Thymosin ß4 a “moonlighting protein” 1
Thymosin ß4 (TB4) is a “moonlighting protein” 1 . Simply put it is multi-functional. While that may seem to be a concept that is simple to grasp, after-all multi-functional simply means it does a bunch of different things, in reality, in the context of biology to label a peptide a “moonlighting protein” adds complexity to our understanding of when, how and to what extent that peptide act. As an analogy, in our world a fork residing in the hand of the hungry will function as a tool for getting food into mouth. Remove that fork from the kitchen and put it in the hand of someone in a life threatening situation and it becomes a weapon. Whether that fork-weapon is used to inflict damage or defend against it depends on the tissue that holds it. In other words the multi-fuction of the fork never depends on the fork itself. The function always depends on where it resides and the hand that holds it, be it a hungry hand, angry hand or a scared hand.
If we move into the world of the cell and look at the concept of moonlighting proteins we’ll bump into one such protein called phosphoglucose isomerase. Most texts will tell you that phosphoglucose isomerase is the enzyme responsible for the second step of glycolysis (degradation of glucose into pyruvate) and is involved in glucogenesis (formation of glucose through the breakdown of glycogen). However the proper way to understand it or rather the moonlighting way is to understand that it’s behavior is permissive not absolute. Phosphoglucose isomerase is a protein that can be a key enzyme in glycolysis but when found moonlighting outside the cell may act as a nerve growth factor.
How a moonlightling protein functions will depend on cellular localization (i.e. where within the cell does it find itself), cell type, oligomeric state (the folding or 3 dimensional protein shape), or the cellular concentration of a ligand, substrate or cofactor (often a threshold amount need be present to trigger action). We are already familiar with these concepts. For example let’s take a quick look at the the oligomeric state (i.e. tertiary structure) as it relates to autocrine Growth Hormone/IGF-1. As you know there exists growth hormone of the type that is released from cells within the pituitary, which flows to the GH-receptors on cells in the liver where it binds and one of the results of this binding is the eventual making and release of IGF-1 which itself flows to tissue where it finds a receptor to bind to. This sort of activity is endocrine or systemic activity.
Now autocrine or local activity in the realm of GH/IGF-1 is where we come in contact with the concept of oligomeric state. Cells everywhere in the body posses various abilities to make a little bit of factors for use therein or use in a way that also affects neighboring cells. In regard to GH, non-liver cells do this by binding a GH molecule to a GH-receptor inside the cell and then birthing the two together. Birthing means they are taken to the cell membrane where the GH/receptor complex is pushed up through the cell membrane. Now the shape of the GH/receptor while it is still inside the cell is turned off. It is off because it’s 3 dimensional shape is not yet the native active state. It is like a closed umbrella and only when it makes it’s way to the cell surface dose the shape become native, active and functional like an open umbrella. The 3 dimensional state of a protein is something that shouldn’t be foreign to readers of this forum.
On the off chance that this concept of muscle cells birthing their own growth factor/receptor is new to you I will add that GH or IGF-1 that is released from the liver or from an exogenous adminstration will not be able to bind to these occupied GH/receptor complexes birthed as autocrine factors. I will also add, welcome to my board. 99% of you never ask the right questions.
If phosphoglucose isomerase has multi-functions what are they? Phosphoglucose isomerase which is secreted by cells (as opposed to remaining within the cells) thereby giving up it’s well known glycolysis/glucogenesis enzymatic role performs several functions when it finds itself outside the cell of it’s birth. One it behaves as a protein identical to neuroleukin which is a cytokine that causes B cells to mature into antibody-secreting cells. Two as a neuroleukin it behaves as a nerve growth factor that promotes the survival of some embryonic spinal neurons and some sensory nerves. Three it becomes the same protein as autocrine motility factor (AMF), which is a cytokine that stimulates cell migration. Four it may also act as a differentiation and maturation mediator (DMM) that brings about differentiation of human myeloid leukemia cells.
This latter differentiation function is growth inhibiting. It is growth inhibiting for the reason that forcing differentiation stops proliferation.
