Prothymosin α immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a β-sheet conformation in a sequence-specific manner
Introduction
Prothymosin α (ProTα) is a glutamic-rich, 109 amino acid (aa) residue long polypeptide (Haritos et al., 1984), principally located in the cell nucleus (Manrow et al., 1991). ProTα is highly conserved among mammals and ubiquitously expressed in all tissues (Haritos, 1987), at levels in excess of 10,000 copies per cell (Eschenfeldt and Berger, 1986). This high level of expression suggests ProTα’s participation in important cellular molecular circuits. Ample experimental evidence highlights the crucial role of ProTα in cell survival and proliferation. With respect to the latter, it has been shown that ProTα regulates DNA remodelling during proliferation (Díaz-Jullien et al., 1996) and modulates nuclear processes by directly interacting with free-core histone H1 (Gomez-Marquez et al., 1989, Karetsou et al., 2002). In addition, ProTα regulates apoptosis through prevention of apoptosome formation (Jiang et al., 2003), while being itself an early target of activated caspase-3 (Enkemann et al., 2000). In cells undergoing apoptosis, ProTα cleavage by caspases generates a carboxyl (C)-terminally truncated polypeptide (aa 1–99), which sequesters cytochrome C (Markova et al., 2003).
Initially isolated from rat thymus (Haritos et al., 1984), ProTα was so-named as the precursor polypeptide of thymosin α1-related peptides and was believed to be the main molecule responsible for the immmunostimulatory capacity of the thymic extract, thymosin fraction 5. Since 1984, studies on ProTα attributed significant immunoenhancing properties to the polypeptide, leading to its classification in the broad family of biologic response modifiers (Pan et al., 1986). Thus, in in vitro experiments, ProTα enhances T-cell proliferation in response to cellular antigens (Baxevanis et al., 1988), increases monocyte HLA-DR molecule expression (Baxevanis et al., 1992), amplifies natural killer (NK) cell cytotoxicity via induction of perforin production and integrin expression (Skopeliti et al., 2006, Voutsas et al., 2000) and augments hydrogen peroxidase-mediated neutrophil antimicrobial responses (Heidecke et al., 1997). ProTα’s immunoactivity is reported to be even more pronounced on lymphocytes of patients with malignancies (Eckert et al., 1997, Garbin et al., 1997) or autoimmune diseases (Reclos et al., 1987) in vitro and most importantly, it can potentiate the deficient responses of immunosuppressed or tumor-bearing animals in vivo (Haritos, 1987, Papanastasiou et al., 1992). These data suggest that its application may be of benefit in clinical protocols.
Investigating the molecular mechanisms responsible for ProTα’s activity, recent data from our group suggest that its immunologically active segment is located at the C-terminus of the polypeptide, spanning the sequences aa(89–102) and aa(103–109) (Skopeliti et al., 2006). Therefore, it seems that different parts of the molecule are responsible for ProTα’s dual mode of action, the central, energy-rich histone-binding region spanning aa(52–82) for its intracellular/proliferative role (Papamarcaki and Tsolas, 1994) and the C-terminal region aa(89–109) for its extracellular/immunomodulating function (Skopeliti et al., 2006). Moreover, via proteomic analysis we were able to provide evidence that ProTα’s immune effect is exerted via toll-like receptor (TLR) signaling (Skopeliti et al., 2007). Interestingly, this information supplemented previous results supporting ProTα’s monocyte-dependent mode of action in unfractionated peripheral blood mononuclear cell (PBMC) cultures (Voutsas et al., 2000), as well as its activity on cells of both the innate (e.g. macrophages, neutrophils, NK cells) (Cordero et al., 1995, Heidecke et al., 1997, Papanastasiou et al., 1992) and the adaptive arms of immunity (e.g. CD4+ and CD8+ T-cells) (Voutsas et al., 2000).
In the context of these pleiotropic activities, here we aimed to elucidate ProTα’s immune-related mechanism of action underlying its immunoenhancing properties. First, in order to identify the exact sequence comprising the ‘immunological core’ of ProTα, we assessed the activity of prolonged synthetic peptides, homologous to the polypeptide’s C-terminus. Second, given that ProTα acts on monocytes via TLR, we investigated the causal connection between monocyte stimulation with ProTα or its active peptide and their subsequent maturation into competent dendritic cells (DC). Finally, we sought to explore the structural characteristics of ProTα and its active peptide attributing their activity to a specific secondary conformation.
