Thomas Huff

beta-Thymosins, small acidic peptides with multiple functions.

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.

The actin binding site on thymosin β4 promotes angiogenesis

Thymosin β4 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 β‐thymosin family in mammalian tissue and is regarded as the main G‐actin sequestering peptide. Thymosin β4 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 β4, proteolytic fragments, and synthetic peptides, we find that a seven amino acid actin binding motif of thymosin β4 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 β4 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 β4 was blocked by this seven amino acid peptide demonstrating it as the major thymosin β4 cell binding site on the molecule. The adhesion and sprouting activity of thymosin β4 was inhibited with the addition of 5–50 nM soluble actin. These results demonstrate that the actin binding motif of thymosin β4 is an essential site for its angiogenic activity.

Nuclear localisation of the G-actin sequestering peptide thymosin β4

Thymosin β4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin β4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin β4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin β4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

Thymosin β4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen

The β‐thymosins constitute a family of highly conserved and extremely water‐soluble 5 kDa polypeptides. Thymosin β4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G‐actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin β4. For example, thymosin β4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin β4 can be cross‐linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin β4 is not cross‐linked to many other proteins and its cross‐linking to fibrin is competed by another family member, thymosin β10. After activation of human platelets with thrombin, thymosin β4 is released and crosslinked to fibrin in a time‐ and calcium‐dependent manner. We suggest that thymosin β4 cross‐linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin β4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.

The Thymosins: Prothymosin α, Parathymosin, and β-Thymosins: Structure and Function

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.

Thymosin β4 serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin1

Thymosin β4 possesses actin‐sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin β4 serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln‐23 and Gln‐36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln‐39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G‐actin and could be cross‐linked to G‐actin by 1‐ethyl‐3‐[3‐(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin β4 may serve as a useful tool for further investigations in cell biology. Thymosin β4 could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin β4 concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin β4 may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase‐related processes.

Plant profilin induces actin polymerization from actin:β‐thymosin complexes and competes directly with β‐thymosins and with negative co‐operativity with DNase I for binding to actin

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin β4 and β9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tβ4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tβ4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle α‐actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross‐linking indicated that birch profilin competes with Tβ4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross‐linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co‐operativity between the profilin and DNase I binding sites on actin.

C-terminal truncation of thymosin β10 by an intracellular protease and its influence on the interaction with G-actin studied by ultrafiltration

Two β‐thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4°C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI‐mass spectroscopy this peptide was identified to be a C‐terminally truncated form of thymosin β10. Having studied the formation in more detail we found that after a 4‐h thaw at 4°C all thymosin β10 was truncated to thymosin β10 1‐41, which was further degraded during the next 20 h. On the other hand, thymosin β4 Ala, the second β‐thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin β10 is formed by a carboxydipeptidase while thymosin β4 Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G‐actin and these three β‐thymosins to be 0.28, 0.72, and 0.94 μM for thymosin β4 Ala, β10, and thymosin β10 1‐41, respectively. The complex with β4 Ala is unambiguously more stable than the complex with β10 or β4 (0.81 μM). The change in the dissociation constant generated by the truncation of the two C‐terminal amino acid residues of β10 is small but statistically significant. This demonstrates that even the very last amino acid residues at the C‐terminus of β‐thymosins are involved in the interaction with G‐actin.

HPLC and post-column derivatization with fluorescamine: isolation of actin-sequestering β-thymosins by reversed-phase HPLC

Abstract Three different procedures are summarized that minimize the possibility of artificial proteolysis during the extraction of small peptides like β-thymosins from tissues or cells. The extracts are desalted and concentrated by solid phase extraction. The peptides can be further purified by isoelectric focusing in a granulated gel, chromatofocusing or conventional ion-exchange chromatography. The final step for purification as well as for quantitative analysis of extracts from tissues or cells is reversephase HPLC. We describe the set-up of a post-column derivatization system with fluorescamine in connection with reversephase or cation-exchange columns. The fluorescence detection offers high sensitivity and permits the use of organic buffering substances, which are volatile but exclude UV detection due to their strong UV absorbance. The isolated β-thymosins are in a saltfree form and thus suitable for studies on biological activities and amino acid sequencing. The procedures summarized here are also applicable to other small peptides.

Oxidation and reduction of thymosin β4 and its influence on the interaction with G-actin studied by reverse-phase HPLC and post-column derivatization with fluorescamine

Abstract Oxidation of methionine residues has been shown to provide a potential regulatory mechanism in protein function in vivo. Thymosin β 4 which forms a one-to-one complex with G-actin is the most abundant member of β-thymosins in mammalian tissues and possesses a methionine residue at position 6. In preparations of mammalian tissues thymosin β 4 is, in most cases, accompanied by a small amount of its sulfoxide. Using reverse-phase HPLC we showed that the oxidation of the methionine residue of thymosin β 4 can be achieved by millimolar concentrations of H 2 O 2 in vitro and is accompanied by an 18-fold increase in the apparent dissociation constant of its complex with G-actin. Peptides were separated by reverse-phase HPLC using a RP-18 column applying a linear gradient of n -propanol in 20 mM pyridine — 0.11 M formic acid — 0.05 M lithiumperchlorate and were detected by fluorescence after postcolumn derivatization with fluorescamine. 50% of thymosin β 4 is oxidized after 3.5 or 6 hours using 3 mM or 1 mM H 2 O 2 , respectively. In the case of 0.5 mM H 2 O 2 , about 45% of the methionine residues are oxidized after 18 hours. The resulting sulfoxide is reduced with aqueous solutions of sodiumbisulfite. The reduction is accompanied by the recovery of the initial affinity to G-actin. With a solution of 90% saturated Na 2 S 2 O 5 we find 50% reduction of the sulfoxide in about 5 hours and 80% after 12 hours while only 30% is reduced with dithiothreitol (0.81 M) after 25 hours. The large amount of sodiumbisulfite necessary for reduction can be separated from the peptide by solid phase extraction.