This article isn’t about phosphoglucose isomerase nor is it meant to explore the concept of “moonlighting proteins” in detail. This article is about Thymosin ß4 which is a “moonlighting protein”. Before we proceed I want you to also take note of the fact that the moonlighting protein we just discussed may act to stimulate cell migration (which is necessary for healing and tissue repair but also for cancer metastis as well). Take note also of it’s ability to be growth inhibiting to cancer in specific tissue. Healing factors that are also moonlightling proteins often have this complexity in their nature. TB4 is no different.
1 – Jeffery, C.J. (1999), Moonlighting proteins. Trends Biochem. Sci. 24, 8–11
Unlike some peptides such as Growth Hormone Releasing Hormone (Mod GRF (1-29)) and partial acyl-Ghrelin mimetics (GHRPs), the peptide TB4 demands caution. It demands caution because in solid tumors some of the functions which are positive functions in the area of healing become negative functions increasing the metastatic capacity and angiogenic response of some cancers.
TB4 is naturally present in all cells except red blood cells, It has been found in blood, plasma and wound fluid. However since TB4 has no secretion signal (in essence an amino acid sequence that would signal that it be sheparded from within the cell across the membrane) it is believed that it is present outside the cell as a result of damaged cells. For the most part it resides in two places – in cell cytoplasm and in cell nucleus.
For the reason that we contemplate exogenous administration of TB4 it is appropriate to begin with at least an attempt to understand how this peptide might enter the cell. We are extremely well versed in the concept of a hormone such as growth hormone never entering a cell, but rather binding to a docking port or receptor on the cell surface. From that docking or binding a relay is initiated which migrates within the cell ending in the nucleus. In essence growth horomone knocks on a door and gives a message to the doorman who then carries it inside the building before passing it on.
We are not well versed in how MGF outside the cell affects a signal inside the cell. No cell membrane receptor has yet been found and the possibility remains that there might not be a receptor since MGF usually remains in the cell of it’s birth and translocates to the nucleus to initiate gene transcription.
Peptides that have no membrane receptor and yet possess the capability of affecting signaling inside the cell usually must enter the cell when some sort of door opens and carry the basics of the message themselves.
There is solid evidence that TB4 does both. In some cells it activates yet to be fully identified receptors, in others it enters the cell where it may either remain in cytoplasm or diffuse across the nuclear membrane and act therein.
How does TB4 enter cells?
As of the end of 2012 the precise mechanism of TB4 transport from outside the cell (extracellular compartment) to the cell cytoplasm is under active study & investigation. The most complete speculative answer may be found in the October 2012 issue of ANNALS OF THE NEW YORK ACADEMY OF SCIENCESreported in Thymosin B4 is rapidly internalized by cells and does not induce intracellular Ca2+ elevation, Czeslaw S. Cierniewski, Ann. N.Y. Acad. Sci. 1269 (2012) 44–52
I quote it below with references in order to document the current state of knowledge, to introduce the concept of cell penetrating peptides and for a casual reader to take away the general understanding that TB4 by “hook or crook” is capable of entering the cell. If this were not the case then the discussion in regard to exogenous TB4 usage in bringing about TB4’s primary function would end. Perhaps however it’s role as a moonlighting protein would remain.
“Previous reports showed that the transmembrane exchange of polypeptides can be done through several different independent mechanisms. TB4 fulfills all criteria of cell-penetrating peptides (CPPs), a group of short, water-soluble and partly hydrophobic and/or polybasic peptides (from 5 to 40 amino acids residues) with a net positive charge at physiological pH.65 They have the ability to penetrate the cell membrane at low micromolar concentrations without using any chiral receptors or causing membrane damage.65,66 However, the mechanism by which CPPs are transported is still not fully understood and is a matter of discussion.67
There are at least two proposed cell entry mechanisms, endocytosis and direct penetration, probably via a transient pore formation. The choice of a transport mechanisms depends on many parameters, including size of peptides, cell type, temperature, and other conditions.68,69 It was suggested that a peptide could pass through the plasma membrane via an energy-independent manner either by formation of micro-micelles at the membrane70 or by direct translocation through the lipid bilayer.71 CPPs with a high content of cationic residues (positive charge) can absorb at the cell surface to the numerous anionic moieties (negatively charged molecules) present in the cell membrane, such as sialic or phospholipic acids and heparin sulfate.72 Subsequent transport by caveolae73 and macropinocythosis74 through a clathrin-dependent pathway,75 and via a cholesterol-dependent, clathrin-mediated pathway,76has been reported. Recent observations suggest that retrograde transport can also be involved in cytosolic entry of some CPPs.77 However, retrograde transport in the recycling endosomes is still poorly described, and only a few reports indicate that Rab11 is probably involved in the sorting machinery.78,79”
65. Jarver, P. & U. Langel. 2006. Cell-penetrating peptides— a brief introduction. Biochim. Biophys. Acta 1758: 260– 263.
