Thymosin

Thymosin beta-4 (aliases: Thymosin β4, Tβ4, β-Thymosin, TMSB4X, FX, PTMB4, TB4X, TMSB4) is a protein that in humans is encoded by the TMSB4X gene (there is also a Y-chromosome copy: TMSB4Y). Thymosin β4 has a molecular weight of 4921 g/mol and the protein consists of 43 amino acids:

• MSDKPDMAEI EKFDKSKLKK TETQEKNPLP SKETIEQEKQ AGES

Thymosin β₄ is a member of the Beta thymosin family of proteins which have in common a sequence of about 40 amino acids (see: Beta Thymosins). Found almost exclusively in multicellular animals, Thymosin β₄ was originally obtained from the thymus, along with in company with several other small proteins.

See also: Actin

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. 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.

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:

Factin ↔ Gactin + Thymosinβ4 ↔ Gactin:Thymosinβ4

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-amino acid 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.

Articles

van Kesteren, et al., Smit • 2006 • JNeuro • FullText

Local Synthesis of Actin-Binding Protein β-Thymosin Regulates Neurite Outgrowth.

Local protein synthesis plays an essential role in the regulation of various aspects of axonal and dendritic function in adult neurons. At present, however, there is no direct evidence that local protein translation is functionally contributing to neuronal outgrowth. Here, we identified the mRNA encoding the actin-binding protein β-thymosin as one of the most abundant transcripts in neurites of outgrowing neurons in culture. β-Thymosin mRNA is not evenly distributed in neurites, but appears to accumulate at distinct sites such as turning points and growth cones. Using double-stranded RNA knockdown, we show that reducing β-thymosin mRNA levels results in a significant increase in neurite outgrowth, both in neurites of intact cells and in isolated neurites. Together, our data demonstrate that local synthesis of β-thymosin is functionally involved in regulating neuronal outgrowth.

Roy, et al. Jacobson • 2001 • J Cell Biol • FullText

Local Photorelease of Caged Thymosin β4 in Locomoting Keratocytes Causes Cell Turning.

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin β4 (Tβ4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tβ4 was produced in an inactive form by “caging” with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tβ4 did not change the normal actin polymerization kinetics, whereas photoactivated Tβ4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tβ4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tβ4 and the consequent reduction in the diffusional spread of the Tβ4–actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tβ4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tβ4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tβ4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tβ4.

Honkura, Matsuzaki, Noguchi, Ellis-Davies, Kasai • 2008 • Cell • FullText

The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines.

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Fischer, Kaech, Knutti, Matus • 1998 • Cell • FullText

Rapid actin-based plasticity in dendritic spines.

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Lang • 2004 • PNAS • FullText

Transient expansion of synaptically connected dendritic spines upon induction of hippocampal long-term potentiation.

We find that induction of long-term potentiation (LTP) of synaptic transmission in acute hippocampal slices of adult mice evokes a reliable, transient expansion in spines that are synaptically activated, as determined with calcium imaging. Similar to LTP, transient spine expansion requires N-methyl- D-aspartate (NMDA) receptor-mediated Ca2 influx and actin polymerization. Moreover, like the early phase of LTP induced by the stimulation protocol, spine expansion does not require Ca2 influx through L-type voltage-gated Ca2 channels nor does it require protein synthesis. Thus, transient spine expansion is a characteristic feature of the initial phases of plasticity at mature synapses and so may contribute to synapse remodeling important for LTP.

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Fujiwara, Vavylonis, Pollard • 2007 • PNAS • FullText

Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy

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Koskinen and Hotulainen • 2014 • Frontiers • FullText

Measuring F-actin properties in dendritic spines

Measurements of actin turnover in dendritic spines: Fitting the data from individual measurements resulted in a mean stable component size of 18% as well as mean time constants of 51 sec for the dynamic component and 840 sec for the stable component.

Bindschadler, Osborn, Dewey, McGrath • 2004 • BiophysicalJ • PMC

A Mechanistic Model of the Actin Cycle

Actin polymerization proceeds until only a small concentration (~0.1 µM) of unpolymerized actin (Gactin) remains. This ‘‘critical concentration’’ is also the minimum concentration required to form filaments (F-actin).

Both regulated and unregulated actin binding proteins modify the actin cycle in cells (Fig. 1). Barbed-end binding proteins block the assembly of G-actin at filament-barbed ends. The most abundant barbed-end binding proteins, capping protein (CP) and gelsolin (Isenberg et al., 1980; Yin et al., 1981), are inactivated by PIP2 and other polyphosphoinositides (Heiss and Cooper, 1991; Janmey and Stossel, 1987). Gelsolin, which also severs actin filaments (Yin and Stossel, 1979), requires micromolar calcium for its activity. CP, gelsolin, and Arp2/3 complex (Mullins et al., 1998), can nucleate new actin filaments. The processes of severing and nucleation help determine the number and length of actin filaments. Arp2/3 complex can also cap pointed ends (Mullins et al., 1998). Arp2/ 3 complex activities are greatly enhanced by the GTPase binding protein N-WASp (Machesky et al., 1999; Yarar et al., 1999). Inhibited by phosphorylation (Morgan et al., 1993), the ADF/cofilin family proteins bind preferentially to ADP containing subunits (Carlier et al., 1997). Cofilin destabilizes filaments by severing them (Maciver et al., 1991), by accelerating the rate of ADP subunit disassembly (Carlier et al., 1997), and by enhancing the rate of Pi release (Blanchoin and Pollard, 1999). Unregulated proteins of the b4-thymosin family bind actin monomers to maintain unpolymerized actin at hundreds of times the critical concentration (Safer et al., 1990). Unlike b4-thymosin, the monomer binding protein profilin has catalytic functions. Profilin accelerates the exchange of ADP for ATP on actin monomer 140-fold (Selden et al., 1999). Also unlike actin complexed with b4-thymosin, profilin-bound G-actin assembles at barbed ends but not pointed ends (Pollard and Cooper, 1984), releasing unbound profilin (Pantaloni and Carlier, 1993).