Beta thymosins Edit Wikipedia article

NMR structure of a β-thymosin. Both thymosin α₁ ^[1] and β-thymosins are intrinsically unstructured proteins, i.e. they lack a stable fold when free in aqueous solution. This structure, mostly alpha helix, was artificially stabilised by an organic solvent.^[2] The thymosin illustrated, originally named β₉ is the cow orthologue of human β₁₀

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.

[edit] See also

• Thymosins
• Thymosin α1

[edit] Single domain β-thymosins

[edit] Distribution

Thymosin beta-4 family
Structure of thymosin beta 9.[3]
Identifiers
Symbol	Thymosin
Pfam	PF01290
InterPro	IPR001152
SMART	SM00152
PROSITE	PDOC00433
SCOP	1hj0
SUPERFAMILY	1hj0
Available protein structures: Pfam structures PDB RCSB PDB; PDBe PDBsum structure summary

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.^[4] The single known exception is in a choanoflagellate, significantly a single-celled organism regarded as the closest living relative of animals.^[5] 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.^[6] 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.^[7]

[edit] 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).^[8][9] 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.^[10] However, sequence similarity searches designed to identify present-day WH2 domains^[11] fail to recognise β-thymosins, (and vice versa) and the sequence and functional similarities may result from convergent evolution.^[12]

[edit] 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.^[13] Following Thymosin α1, β₄ was the second of the biologically active peptides from Thymosin Fraction 5 to be completely sequenced and synthesized.^[14]

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.^[15]

[edit] 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.^[16] 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:^[17]

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.^[18]

[edit] "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.^[19] 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.^[20] 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.^[21]

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.

[edit] 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.^[22]

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,^[23] to induce migration of these cells into heart muscle^[24] and recruit new blood vessels within the muscle.^[25]

[edit] 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.^[26]

[edit] 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.^[14]

[edit] Clinical applications

Thymosin β₄ has been tested in multicenter trials sponsored jointly by RegeneRx Biopharmaceuticals Inc (Rockville, MD, USA) and Sigma Tau (Pomezia, Italy) in the United States and Europe in patients with bed sores, ulcers caused by venostasis, and Epidermolysis bullosa simplex and was found to accelerate bed sore and stasis ulcer repair by one month. The epidermolysis bullosa trial is still enrolling. It has also been tested in patients with chronic neurotrophic corneal epithelial defects and found to promote repair.

Levels of human thymosin β₁₅ in urine have shown promise as a diagnostic marker for prostate cancer which is sensitive to potential aggressiveness of the tumour ^[27]

[edit] Doping in Sports

Thymosin beta-4 was allegedly used by some players in various Australian football codes and is under investigation by the Australian Sports Anti-Doping Authority for anti-doping violations ^[28]

[edit] β-thymosin repeat proteins

[edit] Distribution

These proteins, which typically contain 2-4 repeats of the β-thymosin sequence, are found in all phyla of the animal kingdom, with the probable exception of sponges^[29] The sole mammalian example, a dimer in mice, is synthesised by transcriptional read-through between two copies of the mouse β15 gene, each of which is also transcribed separately.^[30] A uniquely multiple example is the protein thypedin of Hydra which has 27 repeats of a β-thymosin sequence.^[31]

[edit] Biological activities

β-thymosin repeat proteins resemble the monomeric forms in being able to bind to actin, but sequence differences in one example studied, a three-repeat protein Ciboulot of the fruit fly Drosophila, allow binding to ends of actin filaments, an activity which differs from monomer sequestration.^[32]

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 simple forms of learning: both one-trial enhancement of the excitability of sensory neurons in the conditioned stimulus pathway,^[33] and in multi-trial Pavlovian conditioning.^[34] The phosphorylation of Csp24, in common with post-translational modifications of a number of cytoskeleton-related proteins may contribute to actin-filament dynamics underlying structural remodeling of responsive cells.^[35]

[edit] Picture gallery

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  NMR structure of bovine β9-thymosin, the orthologue of human β10.^[2] Rainbow coloured where the N-terminus = blue and the C-terminus = red.

[edit] References