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Vitek M, Zvonar Pobirk A, Roškar R, Matjaž MG. Exploiting the potential of in situ forming liquid crystals: development and in vitro performance of long-acting depots for peptide drug thymosin alpha 1 subcutaneous administration. Drug Deliv 2025; 32:2460708. [PMID: 40066714 PMCID: PMC11899226 DOI: 10.1080/10717544.2025.2460708] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 11/06/2024] [Accepted: 01/24/2025] [Indexed: 03/14/2025] Open
Abstract
The fast-growing filed of long-acting depots for subcutaneous (SC) administration holds significant potential to enhance patient adherence to treatment regimens, particularly in the context of chronic diseases. Among them, injectable in situ forming lyotropic liquid crystals (LCCs) consisting of hexagonal mesophases represent an attractive platform due to their remarkable highly ordered microstructure enabling the sustained drug release. These systems are especially relevant for peptide drugs, as their use is limited by their short plasma half-life and inherent poor stability. In this study, we thus aimed to exploit the potential of a liquid crystalline platform for the sustained release of peptide drug thymosin alpha 1 (Tα1), characterized by a short plasma half-life and with that associated twice-weekly SC administration regimen. We initially selected specified ingredients, with ethanol serving to reduce viscosity and stabilize the peptide drug Tα1, lecithin contributing to LCCs formation and stabilization, and glycerol monooleate or glycerol monolinoleate representing the hexagonal LCCs forming matrix material. The selected studied nonaqueous precursor formulations were characterized by suitable rheological properties for SC injection. A convenient and rapid in situ phase transition of precursor formulations to hexagonal LCCs, triggered by water absorption, was successfully accomplished in vitro. Notably, in situ formed LCCs demonstrated sustained release kinetics of the peptide drug Tα1 for up to 2 weeks of in vitro release testing, offering minimized dosing frequency and thus promoting patient adherence. In summary, the newly developed in situ forming liquid crystalline systems represent prospective injectable long-acting depots for SC administration of the peptide drug Tα1.
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Affiliation(s)
- Mercedes Vitek
- Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
| | - Alenka Zvonar Pobirk
- Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
| | - Robert Roškar
- Department of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
| | - Mirjam Gosenca Matjaž
- Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
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Garaci E, Paci M, Matteucci C, Costantini C, Puccetti P, Romani L. Phenotypic drug discovery: a case for thymosin alpha-1. Front Med (Lausanne) 2024; 11:1388959. [PMID: 38903817 PMCID: PMC11187271 DOI: 10.3389/fmed.2024.1388959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 05/28/2024] [Indexed: 06/22/2024] Open
Abstract
Phenotypic drug discovery (PDD) involves screening compounds for their effects on cells, tissues, or whole organisms without necessarily understanding the underlying molecular targets. PDD differs from target-based strategies as it does not require knowledge of a specific drug target or its role in the disease. This approach can lead to the discovery of drugs with unexpected therapeutic effects or applications and allows for the identification of drugs based on their functional effects, rather than through a predefined target-based approach. Ultimately, disease definitions are mostly symptom-based rather than mechanism-based, and the therapeutics should be likewise. In recent years, there has been a renewed interest in PDD due to its potential to address the complexity of human diseases, including the holistic picture of multiple metabolites engaging with multiple targets constituting the central hub of the metabolic host-microbe interactions. Although PDD presents challenges such as hit validation and target deconvolution, significant achievements have been reached in the era of big data. This article explores the experiences of researchers testing the effect of a thymic peptide hormone, thymosin alpha-1, in preclinical and clinical settings and discuss how its therapeutic utility in the precision medicine era can be accommodated within the PDD framework.
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Affiliation(s)
| | - Maurizio Paci
- Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome, Italy
| | - Claudia Matteucci
- Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Claudio Costantini
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Paolo Puccetti
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Luigina Romani
- San Raffaele Sulmona, L’Aquila, Italy
- Department of Medicine and Surgery, University of Perugia, Perugia, Italy
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Matteucci C, Nepravishta R, Argaw-Denboba A, Mandaliti W, Giovinazzo A, Petrone V, Balestrieri E, Sinibaldi-Vallebona P, Pica F, Paci M, Garaci E. Thymosin α1 interacts with Galectin-1 modulating the β-galactosides affinity and inducing alteration in the biological activity. Int Immunopharmacol 2023; 118:110113. [PMID: 37028279 DOI: 10.1016/j.intimp.2023.110113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/17/2023] [Accepted: 03/18/2023] [Indexed: 04/09/2023]
Abstract
The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.
