TB4 belongs to a group of peptides or family of peptides called thymosins. Thymosins modulate cell migration (normally cells are fixed with no motility), angiogenesis (growing of blood vessels from other blood vessels) and immune responses.4 They have been categorized as biological response modifiers. Usually the term refers to substances that arouse the body’s response to an infection. TB4 specifically is a key regulator of tissue regeneration as it arouses an immune response which leads to regeneration.
This sort of activity is a bit different then another member of the family TB1. TB1 is an immunomodulatory peptide used in patents infected with hepatitus B or C virus as an immune booster (adjuvant) and in melanoma patients combined with chemotherapy. We need to always be careful with adjuvants as there are indications that an adjuvant in the anthrax vaccine is responsible for the autoimmune diseases that have occurred in some solidiers receiving that vaccine. The thought is that squalene was used as an adjuvant to boost the immune system to make antibodies to anthrax when introduced in the vaccine because it became clear that the body wasn’t going to do that in response to a non-adjuvant vaccine. Squalene is naturally present in the body. In some that were given the anthrax vaccine the body developed a response that attacked their own tissue that contained squalene. In other words they developed antibodies to squalene. I understand that I have taken a step away from the topic of this article.
Let’s step to the side again. TB4 is a small acidic amino peptide made up of 43 amino acids with an isoelectric point of 5.1. So what’s an isoelectric point? Well we could alomost say pH and leave it at that. pH is something we are familiar with as it measure the acidity of something. 7 is netral and less then seven is acidic while values above 7 to 14 are basic. Isoelectric charge is the pH value at which molecules carry no electrical charge. TB4 at pH 5.1 carries no electrical charge. Amino acids that make up a peptide are positive, negative or neutral or polar in nature. The collective nature of this group will give a protein its overall charge. In TB4′s case the mixed bag of 43 amino acids that make up the peptide have different characteristics that sum up. Together these characteristics are responsible for TB4‘s isoelectric point of 5.1. You can see that TB4 is acidic. Isoelectric point becomes important when you put a peptide in a particular environment. In general if a protein or peptide is put in an environment with a pH velow their isoelectric point they carry a net positive (+) charge. In environments above their isoelectric points they carry a net negative charge.
As an example, the natural skin surface has a pH below 5. So TB4 if placed on the skin surface will carry a positive charge. In order to drive it into the first layer of skin with a current (such as an iontophoresis machine) you will need to repell it from the electrode which would need to be set to deliver a negative charge.
To reiterate, the net charge on the peptide is affected by the pH of its surrounding environment and can become more positively or negatively charged due to the loss or gain of protons. This loss or gain of protons can over time lead to dgradation of the peptide depending on the extent of proton exchange.
Back to TB4
* - In general much of the following derives from, Stem Cell Regulators, edited by Gerald Litwack, Academic Press, Nov 23, 2011 and Thymosin b4: actin-sequestering protein moonlights to repair injured tissues, Allan L. Goldstein, TRENDS in Molecular Medicine Vol.11 No.9 September 2005
TB4 is the most abundant actin-sequestering protein in human platelets. Actin functions to form microfilaments which provide mechanical support to cells, they can act as scaffold for myosin protein generated muscle contractions and allow for cell motility. Individual subunits (or single units) of microfilaments are called globular actin (G-actin). G-actin subunits assemble into long filamentous polymers (multi-units) called F-actin. Two parallel F-actin strands rotate and layer on top of each other thereby creating the double helix structure of the microfilaments of the cytoskeleton.
TB4 involves itself in this actin filament growth process. It binds to those single subunits called G-actin and acts as a buffer as more subunits are added to form a multi-unit long chain called F-actin. Before this making of long-chained F-actin can get going a protein called profilin also binds to G-actin with a role of exchanging ADP for ATP.
The growth of these long-chained actin filaments is regulated by thymosin and profilin. Thymosin binds to G-actin to buffer the polymerizing (growing) process, while profilin binds to G-actin and brings about the actin-bound exchange of ADP for the energy molecule ATP. This energizes these G-actin subunits. Basically TB4 as a buffer and profilin as a catalyst convert ADP G-actins which normally grow poorly to ATP G-actins which are ready to bind and grow into long chain F-actin structures. They add energy to the process. They bring about cellular movement and cell shape change.
