Thymosin beta 10 loaded ZIF-8/sericin hydrogel promoting angiogenesis and osteogenesis for bone regeneration.

Angiogenesis is pivotal for osteogenesis during bone regeneration. A hydrogel that promotes both angiogenesis and osteogenesis is essential in bone tissue engineering. However, creating scaffolds with the ideal balance of biodegradability, osteogenic, and angiogenic properties poses a challenge. Thymosin beta 10 (TMSB10), known for its dual role in angiogenesis and osteogenesis differentiation, faces limitations due to protein activity preservation. To tackle this issue, ZIF-8 was engineered as a carrier for TMSB10 (TMSB10@ZIF-8), and subsequently integrated into the self-assembled sericin hydrogel. The efficacy of the composite hydrogel in bone repair was assessed using a rat cranial defect model. Characterization of the nanocomposites confirmed the successful synthesis of TMSB10@ZIF-8, with a TMSB10 encapsulation efficiency of 88.21 %. The sustained release of TMSB10 from TMSB10@ZIF-8 has significantly enhanced tube formation in human umbilical vein endothelial cells (HUVECs) in vitro and promoted angiogenesis in the chicken chorioallantoic membrane (CAM) model in vivo. It has markedly improved the osteogenic differentiation ability of MC 3 T3-E1 cells in vitro. 8 weeks post-implantation, the TMSB10@ZIF-8/ Sericin hydrogel group exhibited significant bone healing (86.77 ± 8.91 %), outperforming controls. Thus, the TMSB10@ZIF-8/Sericin hydrogel, leveraging ZIF-8 for TMSB10 delivery, emerges as a promising bone regeneration scaffold with substantial clinical application potential.

Therapeutic applications of thymosin peptides: a patent landscape 2018-present.

INTRODUCTION: Thymosins are small proteins found mainly in the thymus. They are involved in several biological processes, including immunoregulation, angiogenesis, and anti-inflammatory activity. Due to these multiple activities, thymosins are widely used as therapeutics. In fact, these peptides have shown interesting results in the treatment of eye disorders, anticancer therapy, and dysregulated immune disorders. AREA COVERED: We analyzed the thymosins therapeutic patent landscape describing the most significant patents published after 2018 and originally written in English, classified according to the different type of functions and diseases. We searched 'Thymosin' on Patentscope and Espacenet. EXPERT OPINION: Thymalfasin (Zadaxin) is the only FDA-approved thymosine-based drug used to treat chronic hepatitis B and C and as a chemotherapy inducer. This outcome demonstrates how thymosins can be exploited as therapeutics, especially in immunological and anti-cancer therapies. However, the development of modified thymosins could expand their therapeutic interest and application in different diseases. In fact, by chemical modifications, it is possible to increase proteolytic stability in the biological environment, enhance cell permeability, and stabilize the secondary structure of the peptide. Finally, the development of shorter sequences could reduce the cost and production time of these thymosin-based drugs.

Thymosin ß4 and Actin: Binding Modes, Biological Functions and Clinical Applications.

Thymosin ß4 (Tß4) is the ß-thymosin (Tßs) with the highest expression level in human cells; it makes up roughly 70-80% of all Tßs in the human body. Combining the mechanism and activity studies of Tß4 in recent years, we provide an overview of the subtle molecular mechanism, pharmacological action, and clinical applications of Tß4. As a G-actin isolator, Tß4 inhibits the polymerization of G-actin by binding to the matching site of G-actin in a 1:1 ratio through conformational and spatial effects. Tß4 can control the threshold concentration of G-actin in the cytoplasm, influence the balance of depolymerization and polymerization of F-actin (also called Tread Milling of F-actin), and subsequently affect cell's various physiological activities, especially motility, development and differentiation. Based on this, Tß4 is known to have a wide range of effects, including regulation of inflammation and tumor metastasis, promotion of angiogenesis, wound healing, regeneration of hair follicles, promotion of the development of the nervous system, and improving bone formation and tooth growth. Tß4 therefore has extensive medicinal applications in many fields, and serves to preserve the kidney, liver, heart, brain, intestine, and other organs, as well as hair loss, skin trauma, cornea repairing, and other conditions. In this review, we focus on the mechanism of action and clinical application of Tß4 for its main biological functions.

[Research advances on thymosin ß4 in promoting wound healing].

With the aging of population and the development of social economy, the incidence of chronic wounds is increasing day by day, while the incidence of burns and trauma remains at a high level, making wound repair an increasingly concerned area in clinical practice. Thymosin ß4 is a naturally occurring small molecule protein in vivo, which is widely distributed in a variety of body fluids and cells, especially in platelets. Thymosin ß4 has biological activities of promoting angiogenesis, anti-inflammation, anti-apoptosis, and anti-fibrosis, and has many important functions in wound repair. Thymosin ß4 has been observed to promote the healing of various wounds, such as burns, diabetic ulcers, pressure ulcers. This paper will review the molecular structure, mechanism of wound healing promotion, pharmacokinetics, and clinical application of thymosin ß4, aiming to introduce its potential in wound treatment and the shortcomings of current researches.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

An Overview on Functional and Structural Properties of Monomeric and Multimeric ß-Thymosin in Invertebrates.