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Affiliation(s)
- Claudia Matteucci
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy.
| | - Ridvan Nepravishta
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome 00133, Italy
| | - Ayele Argaw-Denboba
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy; European Molecular Biology Laboratory, EMBL, Monterotondo, Rome 00015, Italy
| | - Walter Mandaliti
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome 00133, Italy
| | - Alessandro Giovinazzo
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy; Institute of Biochemistry and Cell Biology, IBBC-CNR, Monterotondo, Rome 00015, Italy
| | - Vita Petrone
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy
| | - Emanuela Balestrieri
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy
| | - Paola Sinibaldi-Vallebona
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy; Institute of Translational Pharmacology, National Research Council, Rome 00133, Italy
| | - Francesca Pica
- Department of Experimental Medicine, University of Tor Vergata, Rome 00133, Italy
| | - Maurizio Paci
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome 00133, Italy
| | - Enrico Garaci
- IRCCS San Raffaele and IRCCS San Raffaele, Rome 00163, Italy; Medical and Experimental BioImaging Center, MEBIC Consortium, Rome 00166, Italy
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Tao N, Xu X, Ying Y, Hu S, Sun Q, Lv G, Gao J. Thymosin α1 and Its Role in Viral Infectious Diseases: The Mechanism and Clinical Application. Molecules 2023; 28:molecules28083539. [PMID: 37110771 PMCID: PMC10144173 DOI: 10.3390/molecules28083539] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Revised: 04/12/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
Thymosin α1 (Tα1) is an immunostimulatory peptide that is commonly used as an immune enhancer in viral infectious diseases such as hepatitis B, hepatitis C, and acquired immune deficiency syndrome (AIDS). Tα1 can influence the functions of immune cells, such as T cells, B cells, macrophages, and natural killer cells, by interacting with various Toll-like receptors (TLRs). Generally, Tα1 can bind to TLR3/4/9 and activate downstream IRF3 and NF-κB signal pathways, thus promoting the proliferation and activation of target immune cells. Moreover, TLR2 and TLR7 are also associated with Tα1. TLR2/NF-κB, TLR2/p38MAPK, or TLR7/MyD88 signaling pathways are activated by Tα1 to promote the production of various cytokines, thereby enhancing the innate and adaptive immune responses. At present, there are many reports on the clinical application and pharmacological research of Tα1, but there is no systematic review to analyze its exact clinical efficacy in these viral infectious diseases via its modulation of immune function. This review offers an overview and discussion of the characteristics of Tα1, its immunomodulatory properties, the molecular mechanisms underlying its therapeutic effects, and its clinical applications in antiviral therapy.
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Affiliation(s)
- Nana Tao
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Xie Xu
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Yuyuan Ying
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Shiyu Hu
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Qingru Sun
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Guiyuan Lv
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
| | - Jianli Gao
- School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
- State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao 999078, China
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Aslam MS, Zaidi SZJ, Toor RH, Gull I, Iqbal MM, Abbas Z, Tipu I, Ahmed A, Athar MA, Harito C, Hassan SU. Interferon α2-Thymosin α1 Fusion Protein (IFNα2-Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect. MATERIALS 2021; 14:ma14123318. [PMID: 34203928 PMCID: PMC8232609 DOI: 10.3390/ma14123318] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Revised: 04/09/2021] [Accepted: 05/09/2021] [Indexed: 12/30/2022]
Abstract
Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2-thymosin α1 fusion protein (IFNα2-Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2-Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2-Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2-Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2-Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.
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Affiliation(s)
- Muhammad Shahbaz Aslam
- Institute of Biochemistry and Biotechnology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (I.G.); (M.M.I.); (M.A.A.)
- Correspondence: (M.S.A.); (S.Z.J.Z.); (S.-u.H.)
| | - Syed Zohaib Javaid Zaidi
- Institute of Chemical Engineering and Technology, University of the Punjab, Lahore 54590, Pakistan
- Correspondence: (M.S.A.); (S.Z.J.Z.); (S.-u.H.)
| | - Rabail Hassan Toor
- School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (R.H.T.); (A.A.)
| | - Iram Gull
- Institute of Biochemistry and Biotechnology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (I.G.); (M.M.I.); (M.A.A.)
| | - Muhammad Mudassir Iqbal
- Institute of Biochemistry and Biotechnology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (I.G.); (M.M.I.); (M.A.A.)
