Beta thymosins

Beta thymosins are a family of proteins which have in common a sequence of about 40 amino acids similar to the small protein thymosin β4. They are found almost exclusively in multicellular animals. Thymosin β4 was originally obtained from the thymus  in company with several other  small proteins which although named collectively  "thymosins" are now known to be structurally and genetically unrelated and present in many different animal tissues.

Distribution
Monomeric β-thymosins, i.e. those of molecular weight similar to those originally isolated from thymus by Goldstein, are found almost exclusively in cells of multicellular animals. The single known exception is in a choanoflagellate, significantly a single-celled organism regarded as the closest living relative of animals. Although found in very early diverged animals such as sponges, monomeric thymosins  are absent from arthropods and nematodes, which do nevertheless possess proteins, known as  β-thymosin repeat proteins, which are constructed from several end-to-end repeats of  β-thymosin sequences. Genomics has shown that tetrapods (land vertebrates) each express three monomeric β-thymosins, which are the animal species' equivalents (orthologues) of human β4, β10 and β15 thymosins, respectively. The human thymosins are encoded by the genes TMSB4X, TMSB10 and TMSB15A and TMSB15B. (In humans, the proteins encoded by the two TMSB15 genes are identical.) Bony fish in general express orthologues of these same three, plus an additional copy of the β4 orthologue.

Relation to the WH2 sequence module
The N-terminal half of β-thymosins bears a strong similarity in amino acid sequence to a very widely distributed sequence module, the WH2 module. (Wasp Homology Domain 2 - the name is derived from Wiskott-Aldrich syndrome protein). Evidence from X-ray crystallography shows that this part of β-thymosins binds to actin in a near-identical manner to that of WH2 modules, both adopting as they bind, a conformation which has been referred to as the β-thymosin/WH2 fold. β-thymosins may therefore have evolved by addition of novel C-terminal sequence to an ancestral WH2 module. However, sequence similarity searches designed to identify present-day WH2 domains fail to recognise β-thymosins, (and vice versa) and the sequence and functional similarities may result from convergent evolution.

Biological activities of thymosin β4
The archetypical β-thymosin is β4 (product in humans of the TMSB4X gene), which is a major cellular constituent in many tissues. Its intracellular concentration may reach as high as 0.5 mM. Following Thymosin α1, β4  was the second of the biologically active peptides from Thymosin Fraction 5 to be completely sequenced and synthesized.

Actin binding
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. 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:

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.

"Moonlighting"
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. 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. 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.

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.

Tissue regeneration
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 world-wide series of on-going clinical trials of potential effectiveness of thymosin β4 in promoting repair of wounds in skin, cornea and heart.

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, to induce migration of these cells into heart muscle and recruit new blood vessels within the muscle.

Anti-inflammatory role for sulfoxide
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.

Terminal deoxynucleotidyl transferase
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.

Clinical applications
Thymosin β4 is currently in multicenter trials in the United States and Europe in patients with bed sores, ulcers caused by venostasis, and Epidermolysis bullosa simplex. It is also about to enter clinical trials in diabetic patients who have been subjected to vitrectomy and in patients who have experienced heart attacks (myocardial infarction).

Levels of human thymosin β15 in urine have shown promise as a diagnostic marker for prostate cancer which is sensitive to potential aggressivenes of the tumour

Distribution
These proteins, which typically contain 2-4 repeats of the β-thymosin sequence, are chiefly found within lower metazoan species such as dipteran flies and nematodes. The one mammalian exception, a dimer in mouse, is synthesised by transcriptional read-through between two copies of the mouse β15 gene, each of which is also transcribed separately. A uniquely multiple example is the protein thypedin of Hydra which has 27 repeats of a β-thymosin sequence.

Biological activities
β-thymosin repeat proteins resemble the monomeric forms in being able to bind to actin, but sequence differences in a studied example, the three-repeat protein Ciboulot of the fruit fly Drosophila, allow binding to ends of actin filaments, an activity which differs from monomer sequestration.

These proteins became of interest in neurobiology with the finding that in the nudibranch (sea slug) Hermissenda crassicornis, the protein Csp24 (conditioned stimulus pathway phosphoprotein-24), with 4 repeats, is involved in one-trial enhancement of the excitability of sensory neurons in the conditioned stimulus pathway, a simple form of learning.