Phosphorylation of Prothymosin α. An Approach to Its Biological Significance.

Prothymosin α (ProTα), the precursor of the thymosin α1 and thymosin α11, is a 109-111 amino acids protein widely distributed in the mammalian tissues that is essential for the cell proliferation and survival through its implication on chromatin remodeling and in the proapoptotic activity. ProTα is phosphorylated at Thr residues by the M2 isoenzyme of the pyruvate kinase in a process that is dependent on the cell proliferation activity, which constitutes a novel dual functionality of this enzyme. The Thr residues phosphorylated are apparently dependent on the carcinogenic transformation of the cells. Thus, in normal lymphocytes residues Thr11 or Thr12 are phosphorylated in addition to a Thr7 residue, while in tumor cells Thr7 is the only residue phosphorylated. Phosphorylation of ProTα seems to be related to its antiapoptotic activity, although other possibilities cannot be discarded.

Binding of 125I-prothymosin alpha to lymphoblasts through the non-thymosin alpha 1 sequence.

The important immunological activities of Thymosin alpha 1 (T alpha 1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin alpha (ProT alpha) is a highly acidic polypeptide, first isolated as the putative precursor of T alpha 1. However ProT alpha is now known to be more immunoreactive than T alpha 1 in certain in vivo and in vitro assays. Recent results indicate that ProT alpha may be useful to design future therapeutic interventions in cancer patients if the mechanisms underlying these effects are puzzled out. With this in mind, we radiolabeled ProT alpha to obtain a high specific activity and a high biological activity for 125I-ProT alpha. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specificity for anti-ProT alpha and anti-T alpha 1. With both tools we studied the presence of binding sites for ProT alpha on the surface of lymphoblast cells. We conclude that ProT alpha binds through the non-T alpha 1 sequence.

On the anomalous behaviour on gel-filtration and SDS-electrophoresis of prothymosin-alpha.

The regulator of T-cell proliferation prothymosin alpha, is a protein with a relative molecular mass of 12 KDa as calculated from its amino acid sequence. This immunoregulator exhibits anomalous behaviour on gel-filtration and SDS-PAGE electrophoresis appearing as oligomers which are 5 or 2 fold larger than the corresponding polypeptide. These results suggest that a dimeric form of prothymosin alpha is stable to dissociation by SDS and reduction by beta-mercapto ethanol.

Prothymosin alpha enhances human natural killer cell cytotoxicity: role in mediating signals for NK activity.

We have investigated the effects of prothymosin alpha (ProT alpha) on the in vitro NK activity of various human cell populations obtained by successive purification of large granular lymphocytes (LGLs) and CD16+ cells. The results of this study indicate that ProT alpha is able to enhance the spontaneous NK activity of cells from normal donors. This effect was time and dose dependent in the range 7 to 350 nM, occurred over a wide range of effector/target cell ratios, and appeared not to require the presence of accessory cells. In addition, ProT alpha exhibited additive effects with recombinant interferon-gamma (rIFN-gamma) and recombinant interleukin-2 (rIL-2). Finally our data suggest that the effect of ProT alpha enhancing the NK cell cytotoxicity appears to involve enhancement of p70 IL-2R expression and internalization of IL-2, and was independent of cell proliferation.

Prothymosin alpha enhances interleukin 2 receptor expression in normal human T-lymphocytes.

We investigated whether the enhancement, by prothymosin alpha (Pro alpha), of the phytohaemagglutinin-stimulated proliferation of human peripheral blood mononuclear cells (PBMC) is due to its affect on the number of cells expressing the interleukin 2 receptor (IL-2R) or the surface density of IL-2R on PBMC. Peripheral blood mononuclear cells were obtained from 21 donors. For both an optimal phytohaemagglutinin (PHA) concentration (H) and a 10-fold dilution (L), their responses fell in two classes, high (h) and low (l), making four dose--response situations. Pro alpha significantly increased the number and IL-2R density of cells expressing IL-2R only when the response in its absence was about half maximal, i.e. for PBMC responding well to the low PHA stimulus (group Lh) or PBMC responding poorly to the optimal stimulus (group Hl). The enhancement of IL-2R expression in group Lh by Pro alpha was dose-dependent and paralleled by increased proliferative response. It appears not to be mediated by IL-2, since it was unaffected when IL-2 production was suppressed by cyclosporin A. The early interaction of Pro alpha with lymphocytes did not require the presence of macrophages, but macrophages were necessary during lymphocyte activation for modulation of PHA-stimulated IL-2R expression to be affected. The immunoregulatory activity of Pro alpha may prove useful for improving the decreased T-cell function associated with immunodeficiency, or for restoration of normal IL-2R expression by the lymphocytes of aged individuals.

Phytohemagglutin-stimulated human T cell: prothymosin alpha as an accessory signal.

Thymic peptide factors are known to modulate proliferation of normal human lymphocytes. In this work, we studied the effect of Prothymosin alpha (Pro alpha) on PHA-stimulated PBMC and PBLC. The observed effects of Pro alpha and thymosin alpha 1 (alpha 1) on PBMC were found to depend on the degree of cell stimulation, dose, and preincubation-time. Thymosin beta 4 (beta 4) had no effect on either cell type, regardless of the degree of stimulation, which shows that beta 4 may be used as a control peptide to work in this area. Induction of lymphoproliferation also depended on the presence of macrophages. Addition of monocytes or a cell-free monocyte culture supernatant (not containing IL-2) to the PHA-stimulated PBLC cultures resulted in T cell proliferation. Although IL-1 could not restore the PHA-induced proliferative response of isolated T cells by itself, it would enhance the helper effect of Pro alpha. Moreover, a polyclonal goat anti-human IL-2R (Tac Ag) did block the proliferative response induced by combined rIL-1 and Pro alpha, suggesting that an IL-2-dependent pathway of T cell proliferation was involved.