Chapter Five - Mechanism of Action of Thymosinα1: Does It Interact with Membrane by Recognition of Exposed Phosphatidylserine on Cell Surface? A Structural Approach
Introduction
The α-thymosins belong to a group of peptides isolated for the first time from calf thymus extracts, denominated Thymosin fraction 5 (TF5) (Goldstein, Guha, Zatz, Hardy, & White, 1972) which showed both in vitro and in vivo systems immune regulatory properties (Low, Thurman, McAdoo, et al., 1979).
Thymosinα1, the most abundant α-Thymosin found in TF5, was the first isolated and sequenced (Low & Goldstein, 1979). It is a 28-amino acid peptide with sequence: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH, where Ac indicates that is acetylated on its N-terminus group. The peptide derives from the N-terminus tract of ProThymosinα (ProTα) from where is cleaved by legumain, a lysosomal asparagine endopeptidase (AEP) present also in mammals (Sarandeses, Covelo, Díaz-Jullien, & Freire, 2003). Both Thymosinα1 (Franco, Diaz, Barcia, & Freire, 1992) and legumain (Chen, Dando, Rawlings, et al., 1997) share a wide distribution in different tissues, suggesting that the ProTα processing to yield Thymosinα1 represents a generalized process in mammalian tissues. Moreover, high legumain activity has been found in lymphoid tissues that also show high levels of Thymosinα1 (Franco et al., 1992), which argue for important biological function of the peptide in these cells.
Thymosinα1 cellular location, in the absence of secretion signals and the incapacity for nuclear migration, is in accord for a cytosolic localization differently from its precursor ProTα (Eschenfeldt et al., 1989, Manrow et al., 1991).
Actually, Thymosinα1 is approved in different countries for treatment of several viral infections (Andreone et al., 2001, Iino et al., 2005, Kullavanuava et al., 2001, You et al., 2006) and as an adjuvant for immune enhancement (Carraro et al., 2012, Goldstein, 2009). Moreover, it is also developed for treatment of non-small cell lung cancer, hepatocellular carcinoma, AIDS, and malignant melanoma (Billich, 2002, Maio et al., 2010). An univocal mechanism of action of Thymosinα1is still unknown since no specific receptors have been identified at the level of the lymphocyte membrane and a growing body of evidence obtained in the last decades attests the clear-cut pleiotropy of this peptide, which targets both normal and tumor cells interacting with multiple cellular components (Garaci et al., 2012, Romani et al., 2012, Serafino et al., 2014).
Previous research showed that Thymosinα1 upregulates the expression of the major histocompatibility complex class I in murine and human tumor cell lines and in primary cultures of human monocyte-enriched peripheral blood mononuclear cells (Giuliani, Napolitano, Mastino, et al., 2000). Thymosinα1 induces T-cell and dendritic cell (DC) maturation and interleukin (IL-2) expression (Knutsen et al., 1999, Romani et al., 2004); it is also capable of upregulating the expression of Toll-like receptors (TLRs) 2 and 9 greater than for 5 and 8 in murine DCs and protects mice from challenge by invasive aspergillosis in the MyD88 (myeloid differentiate factor 88)-dependent way (Romani et al., 2004). Thymosinα1 activates a TRAF6- atypical protein kinase C (PKC)-IkB kinase signaling pathway that leads to the activation of nuclear factor-kB, which in turn initiates cytokine gene expression in murine bone marrow-derived macrophages (Zhang, Chan, Dragoi, et al., 2005). The complex of these findings has generated a renewed interest of the research on this hormone peptide, which is aimed to a better knowledge of its therapeutic potential and to an improvement of its delivery to the target cells, thus implementing its own effectiveness. In this regard, the conjugation of Thymosinα1 with a RGD motif has been attempted very recently to implement Thymosinα1 interaction with tumor cells (Lao, Liu, Chen, & Zheng, 2013). In fact, the amino acidic RGD sequence (Arg-Gly-Asp) is able to recognize integrins avb3, which is specifically associated with upregulated expression of tumor vessels and tumor cells of the endothelium (Zetter, 1997). All these researches would benefit of a greater understanding of the mechanism(s) involved in the Thymosinα1-cell interaction.
