Tropomyosin. We show that Tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. Tpm3.1/3.2 co-localises with non-muscle myosin IIa/IIb heads and are in register but do not overlap with non-muscle myosin IIa/IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.
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co polymers of actin and Tropomyosin account for a major fraction of the human actin cytoskeleton
Current Biology, 2018Co-Authors: Joyce C M Meiring, Edna C Hardeman, Nicole S Bryce, Jeffrey H Stear, Yao Wang, Manuel H Taft, Dietmar J Manstein, Sydney Liu Lau, Peter W GunningAbstract:Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and Tropomyosin and polymers of Tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that Tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with Tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying Tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of Tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that Tropomyosin binding must be inhibited in order to generate Tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with Tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with Tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), Tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of Tropomyosins is not due to a lack of N-terminal acetylation of LMW Tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW Tropomyosins to proteasomal degradation. We conclude that actin-Tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.
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tumor suppressor Tropomyosin tpm2 1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins
Cytoskeleton, 2017Co-Authors: Melissa Desouzaarmstrong, Peter W Gunning, Justine R StehnAbstract:The actin cytoskeleton is a polymer system that acts both as a sensor and mediator of apoptosis. Tropomyosins (Tpm) are a family of actin binding proteins that form co-polymers with actin and diversify actin filament function. Previous studies have shown that elevated expression of the Tropomyosin isoform Tpm2.1 sensitized cells to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. It is not yet known whether Tpm2.1 or other Tropomyosin isoforms regulate sensitivity to apoptosis beyond anoikis. In this study, rat neuroepithelial cells overexpressing specific Tropomyosin isoforms (Tpm1.7, Tpm2.1, Tpm3.1, and Tpm4.2) were screened for sensitivity to different classes of apoptotic stimuli, including both cytoskeletal and non-cytoskeletal targeting compounds. Results showed that elevated expression of Tropomyosins in general inhibited apoptosis sensitivity to different stimuli. However, Tpm2.1 overexpression consistently enhanced sensitivity to anoikis as well as apoptosis induced by the actin targeting drug jasplakinolide (JASP). In contrast the cancer-associated isoform Tpm3.1 inhibited the induction of apoptosis by a range of agents. Treatment of Tpm2.1 overexpressing cells with JASP was accompanied by enhanced sensitivity to mitochondrial depolarization, a hallmark of intrinsic apoptosis. Moreover, Tpm2.1 overexpressing cells showed elevated levels of the apoptosis proteins Bak (proapoptotic), Mcl-1 (prosurvival), Bcl-2 (prosurvival) and phosphorylated p53 (Ser392). Finally, JASP treatment of Tpm2.1 cells caused significantly reduced Mcl-1, Bcl-2 and p53 (Ser392) levels relative to control cells. We therefore propose that Tpm2.1 regulates sensitivity to apoptosis beyond the scope of anoikis by modulating the expression of key intrinsic apoptosis proteins which primes the cell for death.
Kazuo Shiomi - One of the best experts on this subject based on the ideXlab platform.
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Tropomyosins in gastropods and bivalves identification as major allergens and amino acid sequence features
Food Chemistry, 2009Co-Authors: Ai Emoto, Shoichiro Ishizaki, Kazuo ShiomiAbstract:Abstract Tropomyosin appears to be a major cross-reactive allergen of crustaceans and molluscs. In this study, four species of gastropods (disc abalone, turban shell, whelk and Middendorf’s buccinum) and seven species of bivalves (bloody cockle, Japanese oyster, Japanese cockle, surf clam, horse clam, razor clam and short-neck clam) were confirmed to be allergenic by ELISA and their major allergen identified as Tropomyosin by immunoblotting. Inhibition immunoblotting data showed the cross-reactivity of gastropod and bivalve Tropomyosins with one another and also with cephalopod and crustacean Tropomyosins. Then, amino acid sequences of Tropomyosins from 10 species except for Middendorf’s buccinum were elucidated by cDNA cloning. The known amino acid sequence data including our results reveal that molluscan Tropomyosins share low sequence identities (about 60%) with crustacean Tropomyosins and that they are highly homologous with one another within the same group (same family or same order) but not among the groups.
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identification of Tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics
Marine Biotechnology, 2008Co-Authors: Kanna Motoyama, Yota Suma, Shoichiro Ishizaki, Yuji Nagashima, Ying Lu, Hideki Ushio, Kazuo ShiomiAbstract:Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be Tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the Tropomyosins from both species with decapod Tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the Tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp Tropomyosin has high sequence identity (>90% identity) with decapod Tropomyosins, especially with fast-type Tropomyosins. On the other hand, the Antarctic krill Tropomyosin is characterized by diverse alterations in region 13–42, the amino acid sequence of which is highly conserved for decapod Tropomyosins, and hence, it shares somewhat lower sequence identity (82.4–89.8% identity) with decapod Tropomyosins than the mantis shrimp Tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains Tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of Tropomyosin.
