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Ewald Hannappel - One of the best experts on this subject based on the ideXlab platform.
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Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry
2018Co-Authors: Ewald Hannappel, Federica Iavarone, Massimo CastagnolaAbstract:Objectives: We reevaluated a lyophilized sample of Thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in Thymosin fraction 5. Results: We detected more than 100 monoisotopic masses corresponding to Thymosin β4 and truncated forms of ubiquitin, proThymosin α, Thymosin β4, and Thymosin β9. Additionally, we discovered a new polypeptide present in Thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of Thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known Thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of Thymosin fraction 5 is even nowadays highly promising.
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high resolution mass spectrometry for Thymosins detection and characterization
Expert Opinion on Biological Therapy, 2015Co-Authors: Tiziana Cabras, Daniela Delfino, Diana Valeria Rossetti, Claudia Martelli, Ilaria Inserra, Federica Iavarone, Claudia Desiderio, Barbara Manconi, Irene Messana, Ewald HannappelAbstract:Objectives: The aim of this study was to characterize β and α Thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies.Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.Results: In addition to Thymosin β4 (Tβ4) and β10 (Tβ10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ4 and Tβ10 together with other proteolytic fragments. The sulfoxide derivative of both Tβ4 and Tβ10 and the acetylated derivatives at lysine residues of Tβ4 were also characterized. Different proteoforms of proThymosin α, paraThymosin α, ...
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Thymosin β4 and Tissue Transglutaminase. Molecular Characterization of Cyclic Thymosin β4
Protein Journal, 2013Co-Authors: Jana Knop, Thomas Huff, Heinrich Sticht, Ewald HannappelAbstract:Thymosin β4 is the prototype of β-Thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an e-amino group of Thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of Thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of Thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic Thymosin β4). These two amino acid residues are conserved in all β-Thymosins. Cyclic Thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the Thymosin β4 × G-actin complex.
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Thymosin β4 and its posttranslational modifications
Annals of the New York Academy of Sciences, 2010Co-Authors: Ewald HannappelAbstract:Thymosin β 4 as well as the other members of the β-Thymosin family are important G-actin sequestering peptides. The chemical properties, the biosynthesis, and posttranslational modifications (PTMs) of these peptides are discussed. During biosynthesis ofThymosin β 4 the initiator methionine is removed and the N-terminus is acetylated. Research on proteomics revealed several acetylated lysine residues and two phosphorylated threonine residues. The enormous number of phosphorylable and acetylable sites in the human proteome raises the question about the biological significance of these PTMs in the context of β-Thymosins. Presently, this question cannot be answered because neither the concentration of these modified β-Thymosins in cells is known nor the consequences of the modifications on the biological function(s) of β-Thymosins have been studied yet. Thymosin β 4 is also posttranslationally modified by transglutaminase forming covalent bonds with other molecules. Prolyl oligopeptidase generates ac-SDKP from Thymosin β 4 . The concentration of C-terminal peptide fragments of Thymosin β 4 is elevated in the blood of patients with rheumatoid arthritis.
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Thymosin β4 and its posttranslational modifications
Annals of the New York Academy of Sciences, 2010Co-Authors: Ewald HannappelAbstract:Thymosin beta(4) as well as the other members of the beta-Thymosin family are important G-actin sequestering peptides. The chemical properties, the biosynthesis, and posttranslational modifications (PTMs) of these peptides are discussed. During biosynthesis of Thymosin beta(4) the initiator methionine is removed and the N-terminus is acetylated. Research on proteomics revealed several acetylated lysine residues and two phosphorylated threonine residues. The enormous number of phosphorylable and acetylable sites in the human proteome raises the question about the biological significance of these PTMs in the context of beta-Thymosins. Presently, this question cannot be answered because neither the concentration of these modified beta-Thymosins in cells is known nor the consequences of the modifications on the biological function(s) of beta-Thymosins have been studied yet. Thymosin beta(4) is also posttranslationally modified by transglutaminase forming covalent bonds with other molecules. Prolyl oligopeptidase generates ac-SDKP from Thymosin beta(4). The concentration of C-terminal peptide fragments of Thymosin beta(4) is elevated in the blood of patients with rheumatoid arthritis.
Thomas Huff - One of the best experts on this subject based on the ideXlab platform.