| | - Zaigham Abbas
- Department of Microbiology & Molecular Genetics, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan;
| | - Imran Tipu
- Department of Life Sciences, University of Management & Technology, Lahore 54770, Pakistan;
| | - Aftab Ahmed
- School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (R.H.T.); (A.A.)
| | - Muhammad Amin Athar
- Institute of Biochemistry and Biotechnology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; (I.G.); (M.M.I.); (M.A.A.)
| | - Christian Harito
- Industrial Engineering Department, Faculty of Engineering, Bina Nusantara University, Jakarta 11480, Indonesia;
| | - Sammer-ul Hassan
- Bioengineering Research Group, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton SO17 1BJ, UK
- Correspondence: (M.S.A.); (S.Z.J.Z.); (S.-u.H.)
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An J, Dong Y, Li Y, Han X, Sha J, Zou Z, Niu H. Retrospective analysis of T-lymphocyte subsets and cytokines in malignant obstructive jaundice before and after external and internal biliary drainage. J Int Med Res 2021; 49:300060520970741. [PMID: 33641475 PMCID: PMC7917863 DOI: 10.1177/0300060520970741] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Objective To study changes in T lymphocyte subsets, cytokines, and liver enzymes in patients with malignant obstructive jaundice (MOJ) before and after external biliary drainage (percutaneous transhepatic cholangiography drainage, PTCD) and internal biliary drainage (percutaneous transhepatic insertion of biliary stents, PTIBS). Methods MOJ patients undergoing PTCD (n = 44) and PTIBS (n = 38) at our hospital were enrolled in the study from January 2017 until December 2019. Peripheral blood total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), CD3+%, CD4+%, CD4+/CD8+ ratio, interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were measured before and 1 week after biliary drainage. Results There was no significant difference in any parameter between the two groups before biliary drainage. TBIL, DBIL, AST and ALT following PTCD were significantly lower than before PTCD. By contrast, CD3+%, CD4+%, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α showed no significant difference before and 1 week after PTCD. TBIL, DBIL, AST, ALT, IL-6 and TNF-α were significantly lower following PTIBS than before PTIBS. CD3+%, CD4+%, CD4+/CD8+ ratio and IL-2 were significantly higher following PTIBS than before PTIBS. Conclusion Both PTCD and PTIBS were effective for treatment of MOJ, but PTIBS was more beneficial for recovery of immune function.
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Affiliation(s)
- Jianli An
- Department of Interventional Treatment, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Yanchao Dong
- Department of Interventional Treatment, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Yanguo Li
- Department of Radiology, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Xiaoyu Han
- Department of Cardiovascular Medicine, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Junfeng Sha
- Department of Interventional Treatment, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Zibo Zou
- Department of Interventional Treatment, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
| | - Hongtao Niu
- Department of Interventional Treatment, Qinhuangdao Municipal No. 1 Hospital, Qinhuangdao, Hebei Province, PR China
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Development of a Highly Efficient Hybrid Peptide That Increases Immunomodulatory Activity Via the TLR4-Mediated Nuclear Factor-κB Signaling Pathway. Int J Mol Sci 2019; 20:ijms20246161. [PMID: 31817671 PMCID: PMC6940896 DOI: 10.3390/ijms20246161] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 12/02/2019] [Accepted: 12/04/2019] [Indexed: 02/07/2023] Open
Abstract
Immunity is a defensive response that fights disease by identifying and destroying harmful substances or microbiological toxins. Several factors, including work-related stress, pollution, and immunosuppressive agents, contribute to low immunity and poor health. Native peptides, a new class of immunoregulatory agents, have the potential for treating immunodeficiencies, malignancies, and infections. However, the potential cytotoxicity and low immunoregulatory activity and stability of native peptides have prevented their development. Therefore, we designed three hybrid peptides (LTAa, LTAb, and LTAc) by combining a characteristic fragment of LL-37 with an active Tα1 center that included Tα1 (17-24), Tα1 (20-25), and Tα1 (20-27). The best hybrid peptide (LTAa), according to molecule docking and in vitro experiments, had improved immunoregulatory activity and stability with minimal cytotoxicity. We investigated the immunoregulatory effects and mechanisms of LTAa using a cyclophosphamide-immunosuppressed murine model. LTAa effectively reversed immunosuppression by enhancing immune organ development, activating peritoneal macrophage phagocytosis, regulating T lymphocyte subsets, and increasing cytokine (tumor necrosis factor-alpha, interleukin-6, and interleukin-1β) and immunoglobulin (IgA, IgG, and IgM) contents. The immunomodulatory effects of LTAa may be associated with binding to the TLR4/MD-2 complex and activation of the NF-κB signaling pathway. Therefore, LTAa could be an effective therapeutic agent for improving immune function.