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comparative analysis of barnacle Tropomyosin divergence from decapod Tropomyosins and role as a potential allergen
Comparative Biochemistry and Physiology B, 2007Co-Authors: Yota Suma, Shoichiro Ishizaki, Yuji Nagashima, Ying Lu, Hideki Ushio, Kazuo ShiomiAbstract:Abstract Tropomyosin, a myofibrillar protein of 35–38 kDa, represents a major and cross-reactive allergen in decapod crustaceans. This study was initiated to clarify whether decapod-allergic patients also recognize Tropomyosins of barnacles, crustaceans phylogenetically remote from decapods, which are locally consumed as a delicacy. On SDS-PAGE, a 37 kDa protein was observed in all the heated extracts prepared from two species of decapods (American lobster Homarus americanus and black tiger prawn Penaeus monodon) and two species of barnacles (acorn barnacle Balanus rostratus and goose barnacle Capitulum mitella). In immunoblotting, the 37 kDa protein was found to react with monoclonal antibodies against American lobster Tropomyosin and hence identified as Tropomyosin. The patient sera reacted to Tropomyosins from both decapods and barnacles and the reactivity was abolished by preincubation with American lobster Tropomyosin, demonstrating that barnacle Tropomyosins are allergens cross-reactive with decapod Tropomyosins. However, the amino acid sequence of acorn barnacle Tropomyosin, deduced by cDNA cloning experiments, shares higher sequence identity with abalone Tropomyosins than with decapod Tropomyosins. In accordance with this, the phylogenetic tree made for Tropomyosins from various animals showed that the acorn barnacle Tropomyosin is evolutionally classified not into the decapod Tropomyosin family but into the molluscan Tropomyosin family.
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molecular cloning of Tropomyosins identified as allergens in six species of crustaceans
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Kanna Motoyama, Yota Suma, Shoichiro Ishizaki, Yuji Nagashima, Kazuo ShiomiAbstract:Although Tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, Tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of Tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean Tropomyosins including the obtained results reveal that fast Tropomyosins are contained in shrimps (or prawns) and lobsters, slow Tropomyosins in crabs, and both Tropomyosins in crayfishes and hermit crabs. Although fast and slow Tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both Tropomyosins. Keywords: Allergen; cDNA cloning; crab; cross-reactivity; crustacean; prawn; shrimp; Tropomyosin
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cephalopod Tropomyosins identification as major allergens and molecular cloning
Food and Chemical Toxicology, 2006Co-Authors: Kanna Motoyama, Shoichiro Ishizaki, Yuji Nagashima, Kazuo ShiomiAbstract:Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is Tropomyosin in common with the nine species of cephalopods and that the cephalopod Tropomyosins are cross-reactive with one another and also with crustacean Tropomyosins. Molecular cloning experiments first elucidated the primary structures of Tropomyosins from five species of cephalopods. The cephalopod Tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63–64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus Tropomyosin (Pen a 1) are well conserved in the cephalopod Tropomyosins, supporting the cross-reactivity between cephalopod and crustacean Tropomyosins.
Samuel B Lehrer - One of the best experts on this subject based on the ideXlab platform.
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Tropomyosin an invertebrate pan allergen
International Archives of Allergy and Immunology, 1999Co-Authors: Gerald Reese, Rosalia Ayuso, Samuel B LehrerAbstract:Among food allergens, crustaceans, such as shrimp, crab, crawfish and lobster, are a frequent cause of adverse food reactions in allergic individuals. The major allergen has been identified as the muscle protein Tropomyosin. This molecule belongs to a family of highly conserved proteins with multiple isoforms found in both muscle and nonmuscle cells of all species of vertebrates and invertebrates. Its native structure consists of two parallel alpha-helical Tropomyosin molecules that are wound around each other forming a coiled-coil dimer. Allergenic Tropomyosins are found in invertebrates such as crustaceans (shrimp, lobster, crab, crawfish), arachnids (house dust mites), insects (cockroaches), and mollusks (e.g. squid), whereas vertebrate Tropomyosins are nonallergenic. Studies of cross-reactivities among crustaceans and the high degree of sequence identity among them suggest that Tropomyosin is probably the common major allergen in crustaceans. Furthermore, immunological relationships between crustaceans, cockroaches and housedust mites have been established and may suggest Tropomyosin as an important cross-sensitizing pan allergen.
