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

Abstract

Prothymosin α (ProTα) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTα’s C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTα’s immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTα stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTα forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel β-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTα during apoptosis, strengthen our hypothesis that immune response stimulation by ProTα is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific β-sheet conformation and induce DC maturation.

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.