Thymosin beta-4 explained

Thymosin beta-4 is a protein that in humans is encoded by the TMSB4X gene.^{[1] [2] [3]} Recommended INN (International Nonproprietary Name) for thymosin beta-4 is 'timbetasin', as published by the World Health Organization (WHO).^[4]

The protein consists (in humans) of 43 amino acids (sequence: SDKPDMAEI EKFDKSKLKK TETQEKNPLP SKETIEQEKQ AGES) and has a molecular weight of 4921 g/mol.^[5]

Thymosin-β₄ is a major cellular constituent in many tissues. Its intracellular concentration may reach as high as 0.5 mM.^[6] Following Thymosin α1, β₄ was the second of the biologically active peptides from Thymosin Fraction 5 to be completely sequenced and synthesized.^[7]

Function

This gene encodes an actin sequestering protein which plays a role in regulation of actin polymerization. The protein is also involved in cell proliferation, migration, and differentiation. This gene escapes X inactivation and has a homolog on chromosome Y (TMSB4Y).^[3]

Biological activities of thymosin β₄

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.^[8]

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.^[9] 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:^[10]

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.^[11]

"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.^[12] 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.^[13] 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.^[14]

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 worldwide series of on-going clinical trials of potential effectiveness of thymosin β₄ in promoting repair of wounds in skin, cornea and heart.^[15]

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,^[16] to induce migration of these cells into heart muscle^[17] and recruit new blood vessels within the muscle.^[18]

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.^[19]

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.^[7]

Clinical significance

Tβ4 has been studied in a number of clinical trials.^[20]

In phase 2 trials with patients having pressure ulcers, venous pressure ulcers, and epidermolysis bullosa, Tβ4 accelerated the rate of repair. It was also found to be safe and well tolerated.^[21]

In human clinical trials, Tβ4 improves the conditions of dry eye and neurotrophic keratopathy with effects lasting long after the end of treatment.^[22]

Doping in sports

See also: Essendon Football Club supplements saga and Drugs in sport in Australia. Thymosin beta-4 is considered a performance enhancing substance and is banned in sports by the World Anti-Doping Agency due to its effect of aiding soft tissue recovery and enabling higher training loads.^[23] It was central to two controversies in Australia in the 2010s which saw a large proportion of the playing lists from two professional football clubs – the Cronulla-Sutherland Sharks of the National Rugby League and the Essendon Football Club of the Australian Football League – found guilty of doping and suspended from playing; in both cases, the players were administered thymosin beta-4 in a program organised by sports scientist Stephen Dank.^{[24] [25] [26]}