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characterization of recombinant shrimp allergen pen a 1 Tropomyosin
International Archives of Allergy and Immunology, 1997Co-Authors: Gerald Reese, C B Daul, Byeoungju Jeoung, Samuel B LehrerAbstract:Tropomyosin (Pen a 1) from brown shrimp, Penaeus aztecus, has been identified as the only major shrimp allergen. Since beef, pork and chicken are other tropo-myosin-containing foods that are not very allergenic, Tropomyosins can serve to investigate the contribution of the structural properties of a protein to its allergenicity. The aim of this study was to determine the primary structure of Pen a 1 and to identify IgE-binding epitopes. The screening of a unidirectional expression cDNA library from shrimp tail muscle with the Pen-a-1-specific monoclonal antibody 4.9.5 resulted in 4 positive Escherichia coli clones. Immunoblot analysis with human sera from shrimp-allergic subjects demonstrated IgE binding of all 4 recombinant shrimp proteins. Three of 4 expressed recombinant proteins have a molecular weight of approximately 36 kD, consistent with the molecular weight of natural Pen a 1. The DNA sequence analysis identified these recombinant shrimp proteins as Tropomyosin and could be aligned with the sequence of greasyback shrimp (Metapenaeus ensis) Tropomyosin (Met e 1). In order to characterize contiguous IgE-binding epitopes of Pen a 1, a peptide library (Novagen epitope mapping system) expressing 10–30 amino-acid-residue-long recombinant Pen a 1 peptides was constructed and screened with human IgE. Four recombinant, IgE-reactive Pen a 1 peptides were selected and sequenced. They show various degrees of sequence identity with Tropomyosins of other arthropods, such as fruitfly and house dust mite, helminths and vertebrates.
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ige and monoclonal antibody reactivities to the major shrimp allergen pen a 1 Tropomyosin and vertebrate Tropomyosins
Advances in Experimental Medicine and Biology, 1996Co-Authors: Gerald Reese, Deborah Tracey, C B Daul, Samuel B LehrerAbstract:Pen a 1, the major shrimp allergen from brown shrimp Penaeus aztecus has been identified as the muscle protein Tropomyosin. To identify Pen a 1 IgE binding sites, the reactivities of Pen a 1-specific monoclonal antibodies (mAbs) and shrimp-allergic subjects’ IgE to shrimp and homologous mammalian Tropomyosins were analyzed. Pen a 1, purified by preparative SDS-PAGE and commercially obtained porcine, bovine and rabbit Tropomyosin were cleaved by CNBr or digested by endoproteinases Lys-C, Glu-C, trypsin, Arg-C and chymotrypsin. Reactivities of Pen a 1-specific mAbs and IgE to the resulting peptides were analyzed by dot blot and immunoblotting. The dot blot analysis showed that mAbs and IgE antibodies did not react with any of the mammalian Tropomyosins. The immunoblot analysis showed that all Pen a 1 digests bound IgE or mAbs. However, not all peptides in each digest possessed an IgE binding site. IgE binding intensity and frequency varied by subject and peptide digest. IgE and mAb reactivity patterns were similar but no mAb reproduced the IgE binding patterns indicating that subjects’ IgE bound some epitopes that were not recognized by the Pen a 1-specific mAbs. These studies suggest that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule, Pen a 1 may have several similar epitopes, and that Pen a 1 epitopes do not appear to be located in the highly homologous parts of the Tropomyosin molecule.
Sarah E Hitchcockdegregori - One of the best experts on this subject based on the ideXlab platform.
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structure of the n terminus of a nonmuscle alpha Tropomyosin in complex with the c terminus implications for actin binding
Biochemistry, 2009Co-Authors: Norma J. Greenfield, Lucy Kotlyanskaya, Sarah E HitchcockdegregoriAbstract:Tropomyosin is a coiled-coil actin binding protein that stabilizes the filament, protects it from severing, and cooperatively regulates actin’s interaction with myosin. Depending on the first coding exon, Tropomyosins are low molecular weight (LMW), found in the cytoskeleton and predominant in transformed cells, or high molecular weight (HMW), found in muscle and nonmuscle cells. The N- and C-terminal ends form a complex that allows Tropomyosin to associate N terminus-to-C terminus along the actin filament. We determined the structure of a LMW Tropomyosin N-terminal model peptide complexed with a smooth/nonmuscle Tropomyosin C-terminal peptide. Using NMR and circular dichroism we showed that both ends become more helical upon complex formation but that the C-terminal peptide is partially unfolded at 20 °C. The first five residues of the N terminus that are disordered in the free peptide are more helical and are part of the overlap complex. NMR data indicate residues 2−17 bind to the C terminus in the comp...
