RESUMO
The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.
Assuntos
Neoplasias , Timosina , Humanos , Timalfasina , Galectina 1RESUMO
The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.
Assuntos
Amiloidose , Pré-Albumina , Amiloidose/metabolismo , Cálcio/metabolismo , Humanos , Pré-Albumina/química , ProteóliseRESUMO
Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.
Assuntos
Sequência de Aminoácidos , Ácido Hialurônico/química , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Timosina/análogos & derivados , Espectroscopia de Ressonância Magnética , Ligação Proteica , Timalfasina , Timosina/químicaRESUMO
The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.