Section snippets
The NMR Structural Study in Trifluoroethanol Solution
In this regard, the knowledge of the Thymosinα1 conformation when interacting with membranes may represent a useful step toward a better understanding of all the processes involved in its action. In the past, investigations by NMR has been carried out (Elizondo-Riojas et al., 2011, Grottesi et al., 1998) the latter with a limited instrumental NMR resolution and, the former, at much higher magnetic field. Both studies were performed using trifluoroethanol (TFE) as solvent. This solvent was for
The Structural Study by NMR in Micellar Environment
The limitation of the above reported studies is that the chaotropic solvent (TFE) wraps around the peptide structuring it into a helical conformation. Considering that Thymosinα1 may interact with cellular membrane by partial insertion, the determination of these structures in TFE did not give sufficient information to support a feasible hypothesis on the mechanism of action. Thus, the hypothesis that an interaction with membrane might be the possible first step of the mechanism of action was
The Interactions with Phospholipidic Membranes with Negative Regions
These findings led us to investigate the behavior of Thymosinα1 in the presence of membrane models containing phospholipids that are the natural constituents of the membrane. The interaction either with vesicles of dodecylphosphocholine alone or with vesicles of dodecylphosphocholine-sodium dodecylsulfate was performed. The results of the NMR spectroscopy experiments indicated that Thymosinα1 interacts by an aspecific modality with phospholipidic membrane model exposing choline polar heads on
The Conformation of Thymosinα1 in Mixed DPC-d38/SDS-d25 Micelles
The collection of NOEs reported above allowed us to perform a restrained molecular dynamics simulation of Thymosinα1 inserted in the mixed micelles. The results allowed us to report the final structure in Fig. 2 where Thymosinα1 appears structured in helical conformation in two tracts, the first one from residue 1 to 6 which corresponds to the region inserted in mixed micelles and the second one from residue 15 to about 26. A flexible tract in between gives the mobility of the two secondary
The Interaction of Thymosinα1 with Perdeuterated DPC and Perdeuterated DPC–SDS Micelles
The determination of the nuclear magnetic spin-lattice relaxation times of the NMR resonances of Thymosinα1 in the presence of mixed micelles of DPC-d38/SDS-d25 gave for the aliphatic resonances a longitudinal relaxation time (T1) of about 570 ms. The same spectral region in the sample of Thymosinα1 in water the value of the spin-lattice relaxation time obtained was about 813 ms. According to the Bloembergen–Pound–Purcell theory the difference of correlation times of Thymosinα1 in water or in
The Circular Dichroism Study of Thymosinα1 in Perdeuterated DPC and Perdeuterated DPC–SDS Micelles
The circular dichroism spectroscopy of Thymosinα1 in perdeuterated micelles of DPC and DPC–SDS showed spectra with very different shapes. Particularly the spectrum of Thymosinα1 in DPC micelles is similar to that obtained in water with a spectral shape diagnostic of a poorly stable structuration if any. On the other hand the spectrum of Thymosinα1 in DPC–SDS mixed micelles can be attributed to a helical conformation. On the basis of the results of previous studies on circular dichroism
The Structural Study by NMR of the Interaction of Thymosinα1 with Phosphatidylserine in Membranes
Thymosinα1, unstructured in water solution interacts with negative regions of vesicles assuming two tracts of helical conformation with a structural break in between. The study of the interaction of Thymosinα1 with vesicles with mixed dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine, the negative component of the membranes by 1H and natural abundance 15N NMR is herewith reported. The results indicate that the preferred interactions are those where the membrane is negatively
15N NMR Spectroscopy Study of the Interaction
To obtain a clear description of the mechanism of interaction of Thymosinα1 with negative regions of the vesicle, a natural abundance 15N NMR study of the interaction was performed. The possibility of obtaining the 15N HSQC spectra without interference of the numerous phospholipid protons allowed us to study in detail the perturbation of the residues involved in PS interaction. In fact 15N resonances also at natural abundance are sensitive of the change of physico-chemical environment and this
Implications of Thymosinα1 Binding to PS Exposure
Phosphatidylserine (PS) is the negative phospholipid that is generally mainly localized in the inner leaflet of membranes and its exposure is due to the action of enzymes like scramblases (An et al., 2001). Recent studies shed light on the potential function of PS interaction with cytoskeletal proteins to mediate anchorage of actin filaments to the phospholipid bilayer. Moreover, the erythrocyte protein 4.1R that binds to phosphatidylserine interacts with the negative head-group and,
Implication of Thymosinα1 Binding to Membrane and Cells
The binding to membrane of Thymosinα1 together with the assumption of structural elements absent in water solution led us to hypothesize that this can be included among the many pathways proposed for the action of these peptidic hormones. After the proposal of Sargent and Schwyzer (1986), several cases have been reported in literature where an insertion in the membrane, also partial, can be considered an initial step of a biological cascade. Many examples have been proposed of binding to model
Acknowledgments
The technical assistance of Fabio Bertocchi in the skillful maintaining and checking the performances of the NMR instrumentation is gratefully acknowledged.