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the ends of Tropomyosin are major determinants of actin affinity and myosin subfragment 1 induced binding to f actin in the open state
Biochemistry, 1999Co-Authors: Joanna Moraczewska, Kelley Nicholsonflynn, Sarah E HitchcockdegregoriAbstract:Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-Tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to Tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle Tropomyosins, increases actin affinity in Tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d >> 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all Tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with Tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.
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effects of two familial hypertrophic cardiomyopathy causing mutations on alpha Tropomyosin structure and function
Biochemistry, 1997Co-Authors: Nina L Golitsina, Norma J. Greenfield, L Thierfelder, J G Seidman, Kenji Iizuka, Christine E Seidman, Sherwin S Lehrer, Sarah E HitchcockdegregoriAbstract:Missense mutations in alpha-Tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human Tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of Tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of Tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled Tropomyosins differed in their response to myosin S1 binding to the actin-Tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant Tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.
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incorporation of microinjected mutant and wildtype recombinant Tropomyosins into stress fibers in fibroblasts
Cytoskeleton, 1993Co-Authors: Denise Ranucci, Sarah E Hitchcockdegregori, Yoshihiko Yamakita, Fumio MatsumuraAbstract:The structural requirements for assembly of Tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle α-Tropomyosins expressed in E. coli as fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of Tropomyosin using indirect immunofluorescence. The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these Tropomyosins. Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80-residue fusion protein [Hitchcock-DeGregori, S.E., and Heald, R.W. (1987): J. Biol. Chem. 262:9730–9735]. The pattern of incorporation was indistinguishable from that of Tropomyosin isolated from chicken pectoral muscle. The striated α-Tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells. Three recombinant mutant Tropomyosins in which one-half, two-thirds, or one actin binding site was deleted were tested [Hitchcock-DeGregori, S.E., and Varnell, T.A. (1990): J. Mol. Biol. 214:885–896]. Only the fusion protein with a full actin binding site deleted incorporated into stress fibers. However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro. A fourth mutant, in which the conserved amino-terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant [Cho, Y.J., Liu, J., and Hitchcock-DeGregori, S.E. (1990): J. Biol. Chem. 265:538–545]. © 1993 Wiley-Liss, Inc.
C Redwood - One of the best experts on this subject based on the ideXlab platform.
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hypertrophic cardiomyopathy causing asp175asn and glu180gly tpm1 mutations shift Tropomyosin strands further towards the open position during the atpase cycle
Biochemical and Biophysical Research Communications, 2011Co-Authors: Yurii S. Borovikov, Olga E Karpicheva, Nikita A Rysev, C RedwoodAbstract:To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-Tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-Tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type Tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift Tropomyosin strands further towards the open position and to change the affinity of Tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca(2+)-sensitivity caused by these mutations which initiates the disease remodeling.
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dilated cardiomyopathy mutations in alpha Tropomyosin inhibit its movement during the atpase cycle
Biochemical and Biophysical Research Communications, 2009Co-Authors: Yurii S. Borovikov, Olga E Karpicheva, Galina A Chudakova, Paul Robinson, C RedwoodAbstract:The Glu40Lys and Glu54Lys mutations in alpha-Tropomyosin cause dilated cardiomyopathy (DCM). Functional analysis has demonstrated that both mutations decrease thin filament Ca2+-sensitivity and that Glu40Lys reduces maximum activation. To understand the molecular mechanism underlying these changes, we labeled wild type alpha-Tropomyosin and both mutants at Cys190 with 5-iodoacetamide-fluorescein and incorporated the labeled proteins into ghost muscle fibers. Using the polarized fluorimetry, the position of the labeled Tropomyosins on the thin filament and their affinity for actin were measured and the change in these parameters at different stages of the ATPase cycle determined. Both DCM mutations were found to shift Tropomyosin towards the periphery of thin filament and to change the affinity of Tropomyosin for actin; during the ATPase cycle the amplitude of Tropomyosin movement was reduced and at some stages of the cycle even reversed. The correlation of these structural changes with the observed function effects is discussed.
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effects of two hypertrophic cardiomyopathy mutations in alpha Tropomyosin asp175asn and glu180gly on ca2 regulation of thin filament motility
Biochemical and Biophysical Research Communications, 1997Co-Authors: Wu Bing, Hugh Watkins, C Redwood, Giovanna Esposito, Ian Purcell, Steven B MarstonAbstract:Abstract The functional properties of wild type α-Tropomyosin expressed in E. coli with an alanine-serine N-terminal leader (AS-α-Tm) were compared with those of AS-α-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC). Wild type AS-α-Tm and AS-α-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-Tropomyosin whilst the affinity of AS-α-Tm (Glu180Gly) was about threefold weaker. In vitro motility assays were performed with AS-α-Tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin. Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-α-Tropomyosin or mutant Tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to 2+ - regulation of thin filaments, presumably via altered interaction with troponin.
