Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1

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

doi:10.1016/0014-5793(94)00551-6

Cited by 12 publications

(7 citation statements)

References 34 publications

(9 reference statements)

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“…The use of a 96-well plate reader to assay fluorescence also increases assay throughput. While phalloidin can drive the polymerization of ␤-thymosin-actin complexes [Reichert et al, 1994], under our conditions, the fluorescence intensity and F-actin concentration was stable for at least 30 min (data not shown).…”

Section: Actin Monomer Sequestering Assaymentioning

confidence: 69%

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

et al. 2000

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Mammalian cells express several isoforms of beta-thymosin, a major actin monomer sequestering factor, including thymosins beta4, beta10, and beta15. Differences in actin-binding properties of different beta-thymosin family members have not been investigated. We find that thymosin beta15 binds actin with a 2.4-fold higher affinity than does thymosin beta4. Mutational analysis was performed to determine the amino acid differences in thymosin beta15 that specify its increased actin-affinity. Previous work with thymosin beta4 identified an alpha-helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin beta15, but that differences in these domains are not responsible for the variation in actin-binding properties between thymosins beta4 and beta15. Truncation of the unique C-terminal residues in thymosin beta15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin-binding affinity. Replacement of the 10 C-terminal amino acids of thymosin beta15 with those of thymosin beta4 did, however, reduce the actin-binding affinity of the hybrid relative to thymosin beta15. Similarly, replacement of the thymosin beta4 C-terminal amino acids with those of thymosin beta15 led to increased actin binding. We conclude that functional differences between closely related beta-thymosin family members are, in part, specified by the C-terminal variability between these isoforms. Such differences may have consequences for situations where beta-thymosins are differentially expressed as in embryonic development and in cancer.

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Section: Actin Monomer Sequestering Assaymentioning

confidence: 69%

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

et al. 2000

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“…Thymosin β 4 has previously been found to promote angiogenesis (5,13,15,16). Here we have localized the motif for this activity to the central actin binding domain by using seven different synthetic peptides and six different proteolytic fragments.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, antagonistic peptides that block the binding of angiogenin to actin inhibit its activity (28,29). Both thymosin β 4 and angiogenin increase protease activity (13,30,31). Peptides antagonistic to the angiogenin receptor that block its binding to actin also block protease activity and tumor growth and metastasis in vivo (32,33).…”

Section: Discussionmentioning

confidence: 99%

“…Although its role was initially attributed to T cell differentiation (7), it later was found that thymosin β 4 binds to G-actin in a 1:1 complex that prevents monomeric G-actin from polymerizing to filamentous F-actin but supplies a pool of actin monomers inside the cells (12,13). We and others identified thymosin β 4 as an angiogenic factor with activity during HUVEC differentiation in vitro and in vivo (5,(14)(15)(16).…”

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confidence: 99%

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The actin binding site on thymosin β₄promotes angiogenesis

et al. 2003

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Thymosin beta4 is a ubiquitous 43 amino acid, 5 kDa polypeptide that is an important mediator of cell proliferation, migration, and differentiation. It is the most abundant member of the beta-thymosin family in mammalian tissue and is regarded as the main G-actin sequestering peptide. Thymosin beta4 is angiogenic and can promote endothelial cell migration and adhesion, tubule formation, aortic ring sprouting, and angiogenesis. It also accelerates wound healing and reduces inflammation when applied in dermal wound-healing assays. Using naturally occurring thymosin beta4, proteolytic fragments, and synthetic peptides, we find that a seven amino acid actin binding motif of thymosin beta4 is essential for its angiogenic activity. Migration assays with human umbilical vein endothelial cells and vessel sprouting assays using chick aortic arches show that thymosin beta4 and the actin-binding motif of the peptide display near-identical activity at ~50 nM, whereas peptides lacking any portion of the actin motif were inactive. Furthermore, adhesion to thymosin beta4 was blocked by this seven amino acid peptide demonstrating it as the major thymosin beta4 cell binding site on the molecule. The adhesion and sprouting activity of thymosin beta4 was inhibited with the addition of 5-50 nM soluble actin. These results demonstrate that the actin binding motif of thymosin beta4 is an essential site for its angiogenic activity.

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“…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.…”

Section: In~oductionmentioning

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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Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1

Cited by 12 publications

References 34 publications

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

The actin binding site on thymosin β₄promotes angiogenesis

The ternary complex of DNase I, actin and thymosin β4

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Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1

Cited by 12 publications

References 34 publications

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

The actin binding site on thymosin β4promotes angiogenesis

The ternary complex of DNase I, actin and thymosin β4

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The actin binding site on thymosin β₄promotes angiogenesis