Confronto strutturale e studio dell'attività biologica di Angiogenina e Lactogenina per possibili applicazioni di terapia medica e ingegneria tissutale

Ribonucleases (RNases) are proteins involved into many biological processes and hydrolysis of ribonucleic acid. The topic of this study has been the structural and functional characterization of three bovine proteins, namely RNase-A, Angiogenin-1 (bANG) and Lactogenin. The enzymatic, pro-angiogenic and cytotoxic activity has been evaluated. RNase-A is the best known protein showing a strong ribonucleolytic activity and a low cytotoxic action. bANG is a single-chain polypeptide stimulating angiogenesis; it has a weak ribonucleolytic activity necessary, but not sufficient, to induce the neoformation of blood vessels. Lactogenin, also named RNase BL-4, has been relatively poorly studied. In this study the relationship between molecular structure and biological activity of the three RNases has been evaluated by spectroscopy, ribonucleolytic activity analysis and, finally, by their effects on the viability of human endothelial or cancerous cellular cultures. High homology level of primary structure is resulted for all three under study proteins. Moreover, disulfide architecture is preserved except the missing of one bridge in bANG and the presence of a pyroglutamic residue at the N-terminus of Lactogenin. It is known that RNase-A is far more active in cleaving dinucleotide substrates as CpG and UpG in comparison with Lactogenin and bANG. On the other hand, Lactogenin presents a higher specificity for UpG instead of CpG than bANG. Angiogenin has been treated with immobilized trypsin in order to obtain its tryptic peptides and identify which part of protein is more involved into biological activity. The research study has shown that bANG stimulates the proliferation and capillary-like structures formation of Huvec endothelial cells in Matrigel in vitro angiogenic assay. The fragment 1-6 (N-terminal, termed P1) and the fragment 56-61 have stimulated a cellular proliferation response comparable to the native protein's one while the C-terminal fragment 103-124 has exhibited an inhibition effect. Moreover, in the presence of all the fragments P1, 56-61 and C-terminal the cells have demonstrated branch points formation, after seeding on Matrigel. The activity of Lactogenin has been comparable to the one of bANG, even if less strong, while RNase-A have not stimulated HUVEC cells' proliferation or differentation on Matrigel. Angiogenin and Lactogenin have been shown to promote the migration of cultured endothelial cells and the neovascularization in the chicken chorioallantoic membrane. The attachment and growth of HUVEC cells on synthetic materials such as polycaprolactone scaffolds seem to be improved by the addition of Angiogenin and its N-terminal fragment to the culture medium. The cytotoxic properties of RNase-A, bANG and Lactogenin have been valuated by using some tumor cell lines. bANG has expressed a stronger cytotoxic potential, inducing cell death by an apoptotic mechanism. In comparison with RNase A, the major cytotoxicity of bANG could be explained as a minor interaction with the RNAse inhibitor (RI). This hypothesis has been verified modelling bANG structure on the complex human Angiogenin-hRI, as the protein conformation is not so different in the complex from the free form. Many different contacts with hRI have been observed in hANG and bANG. One of the principal anchorage sites of hRI to hANG is resulted the Pro 88 residue, which lies within a hydrophobic pocket defined by three tryptophan residues of hRI. In bANG, the replacing of Pro88, with Ser89 causes a steric and electrostatic strain in the inhibitor enzymatic complex, decreasing the susceptibility of bANG to the inactivation by RI. Further studies will clarify the binding of bANG to hRI by calculating the inhibition constant.