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Zhang L, Wei X, Zhang R, Petitte JN, Si D, Li Z, Cheng J, Du M. Design and Development of a Novel Peptide for Treating Intestinal Inflammation. Front Immunol 2019; 10:1841. [PMID: 31447849 PMCID: PMC6691347 DOI: 10.3389/fimmu.2019.01841] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2019] [Accepted: 07/22/2019] [Indexed: 12/24/2022] Open
Abstract
Intestinal inflammatory disorders, such as inflammatory bowel disease (IBD), are associated with increased pro-inflammatory cytokine secretion in the intestines. Furthermore, intestinal inflammation increases the risk of enteric cancer, which is a common malignancy globally. Native anti-inflammatory peptides are a class of anti-inflammatory agents that could be used in the treatment of several intestinal inflammation conditions. However, potential cytotoxicity, and poor anti-inflammatory activity have prevented their development as anti-inflammatory agents. Therefore, in this study, we designed and developed a novel hybrid peptide for the treatment of intestinal inflammation. Eight hybrid peptides were designed by combining the active centers of antimicrobial peptides, including LL-37 (13-36), YW12D, innate defense regulator 1, and cathelicidin 2 (1-13) with thymopentin or the active center of thymosin alpha 1 (Tα1) (17-24). The hybrid peptide, LL-37-Tα1 (LTA), had improved anti-inflammatory activity with minimal cytotoxicity. LTA was screened by molecule docking and in vitro experiments. Likewise, its anti-inflammatory effects and mechanisms were also evaluated using a lipopolysaccharide (LPS)-induced intestinal inflammation murine model. The results showed that LTA prevented LPS-induced impairment in the jejunum epithelium tissues and infiltration of leukocytes, which are both histological markers of inflammation. Additionally, LTA decreased the levels of tumor necrosis factor-alpha, interferon-gamma, interleukin-6, and interleukin-1β. LTA increased the expression of zonula occludens-1 and occludin, and reduced permeability and apoptosis in the jejunum of LPS-treated mice. Additionally, its anti-inflammatory effect is associated with neutralizing LPS, binding to the Toll-like receptor 4-myeloid differentiation factor 2 (TLR4/MD-2) complex, and modulating the nuclear factor-kappa B signal transduction pathway. The findings of this study suggest that LTA may be an effective therapeutic agent in the treatment of intestinal inflammation.
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Affiliation(s)
- Lulu Zhang
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Xubiao Wei
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Rijun Zhang
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Jim N Petitte
- Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States
| | - Dayong Si
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Zhongxuan Li
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Junhao Cheng
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Mengsi Du
- Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
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Mercurio FA, Scaloni A, Caira S, Leone M. The antimicrobial peptides casocidins I and II: Solution structural studies in water and different membrane-mimetic environments. Peptides 2019; 114:50-58. [PMID: 30243923 DOI: 10.1016/j.peptides.2018.09.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Revised: 09/18/2018] [Accepted: 09/18/2018] [Indexed: 10/28/2022]
Abstract
Antimicrobial peptides (AMPs) represent crucial components of the natural immune defense machinery of different organisms. Generally, they are short and positively charged, and bind to and destabilize bacterial cytoplasmic membranes, ultimately leading to cell death. Natural proteolytic cleavage of αs2-casein in bovine milk generates the antimicrobial peptides casocidin I and II. In the current study, we report for the first time on a detailed structure characterization of casocidins in solution by means of Nuclear Magnetic Resonance spectroscopy (NMR). Structural studies were conducted in H2O and different membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE) and lipid anionic and zwitterionic vesicles. For both peptides, results indicate a mainly disordered conformation in H2O, with a few residues in a partial helical structure. No wide increase of order occurs upon interaction with lipid vesicles. Conversely, peptide conformation becomes highly ordered in presence of TFE, with both casocidins presenting a large helical content. Our data point out a preference of casocidins to interact with model anionic membranes. These results are compatible with possible mechanisms of action underlying the antimicrobial activity of casocidins that ultimately may affect membrane bilayer stability.