Interactions

TMSB4X has been shown to interact with ACTA1^{[27] [28]} and ACTG1.^{[29] [30]}

See also

• Beta thymosins
• Thymosin beta-4, Y-chromosomal
• Thymosins

Further reading

• Huff T, Müller CS, Otto AM, Netzker R, Hannappel E . beta-Thymosins, small acidic peptides with multiple functions . The International Journal of Biochemistry & Cell Biology . 33 . 3 . 205–20 . Mar 2001 . 11311852 . 10.1016/S1357-2725(00)00087-X .
• Book: Bubb MR . Thymosin beta 4 interactions . 66 . 297–316 . 2003 . 12852258 . 10.1016/S0083-6729(03)01008-2 . Vitamins & Hormones . 9780127098661 .
• Goldschmidt-Clermont PJ, Furman MI, Wachsstock D, Safer D, Nachmias VT, Pollard TD . The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells . Molecular Biology of the Cell . 3 . 9 . 1015–24 . Sep 1992 . 1330091 . 275662 . 10.1091/mbc.3.9.1015 .
• Sanders MC, Goldstein AL, Wang YL . Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells . Proceedings of the National Academy of Sciences of the United States of America . 89 . 10 . 4678–82 . May 1992 . 1584803 . 49146 . 10.1073/pnas.89.10.4678 . 1992PNAS...89.4678S . free .
• Safer D, Elzinga M, Nachmias VT . Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable . The Journal of Biological Chemistry . 266 . 7 . 4029–32 . Mar 1991 . 10.1016/S0021-9258(20)64278-8 . 1999398 . free .
• Clauss IM, Wathelet MG, Szpirer J, Islam MQ, Levan G, Szpirer C, Huez GA . Human thymosin-beta 4/6-26 gene is part of a multigene family composed of seven members located on seven different chromosomes . Genomics . 9 . 1 . 174–80 . Jan 1991 . 2004759 . 10.1016/0888-7543(91)90236-8 .
• Soma G, Murata M, Kitahara N, Gatanaga T, Shibai H, Morioka H, Andoh T . Detection of a countertranscript in promyelocytic leukemia cells HL60 during early differentiation by TPA . Biochemical and Biophysical Research Communications . 132 . 1 . 100–9 . Oct 1985 . 2998351 . 10.1016/0006-291X(85)90994-5 .
• Gondo H, Kudo J, White JW, Barr C, Selvanayagam P, Saunders GF . Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes . Journal of Immunology . 139 . 11 . 3840–8 . Dec 1987 . 10.4049/jimmunol.139.11.3840 . 3500230 . 21154742 .
• Friedman RL, Manly SP, McMahon M, Kerr IM, Stark GR . Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells . Cell . 38 . 3 . 745–55 . Oct 1984 . 6548414 . 10.1016/0092-8674(84)90270-8 . 37810920 .
• Erickson-Viitanen S, Ruggieri S, Natalini P, Horecker BL . Distribution of thymosin beta 4 in vertebrate classes . Archives of Biochemistry and Biophysics . 221 . 2 . 570–6 . Mar 1983 . 6838210 . 10.1016/0003-9861(83)90177-7 .
• Pantaloni D, Carlier MF . How profilin promotes actin filament assembly in the presence of thymosin beta 4 . Cell . 75 . 5 . 1007–14 . Dec 1993 . 8252614 . 10.1016/0092-8674(93)90544-Z . free .
• Van Troys M, Dewitte D, Goethals M, Carlier MF, Vandekerckhove J, Ampe C . The actin binding site of thymosin beta 4 mapped by mutational analysis . The EMBO Journal . 15 . 2 . 201–10 . Jan 1996 . 8617195 . 449934 . 10.1002/j.1460-2075.1996.tb00350.x.
• Feinberg J, Heitz F, Benyamin Y, Roustan C . The N-terminal sequences (5-20) of thymosin beta 4 binds to monomeric actin in an alpha-helical conformation . Biochemical and Biophysical Research Communications . 222 . 1 . 127–32 . May 1996 . 8630056 . 10.1006/bbrc.1996.0709 .
• Safer D, Sosnick TR, Elzinga M . Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends . Biochemistry . 36 . 19 . 5806–16 . May 1997 . 9153421 . 10.1021/bi970185v .
• Malinda KM, Goldstein AL, Kleinman HK . Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells . FASEB Journal . 11 . 6 . 474–81 . May 1997 . 9194528 . 10.1096/fasebj.11.6.9194528. free . 39409025 .
• Chen J, Peterson RT, Schreiber SL . Alpha 4 associates with protein phosphatases 2A, 4, and 6 . Biochemical and Biophysical Research Communications . 247 . 3 . 827–32 . Jun 1998 . 9647778 . 10.1006/bbrc.1998.8792 .
• Huff T, Ballweber E, Humeny A, Bonk T, Becker C, Müller CS, Mannherz HG, Hannappel E . Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin . FEBS Letters . 464 . 1–2 . 14–20 . Dec 1999 . 10611475 . 10.1016/S0014-5793(99)01670-1 . free .
• De La Cruz EM, Ostap EM, Brundage RA, Reddy KS, Sweeney HL, Safer D . Thymosin-beta(4) changes the conformation and dynamics of actin monomers . Biophysical Journal . 78 . 5 . 2516–27 . May 2000 . 10777749 . 1300842 . 10.1016/S0006-3495(00)76797-X . 2000BpJ....78.2516D .

