Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

Heintz, Daniela; Reichert, Andreas S.; Mihelić, M.; Voelter, Wolfgang; Faulstich, Heinz

doi:10.1016/0014-5793(93)80181-s

Cited by 53 publications

(44 citation statements)

References 20 publications

(20 reference statements)

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“…Moreover, Leu 6 and Asp 10 in TB9 are replaced by Met 6 and Ala 10 in TB10. We believe that the change of Ala 10 to Asp 10 in the position of residue 10 explains the structural difference between TB9 and TB10, because residue 10 is located at the near of the new turn and because the size difference of the two side chains is remarkable.…”

Section: Resultsmentioning

confidence: 92%

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

Rho

Lee

Chun

et al. 2005

Journal of Biological Chemistry

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Thymosin ␤-10 (TB10) is an actin monomer-sequestering peptide that consists of 43 amino acid residues and that tends to form ␣-helical structures. Previously, we showed that the overexpression of TB10 dramatically increases the frequency of apoptosis in human ovarian cancer cells. To identify the critical residues responsible for TB10-mediated apoptosis, we used a series of computational methods. First, a three-dimensional structure of human TB10 was constructed using the homology modeling method with the calf thymosin ␤-9 NMR structure as a template. Although the sequences of both of these structures are almost identical, 200-ps molecular dynamics simulation results showed that their secondary structures differ. Analyses of molecular dynamics snapshot structures suggested that the TB10 structure is conformationally more complicated than the TB9 structure. The conserved 17 LKKTET 22 motif region of TB10 was tested by Ala and Ser scanning mutagenesis using computational and biochemical methods, and 12 mutants were transfected into cancer cell lines and tested for their effects on growth arrest. Of the 12 mutants examined, only the Thr 20 to Ser 20 mutation showed reduced growth arrest. These results strongly suggest that Thr 20 is specifically required for actin sequestration by TB10 in ovarian cancer cells. These results may provide useful information for the development of a new ovarian cancer therapy.The ␤-thymosins are small (molecular mass Ͻ 5 kDa) highly conserved acidic proteins. They were originally identified in calf thymus and thought to be involved in immunomodulatory functions (1). At least 16 ␤-thymosin isoforms have been identified from various vertebrates and invertebrates, and most, including thymosin ␤-4 (TB4), 3 are constitutively expressed in most tissues (2-5). A majority share a conserved hexapeptide motif, "LKKTET," which is critical for binding actin, and have important roles in maintaining cellular functions, such as cell morphology, proliferation, and locomotion (6). In most mammalian tissues, TB4 and TB10 are major actin monomer-sequestering proteins (7,8). Both form a 1:1 complex with G-actin and inhibit its polymerization to F-actin (9 -11).TB10 is more selectively modulated during embryological development than TB4 and is associated with several diseases (12-15). In addition, TB10 up-regulates the anti-apoptotic protein Bcl-2, which is associated with neoplastic transformation (16). Thymosin ␤-9 (TB9) overexpression was also found to modulate infected bovine macrophage apoptosis (17).In the present study, we employed a combined approach involving molecular modeling and site-directed mutagenesis to better understand the molecular regions of TB10 that control apoptosis. On the basis of its computer model-determined three-dimensional structure, we identified a region of TB10 that might be critical for apoptotic induction. This region was then tested in transfected ovarian cancer cell lines via mutagenesis (Ala and Ser scanning) of the conserved 17 LKKTET 22 motif with respect to actin bi...

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Section: Resultsmentioning

confidence: 92%

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

Rho

Lee

Chun

et al. 2005

Journal of Biological Chemistry

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“…In a previous study the KD value of the actin-TiM complex was measured by fluorescence changes of TMB-actin [20]. After having shown that complexation with DNase I had no influence on the fluorescence of TMB-actin, the same assay was used to determine the KD between TIM and the actinDNase I complex.…”

Section: Resultsmentioning

confidence: 99%

“…The dissociation constant of TIM and the actin-DNase I complex as determined using the actin derivative TMB-actin, prepared acc ~rding to Heintz et al [20]. TMB-actin and DNase I were mixed a: a ratio of 1:2 and fluorescence was measured in a Spex fluorolog (';pex Industries Inc., New York) between 385 and 600 nm (excitation 3 70 nm).…”

