The Experts below are selected from a list of 66 Experts worldwide ranked by ideXlab platform
Ewald Hannappel - One of the best experts on this subject based on the ideXlab platform.
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the β Thymosins intracellular and extracellular activities of a versatile actin binding protein family
Cytoskeleton, 2009Co-Authors: Hans Georg Mannherz, Ewald HannappelAbstract:The Beta-Thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin Beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin Beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that Beta-Thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the Beta-Thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the Beta-Thymosins, i.e., their modulating effect on the actin cytoskeleton.
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The Thymosins. Prothymosin alpha, parathymosin, and Beta-Thymosins: structure and function.
Vitamins and hormones, 2003Co-Authors: Ewald Hannappel, Thomas HuffAbstract: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 immuno-deficiency 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 Beta 1, thymosin alpha 1, prothymosin alpha, parathymosin, and thymosin Beta 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.
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polymerisation of chemically cross linked actin thymosin Beta 4 complex to filamentous actin alteration in helical parameters and visualisation of thymosin Beta 4 binding on f actin
Journal of Molecular Biology, 2002Co-Authors: Edda Ballweber, Thomas Huff, Ewald Hannappel, Harald Stephan, Markus Haener, Nicole Taschner, Daniel Stoffler, Ueli Aebi, Hans Georg MannherzAbstract:The Beta-Thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin Beta(4) with F-actin. Thymosin Beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin Beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin Beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin Beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin Beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin Beta(4). The deduced binding sites of thymosin Beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.
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Beta Thymosins small acidic peptides with multiple functions
The International Journal of Biochemistry & Cell Biology, 2001Co-Authors: Thomas Huff, Christian S G Muller, Roland Netzker, Angela M. Otto, Ewald HannappelAbstract:Abstract The β-Thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin β4 as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. β-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of β-Thymosins appear to be related to the differentiation of cells. Increased expression of β-Thymosins or even the synthesis of a β-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin β4 is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin β4, oxidized thymosin β4, or to the fragment, acSDKP, possibly generated from thymosin β4. Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular β-Thymosins.
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Beta-Thymosins, small acidic peptides with multiple functions
The international journal of biochemistry & cell biology, 2001Co-Authors: Thomas Huff, Christian S G Muller, Roland Netzker, Angela M. Otto, Ewald HannappelAbstract:The Beta-Thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin Beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. Beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of Beta-Thymosins appear to be related to the differentiation of cells. Increased expression of Beta-Thymosins or even the synthesis of a Beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin Beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin Beta(4), oxidized thymosin Beta(4), or to the fragment, acSDKP, possibly generated from thymosin Beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular Beta-Thymosins.
Thomas Huff - One of the best experts on this subject based on the ideXlab platform.
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The Thymosins. Prothymosin alpha, parathymosin, and Beta-Thymosins: structure and function.
Vitamins and hormones, 2003Co-Authors: Ewald Hannappel, Thomas HuffAbstract: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 immuno-deficiency 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 Beta 1, thymosin alpha 1, prothymosin alpha, parathymosin, and thymosin Beta 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.
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polymerisation of chemically cross linked actin thymosin Beta 4 complex to filamentous actin alteration in helical parameters and visualisation of thymosin Beta 4 binding on f actin
Journal of Molecular Biology, 2002Co-Authors: Edda Ballweber, Thomas Huff, Ewald Hannappel, Harald Stephan, Markus Haener, Nicole Taschner, Daniel Stoffler, Ueli Aebi, Hans Georg MannherzAbstract:The Beta-Thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin Beta(4) with F-actin. Thymosin Beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin Beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin Beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin Beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin Beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin Beta(4). The deduced binding sites of thymosin Beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.
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Beta Thymosins small acidic peptides with multiple functions
The International Journal of Biochemistry & Cell Biology, 2001Co-Authors: Thomas Huff, Christian S G Muller, Roland Netzker, Angela M. Otto, Ewald HannappelAbstract:Abstract The β-Thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin β4 as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. β-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of β-Thymosins appear to be related to the differentiation of cells. Increased expression of β-Thymosins or even the synthesis of a β-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin β4 is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin β4, oxidized thymosin β4, or to the fragment, acSDKP, possibly generated from thymosin β4. Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular β-Thymosins.
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Beta-Thymosins, small acidic peptides with multiple functions
The international journal of biochemistry & cell biology, 2001Co-Authors: Thomas Huff, Christian S G Muller, Roland Netzker, Angela M. Otto, Ewald HannappelAbstract:The Beta-Thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin Beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. Beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of Beta-Thymosins appear to be related to the differentiation of cells. Increased expression of Beta-Thymosins or even the synthesis of a Beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin Beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin Beta(4), oxidized thymosin Beta(4), or to the fragment, acSDKP, possibly generated from thymosin Beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular Beta-Thymosins.
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interactions of Beta Thymosins thymosin Beta 4 sulfoxide and n terminally truncated thymosin Beta 4 with actin studied by equilibrium centrifugation chemical cross linking and viscometry
FEBS Journal, 1995Co-Authors: Thomas Huff, Doris Zerzawy, Ewald HannappelAbstract:All Beta-Thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and Beta-Thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several Beta-Thymosins, oxidized thymosin Beta 4 and N-terminally truncated Beta 4. The apparent dissociation constants for actin from bovine heart and Beta-Thymosins were 2.5, 0.1, and 2.7 microM for thymosin Beta 4, [Ala1]Beta 4(Beta Ala4), and Beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin Beta 4 or Beta Ala4. In rabbits thymosin Beta Ala4 replaces Beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin Beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin Beta 4 at Met6 (Beta 4-sulfoxide) as well as truncation of 6 [Beta 4-(7-43)] or 12 [Beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [Beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin Beta 4-sulfoxide or Beta 4-(7-43) or Beta 4-(13-43), only Beta 4-sulfoxide and not the truncated Beta-Thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than Beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin Beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-Beta 4 complex is necessary to observe a comparable dissociation constant.
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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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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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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control of actin dynamics by proteins made of β thymosin repeats the actobindin family
Journal of Biological Chemistry, 2002Co-Authors: Maud Hertzog, Elena G Yarmola, Dominique Didry, Michael R Bubb, Marie-france CarlierAbstract:Actobindin is an actin-binding protein from amoeba, which consists of two Beta-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three Beta-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to Beta-Thymosins. Like Beta-Thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike Beta-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actin-based motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the Beta-thymosin repeats with two terminal subunits.
Dominique Didry - 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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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.
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control of actin dynamics by proteins made of β thymosin repeats the actobindin family
Journal of Biological Chemistry, 2002Co-Authors: Maud Hertzog, Elena G Yarmola, Dominique Didry, Michael R Bubb, Marie-france CarlierAbstract:Actobindin is an actin-binding protein from amoeba, which consists of two Beta-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three Beta-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to Beta-Thymosins. Like Beta-Thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike Beta-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actin-based motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the Beta-thymosin repeats with two terminal subunits.