Notes and References

1. Gómez-Márquez J, Dosil M, Segade F, Bustelo XR, Pichel JG, Dominguez F, Freire M . Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes . Journal of Immunology . 143 . 8 . 2740–4 . Oct 1989 . 10.4049/jimmunol.143.8.2740 . 2677145 .
2. Lahn BT, Page DC . Functional coherence of the human Y chromosome . Science . 278 . 5338 . 675–80 . Oct 1997 . 9381176 . 10.1126/science.278.5338.675 . 1997Sci...278..675L .
3. Web site: Entrez Gene: TMSB4X thymosin, beta 4, X-linked.
4. Web site: Lists of Recommended and Proposed INNs: List 80. https://web.archive.org/web/20080914212821/http://www.who.int/medicines/publications/druginformation/innlists/en/. dead. September 14, 2008. 2018.
5. Web site: protein NP_066932 . NCBI .
6. Hannappel E . beta-Thymosins . Annals of the New York Academy of Sciences . 1112 . 1. 21–37 . September 2007 . 17468232 . 10.1196/annals.1415.018 . 2007NYASA1112...21H . 222082792 .
7. Low TL, Hu SK, Goldstein AL . Complete amino acid sequence of bovine thymosin beta 4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations . Proceedings of the National Academy of Sciences of the United States of America . 78 . 2 . 1162–6 . February 1981 . 6940133 . 319967 . 10.1073/pnas.78.2.1162 . 1981PNAS...78.1162L . free .
8. Banerjee I, Zhang J, Moore-Morris T, Lange S, Shen T, Dalton ND, Gu Y, Peterson KL, Evans SM, Chen J . Thymosin beta 4 is dispensable for murine cardiac development and function . Circ Res . 110 . 3 . 456–64 . Feb 2012 . 22158707 . 3739283 . 10.1161/CIRCRESAHA.111.258616 .
9. Safer D, Elzinga M, Nachmias VT . Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable . J. Biol. Chem. . 266 . 7 . 4029–32 . March 1991 . 10.1016/S0021-9258(20)64278-8 . 1999398 . free .
10. Book: Lodish, Harvey F. . Molecular cell biology . W.H. Freeman . San Francisco . 2000 . 978-0-7167-3706-3 . Chapter 18. Cell Motility and Shape I: Microfilaments. 18.2. The Dynamics of Actin Assembly . https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.section.5145 . registration .
11. Xue B, Aguda AH, Robinson RC . Models of the actin-bound forms of the beta-thymosins . Annals of the New York Academy of Sciences . 1112 . 1. 56–66 . September 2007 . 17468228 . 10.1196/annals.1415.010 . 2007NYASA1112...56X . 26966098 .
12. Jeffery CJ . Moonlighting proteins . Trends Biochem. Sci. . 24 . 1 . 8–11 . January 1999 . 10087914 . 10.1016/S0968-0004(98)01335-8 .
13. Tompa P, Szász C, Buday L . Structural disorder throws new light on moonlighting . Trends Biochem. Sci. . 30 . 9 . 484–9 . September 2005 . 16054818 . 10.1016/j.tibs.2005.07.008 .
14. Freeman KW, Bowman BR, Zetter BR . Regenerative protein thymosin -4 is a novel regulator of purinergic signaling . FASEB J . 25 . 3 . 907–15 . November 2010 . 21106936 . 10.1096/fj.10-169417 . free . 1684588 .
15. Philp D, Kleinman HK . Animal studies with thymosin beta, a multifunctional tissue repair and regeneration peptide . Annals of the New York Academy of Sciences . 1194 . 1. 81–6 . April 2010 . 20536453 . 10.1111/j.1749-6632.2010.05479.x . 2010NYASA1194...81P . 19780581 .
16. Smart N, Bollini S, Dubé KN, Vieira JM, Zhou B, Davidson S, Yellon D, Riegler J, Price AN, Lythgoe MF, Pu WT, Riley PR . De novo cardiomyocytes from within the activated adult heart after injury . Nature . 474 . 7353 . 640–4 . June 2011 . 21654746 . 3696525 . 10.1038/nature10188 .