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Affiliation(s)
- Flavia Anna Mercurio
- Institute of Biostructures and Bioimaging (IBB), National Research Council & Interuniversity Research Centre on Bioactive Peptides (CIRPEB), Via Mezzocannone 16, 80134 Naples, Italy
| | - Andrea Scaloni
- Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Via Argine 1085, 80147 Naples, Italy
| | - Simonetta Caira
- Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Via Argine 1085, 80147 Naples, Italy
| | - Marilisa Leone
- Institute of Biostructures and Bioimaging (IBB), National Research Council & Interuniversity Research Centre on Bioactive Peptides (CIRPEB), Via Mezzocannone 16, 80134 Naples, Italy.
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Mandaliti W, Nepravishta R, Pica F, Vallebona PS, Garaci E, Paci M. Potential mechanism of thymosin-α1-membrane interactions leading to pleiotropy: experimental evidence and hypotheses. Expert Opin Biol Ther 2018; 18:33-42. [DOI: 10.1080/14712598.2018.1456527] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Affiliation(s)
- Walter Mandaliti
- Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome, Italy
| | - Ridvan Nepravishta
- Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome, Italy
- School of Pharmacy, East Anglia University, Norwich, UK
| | - Francesca Pica
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Rome, Italy
| | | | - Enrico Garaci
- San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, Rome, Italy
| | - Maurizio Paci
- Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome, Italy
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Torres MDT, Silva AF, Pedron CN, Capurro ML, de la Fuente‐Nunez C, Junior VXO. Peptide Design Enables Reengineering of an Inactive Wasp Venom Peptide into Synthetic Antiplasmodial Agents. ChemistrySelect 2018. [DOI: 10.1002/slct.201800529] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Marcelo D. T. Torres
- Centro de Ciências Naturais e HumanasUniversidade Federal do ABC
- Synthetic Biology Group
- Research Laboratory of Electronics
- Department of Biological Engineering and Department of Electrical Engineering and Computer ScienceMassachusetts Institute of TechnologyCambridge, MassachusettsUnited States of America
- Broad Institute of MIT and Harvard, Cambridge, MassachusettsUnited States of America
| | - Adriana F. Silva
- Centro de Ciências Naturais e HumanasUniversidade Federal do ABC
| | - Cibele N. Pedron
- Centro de Ciências Naturais e HumanasUniversidade Federal do ABC
| | | | - Cesar de la Fuente‐Nunez
- Synthetic Biology Group
- Research Laboratory of Electronics
- Department of Biological Engineering and Department of Electrical Engineering and Computer ScienceMassachusetts Institute of TechnologyCambridge, MassachusettsUnited States of America
- Broad Institute of MIT and Harvard, Cambridge, MassachusettsUnited States of America
- The Center for Microbiome Informatics and Therapeutics, Cambridge, Massachusetts, United States of America
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Mandaliti W, Nepravishta R, Pica F, Vallebona PS, Garaci E, Paci M. Thymosin α1 Interacts with Hyaluronic Acid Electrostatically by Its Terminal Sequence LKEKK. Molecules 2017; 22:E1843. [PMID: 29077041 PMCID: PMC6150299 DOI: 10.3390/molecules22111843] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2017] [Revised: 10/19/2017] [Accepted: 10/24/2017] [Indexed: 11/17/2022] Open
Abstract
Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.
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Affiliation(s)
- Walter Mandaliti
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", via della Ricerca Scientifica 1, 00133 Rome, Italy.
| | - Ridvan Nepravishta
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", via della Ricerca Scientifica 1, 00133 Rome, Italy.
- School of Pharmacy, East Anglia University, Norwich NR4 7TJ, UK.
| | - Francesca Pica
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", via Montpellier 1, 00133 Rome, Italy.
| | - Paola Sinibaldi Vallebona
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", via Montpellier 1, 00133 Rome, Italy.
| | - Enrico Garaci
- San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, 00163 Rome, Italy.
| | - Maurizio Paci
- Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", via della Ricerca Scientifica 1, 00133 Rome, Italy.
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Torres MDT, Silva AF, Alves FL, Capurro ML, Miranda A, Cordeiro RM, Oliveira Junior VX. Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes. J Pept Sci 2016; 22:132-42. [PMID: 26856687 DOI: 10.1002/psc.2849] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Revised: 11/25/2015] [Accepted: 12/14/2015] [Indexed: 01/02/2023]
Abstract
Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a β-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a β-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.
