Giuseppe Nicastro

Discovery of Subnanomolar Arginine-Glycine-Aspartate-Based αVβ3/αVβ5 Integrin Binders Embedding 4-Aminoproline Residues

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity alpha Vbeta 3/alpha Vbeta 5 integrin binders [IC 50 h (alpha Vbeta 3) 0.03-5.12 nM; IC 50 h (alpha Vbeta 5) 0.88-154 nM]. A total of eight cyclopeptides of type cyclo-[-Arg-Gly-Asp-Amp-], 5- 12, were assembled by a standard solid-phase peptide synthesis protocol that involved the C2-carboxyl and C4-amino functionalities of the proline scaffolds, leaving the N (alpha)-nuclear site untouched. Functionalization of this vacant proline site with either alkyl or acyl substituents proved feasible, with significant benefit to the integrin binding capabilities of the ligands. Notably, six out of eight cyclopeptide inhibitors, 5- 7 and 9- 11, showed moderate yet significant selectivity toward the alpha Vbeta 3 receptor. The three-dimensional structure in water was determined by NMR techniques and molecular dynamics calculations. Docking studies to the X-ray crystal structure of the extracellular segment of integrin alpha Vbeta 3 complexed with reference compound 1 were also performed on selected analogues to highlight the structural features required for potent ligand binding affinity.

Solution structure of the phytotoxic protein PcF: The first characterized member of the Phytophthora PcF toxin family

The PcF protein from Phytophthora cactorum is the first member of the “PcF toxin family” from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix‐loop‐helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.

Activity and NMR structure of synthetic peptides of the bovine ATPase inhibitor protein, IF1

The protein IF(1) is a natural inhibitor of the mitochondrial F(o)F(1)-ATPase. Many investigators have been prompted to identify the shortest segment of IF(1), retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF(1)-(42-58) and IF(1)-(22-46) is correlated to their structure and conformational plasticity determined by CD and [1H]-NMR spectroscopy. Among all the IF(1) segments tested, IF(1)-(42-58) exerts the most potent, pH and temperature dependent activity on the F(o)F(1) complex. The results suggest that, due to its flexible structure, it can fold in helical and/or beta-spiral arrangements that favor the binding to the F(o)F(1) complex, where the native IF(1) binds. IF(1)-(22-46), instead, as it adopts a rigid alpha-helical conformation, it inhibits ATP hydrolysis only in the soluble F(1) moiety.