Thymosin beta-4

Any concepts of the biological role of thymosin β₄ must inevitably be coloured by the demonstration that total ablation of the thymosin β₄ gene in the mouse allows apparently normal embryonic development of mice which are fertile as adults.[10]

Thymosin β₄ was initially perceived as a thymic hormone. However this changed when it was discovered that it forms a 1:1 complex with G (globular) actin, and is present at high concentration in a wide range of mammalian cell types.[11] When appropriate, G-actin monomers polymerize to form F (filamentous) actin, which, together with other proteins that bind to actin, comprise cellular microfilaments. Formation by G-actin of the complex with β-thymosin (= "sequestration") opposes this.

Due to its profusion in the cytosol and its ability to bind G-actin but not F-actin, thymosin β₄ is regarded as the principal actin-sequestering protein in many cell types. Thymosin β₄ functions like a buffer for monomeric actin as represented in the following reaction:[12]

F-actin ↔ G-actin + Thymosin β₄ ↔ G-actin/Thymosin β₄

Release of G-actin monomers from thymosin β₄ occurs as part of the mechanism that drives actin polymerization in the normal function of the cytoskeleton in cell morphology and cell motility.

The sequence LKKTET, which starts at residue 17 of the 43-aminoacid sequence of thymosin beta-4, and is strongly conserved between all β-thymosins, together with a similar sequence in WH2 domains, is frequently referred to as "the actin-binding motif" of these proteins, although modelling based on X-ray crystallography has shown that essentially the entire length of the β-thymosin sequence interacts with actin in the actin-thymosin complex.[13]

In addition to its intracellular role as the major actin-sequestering molecule in cells of many multicellular animals, thymosin β₄ shows a remarkably diverse range of effects when present in the fluid surrounding animal tissue cells. Taken together, these effects suggest that thymosin has a general role in tissue regeneration. This has suggested a variety of possible therapeutic applications, and several have now been extended to animal models and human clinical trials.

It is considered unlikely that thymosin β₄ exerts all these effects via intracellular sequestration of G-actin. This would require its uptake by cells, and moreover, in most cases the cells affected already have substantial intracellular concentrations.

The diverse activities related to tissue repair may depend on interactions with receptors quite distinct from actin and possessing extracellular ligand-binding domains. Such multi-tasking by, or "partner promiscuity" of, proteins has been referred to as protein moonlighting.[14] Proteins such as thymosins which lack stable folded structure in aqueous solution, are known as intrinsically unstructured proteins (IUPs). Because IUPs acquire specific folded structures only on binding to their partner proteins, they offer special possibilities for interaction with multiple partners.[15] A candidate extracellular receptor of high affinity for thymosin β₄ is the β subunit of cell surface-located ATP synthase, which would allow extracellular thymosin to signal via a purinergic receptor.[16]

Some of the multiple activities of thymosin β₄ unrelated to actin may be mediated by a tetrapeptide enzymically cleaved from its N-terminus, N-acetyl-ser-asp-lys-pro, brand names Seraspenide or Goralatide, best known as an inhibitor of the proliferation of haematopoietic (blood-cell precursor) stem cells of bone marrow.

Work with cell cultures and experiments with animals have shown that administration of thymosin β₄ can promote migration of cells, formation of blood vessels, maturation of stem cells, survival of various cell types and lowering of the production of pro-inflammatory cytokines. These multiple properties have provided the impetus for a worldwide series of on-going clinical trials of potential effectiveness of thymosin β₄ in promoting repair of wounds in skin, cornea and heart.[17]

Such tissue-regenerating properties of thymosin β₄ may ultimately contribute to repair of human heart muscle damaged by heart disease and heart attack. In mice, administration of thymosin β₄ has been shown to stimulate formation of new heart muscle cells from otherwise inactive precursor cells present in the outer lining of adult hearts,[18] to induce migration of these cells into heart muscle[19] and recruit new blood vessels within the muscle.[20]

In 1999 researchers in Glasgow University found that an oxidised derivative of thymosin β₄ (the sulfoxide, in which an oxygen atom is added to the methionine near the N-terminus) exerted several potentially anti-inflammatory effects on neutrophil leucocytes. It promoted their dispersion from a focus, inhibited their response to a small peptide (F-Met-Leu-Phe) which attracts them to sites of bacterial infection and lowered their adhesion to endothelial cells. (Adhesion to endothelial cells of blood vessel walls is pre-requisite for these cells to leave the bloodstream and invade infected tissue). A possible anti-inflammatory role for the β₄ sulfoxide was supported by the group's finding that it counteracted artificially-induced inflammation in mice.

The group had first identified the thymosin sulfoxide as an active factor in culture fluid of cells responding to treatment with a steroid hormone, suggesting that its formation might form part of the mechanism by which steroids exert anti-inflammatory effects. Extracellular thymosin β₄ would be readily oxidised to the sulfoxide in vivo at sites of inflammation, by the respiratory burst.[21]

Thymosin β₄ induces the activity of the enzyme terminal deoxynucleotidyl transferase in populations of thymocytes (thymus-derived lymphocytes). This suggests that the peptide may contribute to the maturation of these cells.[9]