Elsevier

Vitamins and Hormones

Volume 102, 2016, Pages 101-119
Vitamins and Hormones

Chapter Five - Mechanism of Action of Thymosinα1: Does It Interact with Membrane by Recognition of Exposed Phosphatidylserine on Cell Surface? A Structural Approach

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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 1H and natural abundance 15N 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.

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.

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