Section: Kd-measurementsmentioning

confidence: 99%

The ternary complex of DNase I, actin and thymosin β4

et al. 1996

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Key words: Actin; Thymosin 134; DNase I; Ternary complex; Thiol-specific crosslinking the differently positioned thiol groups in TIM come close enough to distinct thiol groups in actin to allow the formation of crosslinks. In this way two contact sites between the two proteins could be identified, one between the C-terminus of actin (Cys 374) and position 6 of TIM and a second between the )'-phosphate of the actin-bound nucleotide in actin and the sequence 17 28 in TIM.X-ray analysis of monomeric actin took advantage of a facilitated crystallization of actin when complexed with DNase I. Provided DNase I exerted a similar effect on TIMactin as well, co-crystallization with DNase I would allow the interface of actin and TIM in the ternary complex to be studied. Corresponding complexes have been reported for DNase I, actin and profilin, or ADF/cofilin, respectively [14,15]. However, the ternary complex can be expected to form only if the binding of DNase I and that of TIM to actin do not strongly interfere. We therefore examined whether DNase I affects the affinity of TIM for actin by studying one of the crosslinking reactions mentioned above in the presence of DNase I. Materials and methods In~oductionThymosin 134 (TIM) is one of the small proteins in nonmuscle cells that bind to monomeric actin and thus prevent unregulated polymerization [14]. From the TiM-complexed monomers, polymerization can be started in vitro either by decapping barbed ends of filaments [5,6], or by adding nucleus stabilizers such as myosin S1 or phalloidin [7][8][9][10][11]. The relatively low affinity of T~4 for actin (ca. 1 ~tM [4]) may be functionally significant in allowing the instant release of polymerizable actin into cytoplasm, if required.Since an X-ray analysis of the actin-TiM complex has so far not been achieved we have tried to identify contact sites between the two proteins by a chemical approach [12]. For this, five TIM analogs were synthesized, each of them with one cysteine residue substituted for a hydrophobic amino acid in the TIM chain. Using a set of thiol-specific crosslinkers of varying length, we assayed whether in the complex with actin

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“…As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11). It has been proposed that T␤4 is likely to adopt a unique conformation upon binding actin (12).One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinking of two cysteine residues.…”

mentioning

confidence: 97%

“…One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinking of two cysteine residues.…”

mentioning

confidence: 99%

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

Reichert

Heintz

Echner

et al. 1996

Journal of Biological Chemistry

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Binding sites of actin and thymosin ␤4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys 10 , Cys 374 , or to the sulfur atom of the ATP analog adenosine 5-O-(thiotriphosphate) (ATP␥S), the actin derivatives were reacted with synthetic thymosin ␤4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate crosslinking as followed by UV spectroscopy was found for Cys 374 of actin and Cys 6 of thymosin ␤4, indicating that the N terminus of thymosin ␤4 is in close proximity ( 9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin ␤4 analogs when the cross-linkers were anchored at Cys 10 of actin. A second contact site was identified by cross-linking of Cys 17 and Cys 28 in thymosin ␤4 with the ATP␥S derivative bound to actin, indicating that the hexamotif of thymosin ␤4 (positions 17-22) is in close proximity ( 9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin ␤4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.The physiological conditions within nonmuscle cells favor the assembly of actin monomers. Therefore, the pool of monomeric actin has to be maintained by complexation with small G-actin binding proteins. Particularly thymosin ␤4 (T␤4) 1 , which forms a 1:1 complex with actin monomers, is believed to be involved in preventing actin polymerization (1-6). Dissociation constants of the actin⅐T␤4 complex were found to be in the range of 0.4 -0.7 M for platelet actin and 0.7-2.0 M for muscle actin (4, 7). While complexed with T␤4, the exchange of the bound nucleotide in actin is retarded (8). In a detailed study, Vancompernolle et al. (9) have shown that binding of T␤4 to actin is mainly mediated by the hexamotif LKKTET (17-22), since loss of this sequence is paralleled by an almost complete loss of inhibitory activity. Alterations in the N-terminal part (1-16) of the peptide strongly influence the inhibitory activity of T␤4, whereas alterations in the C-terminal part (31-43) seem to be of minor importance (9). As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11). It has been proposed that T␤4 is likely to adopt a unique conformation upon binding actin (12).One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinki...

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Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

Cited by 53 publications

References 20 publications

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

The ternary complex of DNase I, actin and thymosin β4

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

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