66. El-Andaloussi, S., T. Holm & U. Langel. 2005. Cellpenetrating peptides: mechanisms and applications. Curr. Pharm. Design 11: 3597–3611.
67. Heitz, F., M.C. Morris & G. Divita. 2009. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 157: 195–206.
68. Tunnemann, G., R.M. Martin & S. Haupt. 2006. Cargodependent mode of uptake and bioavailability of TATcontaining proteins and peptides in living cells. FASEB J. 20: 1775–1784.
69. Morris, M.C., J. Depollier&J.Mery. 2001. Peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotech. 19: 1173–1176.
70. Derossi, D., S. Calvet & A. Trembleau. 1996. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271: 18188– 18193.
71. Thoren, P.E., D. Persson & P. Lincoln. 2005. Membrane destabilizing properties of cell-penetrating peptides. Biophys. Chem. 114: 169–179.
72. Vives, E. 2003. Cellular uptake of the Tat peptide: an endocytosis mechanism following ionic interactions. J.Mol. Recogn. 16: 265–271.
73. Fittipaldi, A., A. Ferrari, M. Zoppe, et al. 2003. Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J. Biol. Chem. 278: 34141–34149.
74. Nakase, I., M. Niwa, T. Takeuchi, et al. 2004. Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther. 10: 1011–1022.
75. Saalik, P., A. Elmquist,M. Hansen, et al. 2004. Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug. Chem. 15: 1246–1253.
76. Foerg, C., U. Ziegler, J. Fernandez-Carneado, et al. 2005. Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. Biochemistry 44: 72–81.
77. Fisher, R., K. Kohler, M. Fotin-Mleczek, et al. 2006. A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J. Biol. Chem. 279: 12625– 12635.
78. Chaudhry, A., S.R. Das, S. Jameel, et al. 2008. HIV-1 Nef induces aRab11-dependent routing of endocytosed immune costimulatory proteins CD80 and CD86 to the Golgi. Traffic 9: 1925–1935.
79. Wilcke, M., L. Johannes, T. Galli, et al. 2000. Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-Golgi network. J. Cell. Biol. 151: 1207–1220.
TB4 receptor-mediated activity
To moonlight TB4 probably needs bring about some sort of inside the cell activity from outside.
Is there a TB4 receptor? I mean a receptor on the cell membrane where TB4 can bind and initiate or mediate a signal into the cell. In this scenario TB4 will give a message to a doorman rather then enter the cell itself.
As of the end of 2012 there are indications that TB4 has activity toward different cells via receptor-mediated mechanisms. We have to use the term “mediated” because no specific TB4 receptor has been mapped out and yet for example a few years ago when TB4 was added externally to endothelial cells (cells that line blood vessels and lymph) it started the expression and release of plasminogen activator inhibitor type 1 within the cell. How did it do that? By a mechanism involving activation of an intracellular signalling pathway we have discussed before, mitogen-activated protein kinase cascade. How did it get to that? By acting on an unknown receptor which set signaling in motion through the root of the cell surface receptor which resides within the cells and kicking off signaling inside the cell. 1,2
By 2012 there was quite a bit more clarity in regard to TB4 activity outside the cell mediating activity inside the cell. You may wonder why this is important. Most articles on peptides start off with “Zippy Peptide is a 33 & a half amino acid long peptide, part of the zippy-do-dah family of peptides well known for this and that.” Here we are many paragraphs deep and we haven’t even begun to discuss what it is and what it does other than to say it is a multi-functional moonlighting protein. Simply put I believe where it does is the most important place to begin.
It’s time to marry the where it does with what it does.
What is TB4?
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 ele