Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

Objectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.

Methods: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.

Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia.

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly.

Identification of PDGF-BB binding to thymosin β4 by chemical cross-linking.

Introduction: The purpose of our work was to identify unknown interaction partners of thymosin β4 (Tβ4). It was suggested that Tβ4 could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ4 prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ4 counteracts the PDGF-BB-induced activation of HSCs.

High-resolution mass spectrometry for thymosins detection and characterization.

Objectives: The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies.

Methods: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.

Results: In addition to thymosin β4 (Tβ4) and β10 (Tβ10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively.

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection.

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohn's disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohn's disease led to detection of intestinal mTNF(+) immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF(+) cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF(+) cells (15%).

Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tβ4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tβ4 decreased treadmilling, presumably by sequestering monomers.

Thymosin β4 and tissue transglutaminase. Molecular characterization of cyclic thymosin β4.

Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase.

High-resolution HPLC-ESI-MS characterization of the contact sites of the actin-thymosin β(4) complex by chemical and enzymatic cross-linking.

Thymosin β4 sequesters actin by formation of a 1:1 complex. This transient binding in the complex was stabilized by formation of covalent bonds using the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and a microbial transglutaminase. The localization of cross-linking sites was determined after separating the products using SDS-PAGE by tryptic in-gel digestion and high-resolution HPLC-ESI-MS.

Antibodies in research of thymosin β4: investigation of cross-reactivity and influence of fixatives.

Antibodies against thymosin β4 are available from various sources and have been used in immunohistochemistry, ELISA, and Western blot analyses. None of these antibodies have been fully characterized for specificity and influence of fixation techniques. This presents a difficulty because many tissues express more than one member of the β-thymosin family; in addition, highly homologous sequences are typical elements of β-thymosins.

Identification of interaction partners of β-thymosins: application of thymosin β4 labeled by transglutaminase.

In this review, we identify potential interaction partners of the β-thymosin family. The proteins of this family are highly conserved peptides in mammals and yet only one intracellular (G-actin) and one cell-surface protein (β subunit of F(1) -F(0) ATP synthase) were identified as interaction partners of thymosin β4. Cross-linking experiments may be a possible approach to discover additional proteins that interact with the β-thymosin family.

Thymosin β4: a multi-functional regenerative peptide. Basic properties and clinical applications.

Introduction: Thymosin β(4), a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin β(4), is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin β(4) binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue.

Thymosin beta4 and its posttranslational modifications.

Thymosin beta(4) as well as the other members of the beta-thymosin family are important G-actin sequestering peptides. The chemical properties, the biosynthesis, and posttranslational modifications (PTMs) of these peptides are discussed. During biosynthesis of thymosin beta(4) the initiator methionine is removed and the N-terminus is acetylated.

The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family.

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response.

Thymosin beta4 is an essential paracrine factor of embryonic endothelial progenitor cell-mediated cardioprotection.

Background: Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection.

Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control.

Spreading transmissible spongiform encephalopathies (TSE) have been widely attributed to transmission by ingestion of mammalian central nervous system (CNS) tissue. Reliable exclusion of this epidemiological important route of transmission relies on an effective surveillance of food contamination. Here, myelin proteolipid protein (PLP) is identified as a specific and largely heat-resistant marker for detection of food contaminations by CNS tissue.

Thymosin beta4 upregulates the expression of hepatocyte growth factor and downregulates the expression of PDGF-beta receptor in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are the main producers of type I collagen in the liver, and therefore are responsible, in part, for the fibrous scar observed in cirrhotic livers. Although there is no approved treatment for this deadly disease, drugs inducing HSC apoptosis in animals (gliotoxin) and hepatocyte regeneration in man (hepatocyte growth factor [HGF]), have been used successfully in ameliorating liver fibrosis. In this communication we investigated whether thymosin beta(4) (Tbeta(4)), an actin-sequestering peptide that prevents scarring of the heart after a myocardial infarction and that prevents kidney fibrosis in animals, has the potential to be used to treat liver fibrosis.

Subcellular distribution of thymosin beta4.

The localization of Oregon Green cadaverine-labeled thymosin beta(4), its fragments, and variants was investigated in cytoplasm-depleted A431 cells and in microinjected cells without and with fixation. The studied thymosin beta(4) variants included substitutions of the lysine residues within the basic cluster (14-KSKLKK-19) and the actin-binding motif (17-LKKTETQ-23). In contrast to Oregon Green cadaverine, none of the variants or fragments of thymosin beta(4) could pass the intact nuclear pore of cytoplasm-depleted cells and were hence excluded from the nucleus.

Influence of the N terminus and the actin-binding motif of thymosin beta4 on its interaction with G-actin.

Thymosin beta(4) binds G-actin in a 1:1 ratio and prevents its aggregation to F-actin by sequestration. Substitution or modification of single amino acid residues within the N-terminal sequence 1 to 22 of thymosin beta(4) alters its interaction with G-actin. We generated thymosin beta(4) variants with amino acid substitutions within the N-terminal alpha-helix and the putative actin-binding motif.

Determination of thymosin beta4 and protein in human wound fluid after abdominal surgery.

Wound fluids were collected up to 60 h after abdominal surgery. Immediately after obtaining the wound fluid by Robinson drainage, wound fluid was centrifuged to remove blood cells and inflammatory cells. The concentration of total protein as well as of thymosin beta(4) was determined in the cell-free supernatant solution.

Thymosin beta4 is not always the main beta-thymosin in mammalian platelets.

beta-thymosins constitute a family of highly conserved 5-kDa polypeptides. Thymosin beta(4), the most abundant member of this family, is expressed in most mammalian cell types and is regarded as the main intracellular G-actin sequestering peptide. In addition to this important intracellular function several other activities have been attributed to this peptide.

beta-Thymosins.

The development of thymosin beta(4) from a thymic hormone to an actin-sequestering peptide and back to a cytokine supporting wound healing will be outlined. Thymosin fraction 5 consists of a mixture of polypeptides and improves immune response. Starting with fraction 5, several main peptides (thymosin alpha(1), polypeptide beta(1), and thymosin beta(4)) were isolated and tested for biological activity.