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Affiliation(s)
| | - Adriana Farias Silva
- Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Santo André, SP, Brazil
| | - Flávio Lopes Alves
- Universidade Federal de São Paulo, Departamento de Biofísica, São Paulo, SP, Brazil
| | | | - Antonio Miranda
- Universidade Federal de São Paulo, Departamento de Biofísica, São Paulo, SP, Brazil
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Nepravishta R, Mandaliti W, Vallebona PS, Pica F, Garaci E, Paci M. Mechanism of Action of Thymosinα1: Does It Interact with Membrane by Recognition of Exposed Phosphatidylserine on Cell Surface? A Structural Approach. VITAMINS AND HORMONES 2016; 102:101-19. [PMID: 27450732 DOI: 10.1016/bs.vh.2016.04.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Thymosinα1 is a peptidic hormone with pleiotropic activity, which is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of micelles and vesicles assuming two tracts of helical conformation with a structural flexible break in between. The studies of the interaction of Thymosinα1 with micelles of mixed dipalmitoylphosphatidylcholine and sodium dodecylsulfate and vesicles with mixed dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine, the latter the negative component of the membranes, by (1)H and natural abundance (15)N NMR are herewith reported, reviewed, and discussed. The results indicate that the preferred interactions are those where the surface is negatively charged due to sodium dodecylsulfate or due to the presence of dipalmitoylphosphatidylserine exposed on the surface. In fact the unbalance of dipalmitoylphosphatidylserine on the cellular surface is an important phenomenon present in pathological conditions of cells. Moreover, the direct interaction of Thymosinα1 with K562 cells presenting an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was carried out.
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Affiliation(s)
- R Nepravishta
- University of Rome "Tor Vergata", Rome, Italy; Faculty of Pharmacy Catholic University "Our Lady of Good Counsel", Tirane, Albania
| | - W Mandaliti
- University of Rome "Tor Vergata", Rome, Italy
| | | | - F Pica
- University of Rome "Tor Vergata", Rome, Italy
| | - E Garaci
- University of Rome "Tor Vergata", Rome, Italy; San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, Rome, Italy
| | - M Paci
- University of Rome "Tor Vergata", Rome, Italy.
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16
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Mandaliti W, Nepravishta R, Sinibaldi Vallebona P, Pica F, Garaci E, Paci M. Thymosin α1 Interacts with Exposed Phosphatidylserine in Membrane Models and in Cells and Uses Serum Albumin as a Carrier. Biochemistry 2016; 55:1462-72. [DOI: 10.1021/acs.biochem.5b01345] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Affiliation(s)
- Walter Mandaliti
- Department
of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy
| | - Ridvan Nepravishta
- Department
of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy
- Department
of Chemical Pharmaceutical and Biomolecular Technologies, Faculty of Pharmacy Catholic University “Our Lady of Good Counsel”, Rr. D.
Hoxha, Tirane, Albania
| | - Paola Sinibaldi Vallebona
- Department
of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Francesca Pica
- Department
of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Enrico Garaci
- Department
of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
- San
Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, 00163 Rome, Italy
| | - Maurizio Paci
- Department
of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy
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17
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New studies about the insertion mechanism of Thymosin α1 in negative regions of model membranes as starting point of the bioactivity. Amino Acids 2016; 48:1231-9. [DOI: 10.1007/s00726-016-2169-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2015] [Accepted: 01/05/2016] [Indexed: 10/25/2022]
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18
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19
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Nepravishta R, Mandaliti W, Eliseo T, Vallebona PS, Pica F, Garaci E, Paci M. Thymosin α1 inserts N terminus into model membranes assuming a helical conformation. Expert Opin Biol Ther 2015; 15 Suppl 1:S71-81. [DOI: 10.1517/14712598.2015.1009034] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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20
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21
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Marcelo Der Torossian T, Silva AF, Alves FL, Capurro ML, Miranda A, Vani Xavier O. Highly Potential Antiplasmodial Restricted Peptides. Chem Biol Drug Des 2014; 85:163-71. [DOI: 10.1111/cbdd.12354] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Revised: 04/01/2014] [Accepted: 04/29/2014] [Indexed: 12/17/2022]
Affiliation(s)
| | - Adriana F. Silva
- Centro de Ciências Naturais e Humanas; Universidade Federal do ABC; Santo André Brazil
| | - Flávio L. Alves
- Departamento de Biofísica; Universidade Federal de São Paulo; São Paulo Brazil
| | - Margareth L. Capurro
- Instituto de Ciências Biomédicas II; Universidade de São Paulo; São Paulo Brazil
| | - Antonio Miranda
- Departamento de Biofísica; Universidade Federal de São Paulo; São Paulo Brazil