ß-thymosin 4 (Tß4) is a prototypical actin-monomer sequestering protein that plays an important role in mammalian cells and tissues. In vertebrates, Tß4 is involved in various physiological and pathophysiological processes, such as angiogenesis, hair follicle and hair regeneration, nervous system development, inflammatory response, wound healing, tumour metastasis, and liver and heart protection. Additionally, thymosin domain-containing protein was discovered in invertebrates and was recently shown to be more homologous to Tß4. However, the structural and functional properties are more complex and diverse than those of Tß4. In this review article, we will discuss in detail the structural and functional aspects of ß-thymosin in invertebrates.

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins.

Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 µM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.

Protein-Protein Connections-Oligomer, Amyloid and Protein Complex-By Wide Line 1H NMR.

The amount of bonds between constituting parts of a protein aggregate were determined in wild type (WT) and A53T α-synuclein (αS) oligomers, amyloids and in the complex of thymosin-ß4-cytoplasmic domain of stabilin-2 (Tß4-stabilin CTD). A53T αS aggregates have more extensive ßsheet contents reflected by constant regions at low potential barriers in difference (to monomers) melting diagrams (MDs). Energies of the intermolecular interactions and of secondary structures bonds, formed during polymerization, fall into the 5.41 kJ mol-1 ≤ Ea ≤ 5.77 kJ mol-1 range for αS aggregates. Monomers lose more mobile hydration water while forming amyloids than oligomers. Part of the strong mobile hydration water-protein bonds break off and these bonding sites of the protein form intermolecular bonds in the aggregates. The new bonds connect the constituting proteins into aggregates. Amyloid-oligomer difference MD showed an overall more homogeneous solvent accessible surface of A53T αS amyloids. From the comparison of the nominal sum of the MDs of the constituting proteins to the measured MD of the Tß4-stabilin CTD complex, the number of intermolecular bonds connecting constituent proteins into complex is 20(1) H2O/complex. The energies of these bonds are in the 5.40(3) kJ mol-1 ≤ Ea ≤ 5.70(5) kJ mol-1 range.

Furan warheads for covalent trapping of weak protein-protein interactions: cross-linking of thymosin ß4 to actin.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin ß4 (Tß4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tß4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

In Vitro-Evolved Peptides Bind Monomeric Actin and Mimic Actin-Binding Protein Thymosin-ß4.

Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. The filamentous form of actin (F-actin) can be studied with help of natural products that specifically recognize it, as for example fluorophore-labeled probes of the bicyclic peptide phalloidin, but no synthetic probes exist for the monomeric form of actin (G-actin). Herein, we have panned a phage display library consisting of more than 10 billion bicyclic peptides against G-actin and isolated binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity to a region of thymosin-ß4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between actin subdomains 1 and 3. Together with F-actin-specific peptides that we also isolated, we evaluated the G-actin peptides as probes in pull-down, imaging, and competition binding experiments. While the F-actin peptides were applied successfully for capturing actin in cell lysates and for imaging, the G-actin peptides did not bind in the cellular context, most likely due to competition with thymosin-ß4 or related endogenous proteins for the same binding site.

Multiple potential roles of thymosin ß4 in the growth and development of hair follicles.

The hair follicle (HF) is an important mini-organ of the skin, composed of many types of cells. Dermal papilla cells are important signalling components that guide the proliferation, upward migration and differentiation of HF stem cell progenitor cells to form other types of HF cells. Thymosin ß4 (Tß4), a major actin-sequestering protein, is involved in various cellular responses and has recently been shown to play key roles in HF growth and development. Endogenous Tß4 can activate the mouse HF cycle transition and affect HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. In addition, exogenous Tß4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs (hair follicles) in cashmere goats. However, the molecular mechanisms through which Tß4 promotes HF growth and development have rarely been reported. Herein, we review the functions and mechanisms of Tß4 in HF growth and development and describe the endogenous and exogenous actions of Tß4 in HFs to provide insights into the roles of Tß4 in HF growth and development.

Molecular Motions and Interactions in Aqueous Solutions of Thymosin-ß4 , Stabilin CTD and Their 1 : 1 Complex, Studied by 1 H-NMR Spectroscopy.

Wide-line 1 H NMR measurements were extended and all results were interpreted in a thermodynamics-based new approach on aqueous solutions of thymosin-ß4 (Tß4 ), stabilin cytoplasmic domain (CTD), and their 1 : 1 complex. Energy distributions of potential barriers controlling the motion of protein-bound water molecules were determined. Heterogeneous and homogeneous regions were found in the protein-water interface. The measure of heterogeneity of this interface gives quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to ∼20 % of the individual whole proteins. About 40 % of the binding sites of free Tß4 get involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Electrospun thymosin Beta-4 loaded PLGA/PLA nanofiber/ microfiber hybrid yarns for tendon tissue engineering application.

