Biomedical subjects

Ewald Hannappel

Publications and source records attributed to Ewald Hannappel.

8 recordsLinked to original sources

PubMed2005

Thymosin beta4: actin-sequestering protein moonlights to repair injured tissues.

Here, we review the biochemical and molecular properties of thymosin beta(4) (Tbeta(4)), the major actin-sequestering molecule in eukaryotic cells, and its key role in dermal- and corneal-wound healing. Tbeta(4) has several, novel, potential clinical applications in the repair and remodeling of ulcerated tissues and solid organs following hypoxic injuries, such as myocardial infarction and stroke. It might also have important repair functions in the pathophysiologic sequelae that are associated with actin toxicity and with septic shock, such as respiratory distress syndrome, multi-organ failure and severe tissue trauma.

Actins↗

PubMed2004

Nuclear localisation of the G-actin sequestering peptide thymosin beta4.

Thymosin beta4 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 beta4 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 beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 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 beta4 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 beta4 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.

Actins↗

PubMed2004

Thymosin beta 4 and its N-terminal tetrapeptide, AcSDKP, inhibit proliferation, and induce dysplastic, non-apoptotic nuclei and degranulation of mast cells.

Thymosin beta4 (Tbeta4), a 5 kDa polypeptide, is a member of the beta-thymosin family. It acts as the principal intracellular G-actin sequestering peptide and exhibits extracellular functions in angiogenesis and wound healing. The N-terminus of Tbeta4 contains a bioactive tetrapeptide, acSDKP, a negative regulator of hematopoietic stem-cell proliferation. Here, we show that both peptides inhibit mast-cell proliferation over the concentration range of 10(-6) to 10(-17) M with the maximum effect of both at 10(-14) M. Both Tbeta4 and acSDKP caused dysplastic mast-cell nuclei that were confirmed by DAPI fluorescent staining. Flow-cytometric analysis of ploidy revealed that the dysplastic nuclei were not multinucleated, but fragmented nuclei in G2 growth arrest. We could further demonstrate that 10(-8) or 10(-14) M Tbeta4 or acSDKP induce mast-cell degranulation. A concentration of 10(-8) M Tbeta4 or acSDKP caused 57 or 89% degranulation, respectively. A number of tryptic fragments of Tbeta4 were assayed beside intact Tbeta4 and the tetrapeptide, and found to be inactive.

Animals↗

PubMed2003

The actin binding site on thymosin beta4 promotes angiogenesis.

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.

Actins↗

PubMed2003

The thymosins. Prothymosin alpha, parathymosin, and beta-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.

Actins↗

PubMed2003

Reduction of thymosin beta4 and actin in HL60 cells during apoptosis is preceded by a decrease of their mRNAs.

Thymosin beta4 (Tbeta4) is the most prominent representative of the beta-thymosins, a family of highly conserved polar 5 kDa peptides. This peptide is now regarded to be the main G-actin sequestering peptide in mammals and therefore plays an important role in organization of the microfilamental system. During apoptosis of cells this microfilamental system is disrupted. Therefore we studied changes in thymosin beta4 and actin content of HL60 cells after induction of apoptosis using cytosine arabinoside (araC). Thymosin beta4 content decreased to about 30% of the control value after incubation for 48 h in the presence of araC. Also the amount of total actin decreased to about half of the control, while total cellular protein remained constant. To further elucidate if the changes of thymosin beta4 and actin content correlate with the gene expression the relative mRNA content of thymosin beta4 and beta-actin was determined using the ribonuclease protection assay (RPA). Already after 24 h the relative amount of mRNA of thymosin beta4 and beta-actin was greatly reduced to 71 and 58%, respectively. Upon a 48 h araC treatment, the mRNA of these two proteins decreased to 15 and 10% compared to the control, whereas the content of total RNA and protein per cell was nearly unchanged. According to our data araC has a significant influence on the transcriptional level of thymosin beta4 and actin.

Actins↗

PubMed2002

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.

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.

Actins↗

PubMed2002

Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen.

The beta-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 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 beta4. For example, thymosin beta4 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 beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, thymosin beta10. After activation of human platelets with thrombin, thymosin beta4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. We suggest that thymosin beta4 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 beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.

Actins↗