Section snippets
Peptide synthesis
Peptides were synthesized by the Fmoc (9-fluorenylmethoxycarbonyl)/tBu chemistry utilizing a multiple peptide synthesizer Syro II (MultiSynTech, Witten, Germany). After cleavage, crude peptides were purified by HPLC on a reverse phase C18 Nucleosil 100-5C column (HPLC Technologies, UK) to a purity of >95%, using a linear gradient of 5–80% acetonitrile in 0.05% trifluoroacetic acid for 45 min. All peptides were characterized by matrix-assisted laser desorption ionization-time of flight mass
Identification of the active peptide segment (TKKQKTDEDD) of ProTα
Previous data support the localization of ProTα’s immunoreactive fragment at the C-terminus of the polypeptide, specifically within the sequences aa(89–102) and aa(103–109) (Skopeliti et al., 2006). This was the first indication that ProTα’s immunological role can be attributed to the activity of a defined segment, immunologically overlooked to date and considered important only for the polypeptide’s nuclear targeting (Manrow et al., 1991). In the present study, we sought to precisely define
Discussion
ProTα was initially isolated as the precursor molecule of the immunostimulatory Tα1-related peptides (Haritos et al., 1984). However, it was shown to be more effective in stimulating various leukocyte reactions than other members of the α-thymosin family (Haritos, 1987). Accumulating evidence yet attributes a different, albeit equally significant, role to ProTα. The polypeptide sustains nuclear events related to stimulation of cell proliferation (Karetsou et al., 1998) and regulation of
Acknowledgements
This work was partly supported by the GSRT, Ministry of Development (Grant 05-NON EU-404) to OET and by the Deutsche Forschungsgemeinschaft (SFB 685 B2, B4).
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2021, MitochondrionCitation Excerpt :As a member of the α-thymosin family, prothymosin α (PTMA), comprising 109–110 highly acidic amino acids, is ubiquitously expressed in various types of cells and highly conserved in mammals, which has been generally considered as a nuclear protein with a nuclear localization signal (NLS) (Kijogi et al., 2016; Su et al., 2013). PTMA plays important roles in various biological processes, such as cell cycle, proliferation, apoptosis, gene transcription and immunomodulation (Karetsou et al., 2002; Kijogi et al., 2016; Martini et al., 2000; Skopeliti et al., 2009). For example, PTMA promotes gene transcription by interacting with the CREB-binding protein (Karetsou et al., 2002).
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2016, Vitamins and HormonesCitation Excerpt :The C-terminal decapeptide proTα(100–109) (TKKQKTDEDD) was identified by our research team as the immunoactive area of the polypeptide and a potent lymphocyte stimulator (Skopeliti et al., 2006). ProTα(100–109) has been shown to stimulate PBMC proliferation and cytotoxicity and to promote the phenotypic maturation of DCs (Skopeliti et al., 2009), and, consequently, it improved the functionality of immunogenic peptide-pulsed DCs, induced TH1-type immune response polarization (Ioannou et al., 2013), augmented basic properties of human neutrophils (Samara, Ioannou, et al., 2013), enhanced the depressed cytotoxicity of tumor-associated lymphocytes against autologous tumor cells in vitro, and retarded tumor growth in vivo (Voutsas et al., 2013). Using as control a scrambled decapeptide with the same amino acid composition but a different primary structure, the immunoenhancing activity of proTα(100–109) was shown to be sequence-specific and comparable to that of intact proTα.
Structures of Thymosin Proteins
2016, Vitamins and HormonesCitation Excerpt :Due to the apparent immunomodulating role of the ProTα C-terminal tail elucidated by Tsitsilonis and coworkers (Skopeliti et al., 2006), they sought to investigate the structure of the ProTα carboxy-terminus, residues 100–109 (TKKQKTDEDD) which includes the nuclear localization signal (NLS) TKKQKT (Manrow, Sburlati, Hanover, & Berger, 1991). By comparing attenuated total reflectance Fourier-transform infrared (ATF FT-IR) spectra of the C-terminus, truncated versions of it, including the NLS, and a scrambled version of it, they (Skopeliti et al., 2009) observed wave number ranges for the amide I, amide II, and amide III regions of the ATF FT-IR spectra that were most consistent with the formation of antiparallel β-pleated sheets. Their model structures based on similar hairpin peptides deposited in the Protein Data Bank (Berman et al., 2000) were also consistent with these structures (Fig. 1).
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