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

Prothymosin (ProT) 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 ProT's C-terminus to stimulate lymphocyte functions, in particu- lar 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 activ- ity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProT's immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProT 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 spe- cific conditions, ProT forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative stain- ing). Although the peptide TKKQKTDEDD adopts an antiparallel -sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in con- junction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProT during apoptosis, strengthen our hypothesis that immune response stimulation by ProT is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific -sheet conformation and induce DC maturation.