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Thymosin β4 and Tissue Transglutaminase. Molecular Characterization of Cyclic Thymosin β4
Protein Journal, 2013Co-Authors: Jana Knop, Thomas Huff, Heinrich Sticht, Ewald HannappelAbstract:Thymosin β4 is the prototype of β-Thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an e-amino group of Thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of Thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of Thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic Thymosin β4). These two amino acid residues are conserved in all β-Thymosins. Cyclic Thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the Thymosin β4 × G-actin complex.
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Thymosin β4 Is Not Always the Main β‐Thymosin in Mammalian Platelets
Annals of the New York Academy of Sciences, 2007Co-Authors: Thomas Huff, Christian S G Muller, Ewald HannappelAbstract:p-Thymosins constitute a family of highly conserved 5-kDa polypeptides. Thymosin β 4 , the most abundant member of this family, is expressed in most mammalian cell types and is regarded as the main intracellular G-actin sequestering peptide. In addition to this important intracellular function several other activities have been attributed to this peptide. Thymosin β 4 is released from human platelets and cross-linked to fibrin after activation of platelets with thrombin. While in most mammalian tissues Thymosin β 4 is accompanied by a second member of this peptide family, in human platelets only Thymosin p4 is present. To elucidate if it is common to mammalian platelets that only one β-Thymosin is present, we analyzed platelets from several mammals for their p-Thymosin content. In human platelets only Thymosin β 4 could be detected, whereas in bovine platelets Thymosin β 9 , which is normally the minor p-Thymosin in bovine tissues, was identified as the main β-Thymosin. In rabbit platelets, Thymosin β 4 is not simply replaced by the most homologous Thymosin β 4 Ala , as might be expected from sequence homology. Thymosin β 4 Ala and Thymosin β 10 were found, but Thymosin β 10 is present in about 2.5-fold higher amounts. Because Thymosin β 4 Ala possesses about threefold higher affinity to G-actin, compared to Thymosin β 4 , β 10 , and β 9 , we suggest that expression of p-Thymosins is triggered by functional requirements and not sequence homology.
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Thymosin β4 Is Not Always the Main β‐Thymosin in Mammalian Platelets
Annals of the New York Academy of Sciences, 2007Co-Authors: Thomas Huff, Christian S G Muller, Ewald HannappelAbstract:beta-Thymosins constitute a family of highly conserved 5-kDa polypeptides. Thymosin beta(4), the most abundant member of this family, is expressed in most mammalian cell types and is regarded as the main intracellular G-actin sequestering peptide. In addition to this important intracellular function several other activities have been attributed to this peptide. Thymosin beta(4) is released from human platelets and cross-linked to fibrin after activation of platelets with thrombin. While in most mammalian tissues Thymosin beta(4) is accompanied by a second member of this peptide family, in human platelets only Thymosin beta(4) is present. To elucidate if it is common to mammalian platelets that only one beta-Thymosin is present, we analyzed platelets from several mammals for their beta-Thymosin content. In human platelets only Thymosin beta(4) could be detected, whereas in bovine platelets Thymosin beta(9), which is normally the minor beta-Thymosin in bovine tissues, was identified as the main beta-Thymosin. In rabbit platelets, Thymosin beta(4) is not simply replaced by the most homologous Thymosin beta(4)(Ala), as might be expected from sequence homology. Thymosin beta(4)(Ala) and Thymosin beta(10) were found, but Thymosin beta(10) is present in about 2.5-fold higher amounts. Because Thymosin beta(4)(Ala) possesses about threefold higher affinity to G-actin, compared to Thymosin beta(4), beta(10), and beta(9), we suggest that expression of beta-Thymosins is triggered by functional requirements and not sequence homology.