References (67)
- et al.
Structural and functional characterization of protein 4.1R-phosphatidylserine interaction potential role in 4.1R sorting within cells
The Journal of Biological Chemistry
(2001) - et al.
Reduction-of-dimensionality kinetics at reaction-limited cell surface receptors
Biophysical Journal
(1994) - et al.
Thymosin-alpha 1 (Zadaxin) enhances the immunogenicity of an adjuvated pandemic H1N1v influenza vaccine (Focetria) in hemodialyzed patients: A pilot study
Vaccine
(2012) - et al.
Potential role of the membrane in hERG channel functioning and drug-induced long QT syndrome
Biochimica et Biophysica Acta
(2010) - et al.
Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase
The Journal of Biological Chemistry
(1997) - et al.
Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor
The Journal of Biological Chemistry
(2004) - et al.
Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: An electrostatic model and experimental results
Biophysical Journal
(1998) - et al.
NMR structure of human Thymosinalpha-1
Biochemical and Biophysical Research Communications
(2011) - et al.
Isolation and partial sequencing of the human proThymosinalpha gene family. Evidence against export of the gene products
The Journal of Biological Chemistry
(1989) - et al.
Thymosinalpha 1 is a native peptide in several tissues
Biochimica et Biophysica Acta
(1992)
Oxidative stress causes membrane phospholipid rearrangement and shedding from RBC membranes—An NMR study
Biochimica et Biophysica Acta
The conformation of peptide Thymosinalpha 1 in solution and in a membrane like environment by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane
Peptides
Thymosin-alpha1 stimulates maturation of CD34 + stem cells into CD3+4 + cells in an in vitro thymic epithelia organ coculture model
International Journal of Immunopharmacology
Annexin 5 and apolipoprotein E2 protect against Alzheimer's amyloid-beta-peptide cytotoxicity by competitive inhibition at a common phosphatidylserine interaction site
Peptides
Exposure of platelet membrane phosphatidylserine regulates blood coagulation
Progress in Lipid Research
Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor
Biophysical Journal
The chemistry and biology of thymosin. II. Amino acid sequence analysis of Thymosinalpha1 and polypeptide beta1
The Journal of Biological Chemistry
The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of Thymosinalpha1 and polypeptide beta1 from calf thymus
The Journal of Biological Chemistry
Solution NMR studies of cell-penetrating peptides in model membrane systems
Advanced Drug Delivery Reviews
Nuclear targeting of proThymosinalpha
The Journal of Biological Chemistry
Evidence for a phosphatidylserine binding site in factor v; the second c-type domain
The Journal of Biological Chemistry
Thermodynamics of the interaction between oxytocin and its myometrial receptor in sheep: A stepwise binding mechanism
Biochemical Pharmacology
Thymosinalpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling
Blood
ProThymosinalpha is processed to Thymosinalpha 1 and Thymosinalpha 11 by a lysosomal asparaginyl endopeptidase
The Journal of Biological Chemistry
Acylated and unacylated ghrelin binding to membranes and to ghrelin receptor: Towards a better understanding of the underlying mechanisms
Biochimica et Biophysica Acta, Biomembranes
Chronic ethanol administration induced an increase in phosphatidylserine in guinea pig synaptic plasma membranes
Biochemical and Biophysical Research Communications
Ligands, their receptors and plasma membranes: A review
Molecular and Cellular Endocrinology
Reaction rate enhancement by surface diffusion of adsorbates
Biophysical Chemistry
In vitro effect of thymosin-alpha1 and interferon-alpha on Th1 and Th2 cytokine synthesis in patients with chronic hepatitis C
Journal of Viral Hepatitis
Thymosin alpha1 SciClone Pharmaceuticals
Current Opinion in Investigational Drugs
Lipid membrane-induced optimization for ligand–receptor docking: Recent tools and insights for the “membrane catalysis” model
European Biophysical Journal
Thymosinα1 and cancer: Action on immune effector and tumor target cells
The Annals of the New York Academy of Sciences
Thymosin-alpha1 regulates MHC class I expression in FRTL-5 cells at transcriptional level
European Journal of Immunology
Cited by (3)
Thymosin α1 interacts with Galectin-1 modulating the β-galactosides affinity and inducing alteration in the biological activity
2023, International ImmunopharmacologyPotential mechanism of thymosin-α1-membrane interactions leading to pleiotropy: experimental evidence and hypotheses
2018, Expert Opinion on Biological Therapy