TB4 must have evolved from another protein. It didn’t just happen. Nature seemed to deem it vital. The N-terminal half of the TB4 peptide is very similiar to another peptide called Wasp Homology Domain 2 (WH2) whose function is also the binding of actin. Since TB4 binds to G-actin through the LKKTET sequence of amino acids that make up it’s peptide structure which is similiar to WH2′s actin binding structure it is possible that TB4 evolved from WH2 by acquiring an addition of C-terminal sequence. This is not specifically germane to the topic of potential usage of TB4 but it is interesting to know that TB4 may have an ancesteral peptide. I am bringing this up mostly so you can see which amino acid sequence in TB4′s 43 aamino acid structure is responsible for binding actin. It is the LKKTET portion.
In water TB4 is unstructured (closed umbrella) and folds (activates) when it binds to G-actin.5 In addition TB4 forms different secondary structures (which are distinct from the actin binding shape it takes) upon interaction with other proteins. This fits the proper definition of a moonlighting peptide. In fact if the analogy were proper I wouldn’t use the open/closed umbrella analogy (which works for autocrine receptor-GH complexes). Instead the analogy might be the Bat Mobile (Batman’s shape changing multi-functional ride.) This oligomer morphing is what is responsible in part for TB4′s multi-taking.
As a very important example of TB4 morphing we have the methionine at amino acid position six. This amino acid is easily oxidized and in monocytes (type of immune cell) the resulting TB4 sulphoxized functions to inhibit the chemotactic response of neutrophils (cells that make up part of the first line of defense in immunity). Chemotaxis is basically purposeful cellular movement.6So TB4 and the sulfoxide version will prevent movement. However if we move TB4 or TB4 sulfoxide into myoblasts both will stimulate migratory response. 7 In different tissue we see the same peptide TB4 and it’s sulphoxized form acting in a different manner by promoting the migration of myoblasts to facilitate skeletal muscle regeneration.
Location or “where does TB4 act” as well as the shape of the peptide determine the function performed.
If we take a look at the full 43 amino acid structure of TB4 we will find that the front end amino acid sequence is functional and in fact “lives” alone and becomes it’s own peptide. The peptide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) is cleaved/released from the N-terminus of the TB4 peptide by the action of an endopeptidase. This peptide (Ac-SDKP)is plentiful in serum and other body fluids. One of it’s actions is inhibition of hematopoietic stem cell proliferation. Hematopoietic stem cells give rise to all the blood cell types. Another of it’s actions appears to be endothelial cell (line vessels and lymph) migration and decreased inflammation. Outside the cell, concentrations of this Ac-SDKP cleaved peptide are regulated through degradation by an angiotensin-converting enzyme.
Here is a visual of the structural description.
Role in Skeletal Muscle Regeneration
Skeletal muscle repair is an ongoing daily event and only varies by degree. During regenration various proteins and cytokins (signaling molecules whether they take a peptide structure or not) are produced by regenerating muscle fibers and surrounding immune cells which stimulate the cell cycle entry of satelite cells (proliferation), the recruitment and differentiation of myoblasts and myotube formation and fusion.
Expression of TB4 mRNA (messenger RNA) is upregulated in injured skeletal muscle compared to health uninjured muscle. The appearance of mRNA often indicates the making of that protein. The expression of mRNA for TB4 indicates that TB4 is created. The cells responsible for the creation of TB4 are regenerating muscle fibers and inflammatory hematopoietic cells. Macrophages and T cells infiltrate the damaged area and secrete a number of cytokins including TB4. The specifics of secretion are not yet known but are belived to come from damaged permeable muscle tissue at the beginning of the repair process.
TB4 and sulfoxide TB4 promote migration and mobilization of myoblasts and myocytes into regenrating skeletal muscles.
These represent proteins that are important partners in the cell-migration and anti-inflammatory activities of TB4.
Extracellularly TB4′s actions must be mediated by cell surface receptors and two possibilities have been proposed.
- adenosine receptors8
- F1-F0 ATP synthase9
Freeman in the F1-F0 ATP synthase study found a unique signal transduction mechanism with ATP as a second messenger. In this model TB4 binds to F1-F0 ATP synthase on the plasma membrane to inhibit its ATP hydrolysis (degradation) activity thereby increasing the cell surface ATP concentration. Increased ATP levels then stimulated the purinergic receptor P2X4 to bring about cell migration of umbilical vein endothelial cells.
Extracellular TB4 seems to induce cell migration while transient increases in intracellular cytoplasmic TB4 levels are anti-migratory. TB4 extracellularly is a regulator of wound healing and a potent regenerative peptides in a variety of tissues. Extracellular administration of TB4 improves corneal healing, dermal wound healing and cardiac repair after ischemic injury.
Whether TB4′s extracellular actions come solely through the receptor mechanism identified by Freeman is unclear at this time. The Freeman study summarized some of these healing effects as follows