17. Book: Smart N, Riley PR . Current Protocols in Stem Cell Biology . Derivation of epicardium-derived progenitor cells (EPDCs) from adult epicardium . Curr Protoc Stem Cell Biol . Chapter 2 . Unit2C.2 . February 2009 . 8 . 19235142 . 10.1002/9780470151808.sc02c02s8 . 978-0470151808 .
18. Riley PR, Smart N . Thymosin beta4 induces epicardium-derived neovascularization in the adult heart . Biochem. Soc. Trans. . 37 . Pt 6 . 1218–20 . December 2009 . 19909250 . 10.1042/BST0371218 .
19. Young JD, Lawrence AJ, MacLean AG, Leung BP, McInnes IB, Canas B, Pappin DJ, Stevenson RD . Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids . Nature Medicine . 5 . 12 . 1424–7 . December 1999 . 10581087 . 10.1038/71002 . 19680965 .
20. Crockford D, Turjman N, Allan C, Angel J . Thymosin beta4: structure, function, and biological properties supporting current and future clinical applications . Annals of the New York Academy of Sciences . 1194 . 1. 179–89 . April 2010 . 20536467 . 10.1111/j.1749-6632.2010.05492.x . 2010NYASA1194..179C . 29360082 .
21. Book: Kleinman HK, Sosne G . Thymosins . Thymosin β4 Promotes Dermal Healing . Vitamins and Hormones . 102 . 251–75 . 2016 . 27450738 . 10.1016/bs.vh.2016.04.005 . review . 9780128048184 .
22. Sosne G, Kleinman HK . Primary Mechanisms of Thymosin β4 Repair Activity in Dry Eye Disorders and Other Tissue Injuries . Investigative Ophthalmology & Visual Science . 56 . 9 . 5110–7 . August 2015 . 26241398 . 10.1167/iovs.15-16890 . free .
23. Web site: CAS 2015/A/4059 World Anti-Doping Agency v. Thomas Bellchambers et al., Australian Football League, Australian Sports Anti-Doping Authority. 2016. 20 March 2016. 4 March 2016. https://web.archive.org/web/20160304094609/http://www.tas-cas.org/fileadmin/user_upload/Arbitral_Award_WADA_ESSENDON.pdf. dead.
24. Web site: Cronulla Sharks and thymosin beta-4 ... is it doping? . Benjamin . Koh . The Conversation . 7 March 2013 .
25. Ho EN, Kwok WH, Lau MY, Wong AS, Wan TS, Lam KK, Schiff PJ, Stewart BD . Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry . Journal of Chromatography A . 1265 . 57–69 . Nov 2012 . 23084823 . 10.1016/j.chroma.2012.09.043 .
26. News: Essendon supplements saga: ASADA backs Court of Arbitration for Sport decision to upheld WADA appeal . ABC News (Australian Broadcasting Corporation) . 11 January 2016 .
27. Ballweber E, Hannappel E, Huff T, Stephan H, Haener M, Taschner N, Stoffler D, Aebi U, Mannherz HG . 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 . Journal of Molecular Biology . 315 . 4 . 613–25 . Jan 2002 . 11812134 . 10.1006/jmbi.2001.5281 .
28. Safer D, Sosnick TR, Elzinga M . Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends . Biochemistry . 36 . 19 . 5806–16 . May 1997 . 9153421 . 10.1021/bi970185v .
29. Hertzog M, van Heijenoort C, Didry D, Gaudier M, Coutant J, Gigant B, Didelot G, Préat T, Knossow M, Guittet E, Carlier MF . The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly . Cell . 117 . 5 . 611–23 . May 2004 . 15163409 . 10.1016/S0092-8674(04)00403-9 . free .
30. Van Troys M, Dewitte D, Goethals M, Carlier MF, Vandekerckhove J, Ampe C . The actin binding site of thymosin beta 4 mapped by mutational analysis . The EMBO Journal . 15 . 2 . 201–10 . Jan 1996 . 8617195 . 449934 . 10.1002/j.1460-2075.1996.tb00350.x .