Microfiber yarns (MY) have been widely employed to construct tendon tissue grafts. However, suboptimal ultrastructure and inappropriate environments for cell interactions limit their clinical application. Herein, we designed a modified electrospinning device to coat poly(lactic-co-glycolic acid) PLGA nanofibers onto polylactic acid (PLA) MY to generate PLGA/PLA hybrid yarns (HY), which had a well-aligned nanofibrous structure, resembling the ultrastructure of native tendon tissues and showed enhanced failure load compared to PLA MY. PLGA/PLA HY significantly improved the growth, proliferation, and tendon-specific gene expressions of human adipose derived mesenchymal stem cells (HADMSC) compared to PLA MY. Moreover, thymosin beta-4 (Tß4) loaded PLGA/PLA HY presented a sustained drug release manner for 28 days and showed an additive effect on promoting HADMSC migration, proliferation, and tenogenic differentiation. Collectively, the combination of Tß4 with the nano-topography of PLGA/PLA HY might be an efficient strategy to promote tenogenesis of adult stem cells for tendon tissue engineering.

Metallacarboranes as a tool for enhancing the activity of therapeutic peptides.

Metallacarboranes are anionic boron clusters with high affinity to serum albumin, ability to cross biological membranes, and no apparent toxicity in vitro and in vivo. Thus, conjugation with cobalt bis(1,2-dicarbollide), [COSAN]- , ([3,3'-Co(1,2-C2 B9 H11 )2 ]- ) may improve the properties of therapeutic peptides or proteins at both molecular and systemic levels. Here, we conjugated [COSAN]- with the therapeutic peptide thymosin ß4 (Tß4), which has a pleiotropic activity that results in enhanced healing and regeneration of injured tissues. Using fluorescence quenching of human serum albumin and surface plasmon resonance techniques, we showed that the conjugates have a high affinity to human serum albumin. Using an in vitro wound closure assay, we showed that conjugation with [COSAN]- enhances the activity of Tß4 toward fibroblasts (MSU1.1 cell line). These results indicate an application of metallacarboranes in the development of analogs of various therapeutic peptides/proteins with superior pharmacological properties.

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α.

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 µM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R1, R1ρ, R2 and ηxy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.

Thymosins participate in antibacterial immunity of kuruma shrimp, Marsupenaeus japonicus.

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.

Comparative study of ß-thymosin in two scallop species Argopecten irradians and Chlamys farreri.

The ß-thymosin (Tß) proteins participate in numerous biological processes, such as cell proliferation and differentiation, anti-inflammatory and antimicrobial mechanism. To date, Tß proteins have been well studied in vertebrates, especially mammals. While limited Tß or Tß-like proteins have been reported in invertebrates. Moreover, rare information of Tß or Tß-like proteins is available in scallop species yet. In the present study, two Tß homologues, AiTß and CfTß, were identified and characterized from two scallop species bay scallop Argopecten irradians and Zhikong scallop Chlamys farreri. They were both 41 amino acid peptide and contained one THY domain, a highly conserved actin-binding motif and two conserved helix forming regions. Tissue distribution and expression profiles of their mRNA transcripts were roughly similar yet different in detail, while their recombinant proteins exhibited different immunomodulation activity on the downstream immune parameters. These results collectively indicated that the function of Tß family in scallop were functionally differentiated.

Binding Affinity and Function of the Extremely Disordered Protein Complex Containing Human Linker Histone H1.0 and Its Chaperone ProTα.

It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of ∼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.

Synthesis and Biological Activity of Thymosin ß4-Anionic Boron Cluster Conjugates.

Anionic boron clusters are man-made, inorganic compounds with potential applications in therapeutic peptides modification to improve their biological activity and pharmacokinetics, e.g., by enabling complexation with serum albumin. However, the conjugation of anionic boron clusters and peptides remains poorly understood. Here, we report a solid-state, thermal reaction to selectively conjugate carboxylic groups in the peptide thymosin ß4 (Tß4) with cyclic oxonium derivatives of anionic boron clusters (dodecaborate anion [B12H12]2- and cobalt bis(1,2-dicarbollide), [COSAN]- [3,3'-Co(1,2-C2B9H11)2]-). Modification of the carboxylic groups retains the negative charge at the modification site and leads to the formation of ester bonds. The ester bonds in the conjugates undergo hydrolysis at different rates depending on the site of the modification. We obtained conjugates with dramatically different stabilities (τ1/2 from 3-836 h (Tß4-[B12H12]2- conjugates) and 9-1329 h (Tß4-[COSAN]- conjugates)) while retaining or improving the prosurvival activity of Tß4 toward cardiomyocytes (H9C2 cell line).