Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as “thymic hormones,” are produced by this gland. Although the majority of them have not been proven to be thymus-speciWc, thymic peptides comprise an eVective group of regulators, mediating important immune functions. Thymosin fraction Wve (TFV) was the Wrst thymic extract shown to stimulate lymphocyte proliferation and diVerentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin (proT) and thymosin 1 (T1) showed that they are of clinical signiWcance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeWciencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their eVect are yet not fully elucidated proT and T1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proT, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of T1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proT into the clinical setting

Solid-phase synthesis of a biotin derivative and its application to the development of anti-biotin antibodies

A biotin derivative, namely biotin-aminocaproic acid-lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid-lysine "long-arm" was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its Nε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic "lysine-dendrimer" was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten. © 2009 Springer Science+Business Media, LLC

Solid-phase synthesis of a peptide derivative of thymosin alpha1 and initial studies on its 99mTc-radiolabelling

A derivative (1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be 99mTc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment Tα1[1-14] as a bioactive segment, at the C-terminus of which a 99mTc-chelating moiety consisting of Nα,Nα-dimethylglycine, serine and cysteine is linked through the Nε-amino group of a 'bifunctional' lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid (dmGSCK{N ε-Tα1[1-14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with 99mTc, thus resulting in the first, to our knowledge, so far reported 99mTc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide. © 2007 The Authors

Thermal unfolding of human BRCA1 BRCT-domain variants

Missense mutations at the BRCT domain of human BRCA1 protein have been associated with an elevated risk for hereditary breast/ovarian cancer. They have been shown to affect the binding site and they have also been proposed to affect domain stability, severely hampering the protein's tumor suppressor function. In order to assess the impact of various such mutations upon the stability and the function of the BRCT domain, heat-induced denaturation has been employed to study the thermal unfolding of variants M1775R and R1699W, which have been linked with the disease, as well as of V1833M, which has been reported for patients with a family history. Calorimetric and circular dichroism results reveal that in pH 9.0, 5 mM borate buffer, 200 mM NaCl, analogously to wild type BRCT, all three variants undergo partial thermal unfolding to a denatured state, which retains most of the native's structural characteristics. With respect to wild-type BRCT, the mutation M1775R induces the most severe effects especially upon the thermostability, while R1699W also has a strong impact. On the other hand, the thermal unfolding of variant V1833M is only moderately affected relative to wild-type BRCT. Moreover, isothermal titration calorimetric measurements reveal that contrary to M1775R and R1699W variants, V1833M binds to BACH1 and CtIP phosphopeptides. © 2007 Elsevier B.V. All rights reserved

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTα), i.e. against either non-conjugated ProTα (isolated from bovine thymus) or ProTα conjugated to keyhole limpet hemocyanin (ProTα/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTα, while antibodies Y against ProTα/KLH had a better apparent titer, could better discriminate between ProTα and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G. © 2005 Elsevier Inc. All rights reserved

Development of a specific IgY-based ELISA for prothymosin alpha, a bioactive polypeptide with diagnostic and therapeutic potential

Cell biology; Immunology; Proteins; Biochemistry; Cell necrosis; ELISA; HeLa cell culture supernatants; Immunoglobulins Y (IgY); Prothymosin alpha (ProTα) © 2019 The Author(s) Prothymosin alpha (ProTα) is a highly conserved polypeptide (109 amino acids in humans) with diagnostic and therapeutic potential; ProTα exerts intra- and extra-cellular biological functions associated with cell proliferation, apoptosis and immune regulation, while it has been suggested to act as a damage-associated molecular pattern (DAMP) or alarmin. In this work, chicken polyclonal anti-ProTα antibodies that had been developed several years ago were immunochemically evaluated and proven to retain immunoreactivity for ProTα, with remarkable thermal and pH stability. Moreover, the antibodies showed practically no cross-reactivity with a series of ProTα-fragments, eventually intracellularly produced -such as ProTα[1-28] (also known as Tα1) and ProTα[100-109], which exert per se biological activity and might be present in biological samples along with the intact molecule, being therefore highly specific for whole-length ProTα. Based on the above antibodies (IgYs-3e), a highly specific competitive ProTα-ELISA with well-studied analytical characteristics (intra- and inter-assay CVs: ≤5% and ≤12%, respectively, limit of detection: 2.1 ng/mL, recovery: 88–104%) was developed. The new ProTα-ELISA was applied to the analysis of supernatants of HeLa cells driven to necrosis; intact ProTα was measured in cell culture supernatants, at levels that seemed to depend on % cell necrosis. © 2019 The Author(s

The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα was trypsinised, and the five fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTα(89-102) and proTα(103-109) significantly fortified healthy donor-lymphocytes' immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89-102) and proTα(103-109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1-14), proTα(21-30) and proTα(31-87) marginally upregulated lymphocyte activation. This is the first report showing that proTα's immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients. © Springer-Verlag 2006