| | - Oliveira Vani Xavier
- Centro de Ciências Naturais e Humanas; Universidade Federal do ABC; Santo André Brazil
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22
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Volk DE, Tuthill CW, Elizondo-Riojas MA, Gorenstein DG. NMR structural studies of thymosin α1 and β-thymosins. Ann N Y Acad Sci 2012; 1270:73-8. [DOI: 10.1111/j.1749-6632.2012.06656.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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23
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Benaki D, Zikos C, Karachaliou CE, Tsitsilonis O, Leondiadis L, Kalbacher H, Voelter W, Papadopoulos M, Pirmettis I, Pelecanou M, Livaniou E. Complexes of an Alpha Thymosin Derivative with185/187Re and99mTc: Structural Analysis and Initial Biological Evaluation. Chem Biol Drug Des 2012; 80:545-53. [DOI: 10.1111/j.1747-0285.2012.01425.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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24
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Elizondo-Riojas MA, Chamow SM, Tuthill CW, Gorenstein DG, Volk DE. NMR structure of human thymosin alpha-1. Biochem Biophys Res Commun 2011; 416:356-61. [PMID: 22115779 DOI: 10.1016/j.bbrc.2011.11.041] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2011] [Accepted: 11/09/2011] [Indexed: 02/07/2023]
Abstract
800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double β-turns in the N-terminal twelve residues which form a distorted helical structure.
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Affiliation(s)
- Miguel-Angel Elizondo-Riojas
- Center for Proteomics and Systems Biology, Institute of Molecular Medicine for Prevention of Human Diseases, Department of NanoMedicine and Biomedical Engineering, University of Texas Health Science Center-Houston, 1825 Pressler, Houston, TX 77030, United States
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25
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QIE J, MA J, WANG L, XU X, ZHOU W, QI C, ZHAO X, ZHANG Y, LIU K. Site-directed PEGylations of Thymosin α1 Analogs and Evaluation of Their Immunoactivity. CHINESE J CHEM 2009. [DOI: 10.1002/cjoc.200990134] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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26
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Gao D, Zhang X, Zhang J, Cao J, Wang F. Expression of thymosin alpha1-thymopentin fusion peptide in Pichia pastoris and its characterization. Arch Pharm Res 2008; 31:1471-6. [PMID: 19023544 DOI: 10.1007/s12272-001-2132-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2008] [Revised: 10/25/2008] [Accepted: 10/27/2008] [Indexed: 11/26/2022]
Abstract
Thymopentin plays an important role in improving imbalanced immune systems of patients, however, it has a limited half-life in plasma. To get more stable and active thymopentin analogs, a fusion thymosin alpha1-thymopentin (Talpha1-TP5) gene was synthesized and cloned into vector pGAPZalphaA. Talpha1-TP5 fusion peptide was expressed in pichia pastoris and purified by metal chelating chromatography and gel filtration chromatography. The circular dichroism spectra (CD) indicated that the secondary structure of Talpha1-TP5 fusion peptide is dominated by a-helix and random coil. In vitro analysis showed that the plasma half-life of Talpha1-TP5 fusion peptide is 140 +/- 14 min, which is longer than that of TP5 (5.6+/-0.7 min) and Talpha1 (127+/-11 min). The in vitro activity assay presented that Talpha1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes, and in vivo experiment it showed better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Talpha1 and TP5. Our findings suggest that Talpha1-TP5 fusion peptide might be a potential therapeutic agent.
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Affiliation(s)
- Demin Gao
- Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China
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27
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Fan YZ, Chang H, Yu Y, Liu J, Wang R. Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines. Peptides 2006; 27:2165-73. [PMID: 16644063 DOI: 10.1016/j.peptides.2006.03.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/03/2006] [Revised: 03/13/2006] [Accepted: 03/13/2006] [Indexed: 11/24/2022]
Abstract
Thymosin alpha1 (Talpha1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Talpha1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Talpha1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Talpha1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Talpha1 than all the human leukemia cell lines. D-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Talpha1, whereas atropine, a muscarinic acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Talpha1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.
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Affiliation(s)
- Ying-zhe Fan
- Department of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou 73000, PR China
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28
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Yang YF, Yuan HY, Liu NS, Chen XL, Gao BY, Lu H, Li YY. Construction, expression and characterization of human interferon α2b-(G4S) n-thymosin α1 fusion proteins in Pichia pastoris. World J Gastroenterol 2005; 11:2597-602. [PMID: 15849818 PMCID: PMC4305750 DOI: 10.3748/wjg.v11.i17.2597] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris.
METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S)n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex™ 75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.
RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex™ 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.
CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins’ function in vivo.
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Affiliation(s)
- You-Feng Yang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
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Romani L, Bistoni F, Gaziano R, Bozza S, Montagnoli C, Perruccio K, Pitzurra L, Bellocchio S, Velardi A, Rasi G, Di Francesco P, Garaci E. Thymosin alpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling. Blood 2004; 103:4232-9. [PMID: 14982877 DOI: 10.1182/blood-2003-11-4036] [Citation(s) in RCA: 162] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Dendritic cells (DCs) show a remarkable functional plasticity in the recognition of Aspergillus fumigatus and orchestrate the antifungal immune resistance in the lungs. Here, we show that thymosin alpha 1, a naturally occurring thymic peptide, induces functional maturation and interleukin-12 production by fungus-pulsed DCs through the p38 mitogen-activated protein kinase/nuclear factor (NF)-kappaB-dependent pathway. This occurs by signaling through the myeloid differentiation factor 88-dependent pathway, involving distinct Toll-like receptors. In vivo, the synthetic peptide activates T-helper (Th) cell 1-dependent antifungal immunity, accelerates myeloid cell recovery, and protects highly susceptible mice that received hematopoietic transplants from aspergillosis. By revealing the unexpected activity of an old molecule, our finding provides the rationale for its therapeutic utility and qualify the synthetic peptide as a candidate adjuvant promoting the coordinated activation of the innate and adaptive Th immunity to the fungus.
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Affiliation(s)
- Luigina Romani
- Department of Experimental Medicine and Biochemical Sciences Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy.
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30
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Hannappel E, Huff T. The thymosins. Prothymosin alpha, parathymosin, and beta-thymosins: structure and function. VITAMINS AND HORMONES 2003; 66:257-96. [PMID: 12852257 DOI: 10.1016/s0083-6729(03)01007-0] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The studies on thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immuno-deficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide beta 1, thymosin alpha 1, prothymosin alpha, parathymosin, and thymosin beta 4) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the thymosins is discussed in this review.
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Affiliation(s)
- Ewald Hannappel
- Institute for Biochemistry/Faculty of Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany
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31
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Abstract
The experimental material accumulated in the literature on the conformational behavior of intrinsically unstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity. Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premolten globules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poor solvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second group are essentially more compact, exhibiting some amount of residual secondary structure, although they are still less dense than native or molten globule proteins. An important feature of the intrinsically unstructured proteins is that they undergo disorder-order transition during or prior to their biological function. In this respect, the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globules, premolten globules, and random coils) and transitions between any two of the states, is discussed.
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Affiliation(s)
- Vladimir N Uversky
- Institute for Biological Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia.
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32
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Abstract
Prothymosin alpha (ProTalpha) is a highly acidic and small protein of only 111 amino acids with an unusual primary structure. One would expected it to play an essential role in the organism, as it has a wide distribution and is high conserved among mammals, yet its exact function remains elusive. Despite the number of effects described for ProTalpha, intracellular and extracellular, none are accepted as its physiological role. Furthermore, many other aspects of its biology still remain obscure. In this review, we discuss the structural properties, location, gene family, functions and immunomodulatory activities of and cellular receptors for ProTalpha. These topics are addressed in an attempt to reconcile opposing outlooks while emphasizing those points where scant investigations do exist. We have also re-evaluated some previous results in light of the structural properties of ProTalpha and have found that molecular mimetism could be the underlying basis. This molecular mimicry hypothesis provides a clue that must not be overlooked for a realistic appraisal of future results.
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Affiliation(s)
- A Piñeiro
- Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela. 15706, Santiago de Compostela, Spain
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33
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Chichkova NV, Evstafieva AG, Lyakhov IG, Tsvetkov AS, Smirnova TA, Karapetian RN, Karger EM, Vartapetian AB. Divalent metal cation binding properties of human prothymosin alpha. EUROPEAN JOURNAL OF BIOCHEMISTRY 2000; 267:4745-52. [PMID: 10903508 DOI: 10.1046/j.1432-1327.2000.01529.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.
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Affiliation(s)
- N V Chichkova
- Belozersky Institute of Physico-Chemical Biology and Center of Molecular Medicine, Moscow State University, Russia
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