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intracellular β Thymosins
2007Co-Authors: Ewald Hannappel, Thomas Huff, Daniel SaferAbstract:The β-Thymosins are a family of highly conserved polar peptides consisting of 40 to 44 amino acid residues. All β-Thymosins bind monomeric G-actin in a 1:1 complex. The dissociation constant of the complex is in the micromolar range and allows for fast binding and release of G-actin. Because of the high intracellular concentration of β-Thymosins (up to 500 µM) in most vertebrate cells, β-Thymosins are considered the main intracellular G-actin sequestering peptides. Thymosin β4 binds to G-actin in an extended conformation, and folds into a stable conformation upon binding. The N- and C-termini of Thymosin β4 contact the barbed and pointed ends of the monomeric actin. Thymosin β4 is present in the nucleus as well as the cytoplasm and might be responsible for sequestering nuclear actin. Even minor cell damage might be responsible for the release of β-Thymosins detectable in the extracellular fluids. Extracellular β-Thymosins affect matrix metallo-proteinases, chemotaxis, angiogenesis and wound healing. However, only very little is known about the molecular mechanisms mediating the effects attributed to extracellular β-Thymosins.
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the Thymosins proThymosin α paraThymosin and β Thymosins structure and function
Vitamins and Hormones Series, 2003Co-Authors: Ewald Hannappel, Thomas HuffAbstract:Abstract The studies on Thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immunodeficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called Thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that Thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide β 1 , Thymosin α 1 , proThymosin α, paraThymosin, and Thymosin β 4 ) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the Thymosins is discussed in this review. © 2003, Elsevier Science (USA).
Carine Van Heijenoort - One of the best experts on this subject based on the ideXlab platform.
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Multifunctionality of the β-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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multifunctionality of the β Thymosin wh2 module g actin sequestration actin filament growth nucleation and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Multifunctionality of the beta-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing.
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Structure, function, and evolution of the beta-Thymosin/WH2 (WASP-Homology2) actin-binding module.
Annals of the New York Academy of Sciences, 2007Co-Authors: Marie-france Carlier, Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Marcel Knossow, François-xavier Cantrelle, Louis Renault, Eric GuittetAbstract:beta-Thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-Thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using Thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-Thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.
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the β Thymosin wh2 domain structural basis for the switch from inhibition to promotion of actin assembly
Cell, 2004Co-Authors: Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Martin Gaudier, Jerome Coutant, Benoit Gigant, Gerard Didelot, Thomas Preat, Marcel Knossow, Eric GuittetAbstract:The widespread beta-Thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-Thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-Thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-Thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-Thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-Thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.
Marie-france Carlier - One of the best experts on this subject based on the ideXlab platform.
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Multifunctionality of the β-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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multifunctionality of the β Thymosin wh2 module g actin sequestration actin filament growth nucleation and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Multifunctionality of the beta-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing.
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Structure, function, and evolution of the beta-Thymosin/WH2 (WASP-Homology2) actin-binding module.
Annals of the New York Academy of Sciences, 2007Co-Authors: Marie-france Carlier, Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Marcel Knossow, François-xavier Cantrelle, Louis Renault, Eric GuittetAbstract:beta-Thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-Thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using Thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-Thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.
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coupling of folding and binding of Thymosin β4 upon interaction with monomeric actin monitored by nuclear magnetic resonance
Journal of Biological Chemistry, 2004Co-Authors: Michael Domanski, Marie-france Carlier, Maud Hertzog, Jerome Coutant, Eric Guittet, Irina Gutscheperelroizen, Francois Bontems, Carine Van HeijenoortAbstract:Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of Thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of Thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that Thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of Thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of Thymosins on G-actin.
Eric Guittet - One of the best experts on this subject based on the ideXlab platform.
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Multifunctionality of the β-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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multifunctionality of the β Thymosin wh2 module g actin sequestration actin filament growth nucleation and severing
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Multifunctionality of the beta-Thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing.
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france CarlierAbstract:The beta-Thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-Thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-Thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.
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Structure, function, and evolution of the beta-Thymosin/WH2 (WASP-Homology2) actin-binding module.
Annals of the New York Academy of Sciences, 2007Co-Authors: Marie-france Carlier, Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Marcel Knossow, François-xavier Cantrelle, Louis Renault, Eric GuittetAbstract:beta-Thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-Thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using Thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-Thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.
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the β Thymosin wh2 domain structural basis for the switch from inhibition to promotion of actin assembly
Cell, 2004Co-Authors: Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Martin Gaudier, Jerome Coutant, Benoit Gigant, Gerard Didelot, Thomas Preat, Marcel Knossow, Eric GuittetAbstract:The widespread beta-Thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-Thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-Thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-Thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-Thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-Thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.
