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The fibroblast growth factor (FGF)/FGF receptor (FGFR) system plays a crucial role in cancer by affecting tumor growth, angiogenesis, drug resistance, and escape from anti-angiogenic anti-vascular endothelial growth factor therapy. The soluble pattern recognition receptor long-pentraxin 3 (PTX3) acts as a multi-FGF antagonist. Here we demonstrate that human PTX3 overexpression in transgenic mice driven by the Tie2 promoter inhibits tumor growth, angiogenesis, and metastasis in heterotopic, orthotopic, and autochthonous FGF-dependent tumor models. Using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2, we identified a small-molecule chemical (NSC12) that acts as an extracellular FGF trap with significant implications in cancer therapy.

Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-and two-dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured [~-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of [3-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.

The use of spectroscopy in the study of fatty acids binding to bovine ␤-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Bovine b-lactoglobulin~BLG! in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13 C enriched palmitic acid~PA! and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13 C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.Keywords: bovine b-lactoglobulin; lipocalin binding sites; nuclear magnetic resonance; palmitic acid; T 1 measurements; Tanford transition Bovine b-lactoglobulin~BLG! is a 18 kDa protein belonging to the lipocalin superfamily, a big family of proteins with a variety of biological functions related to the binding and transport of metabolites. Although lipocalins show low sequence similarity, they share a common b-barrel topology, as the major structural motif. Structure determination of bovine BLG has been performed by X-ray and NMR at neutral and low pH, respectively~Brownlow et al

Bovine beta-Lactoglobulin (BLG) has been studied for many decades, but only recently structural data have been obtained, making it possible to simulate its molecular properties. In the present study, electrostatic properties of BLG are investigated theoretically using Poisson-Boltzmann calculations and experimentally following pH titration via NMR. Electrostatic properties are determined for several structural models, including an ensemble of NMR structures obtained at low pH. The changes in electrostatic forces upon changes in ionic strength, solvent dielectric constant, and pH are calculated and compared with experiments. pK(a)s are computed for all titratable sites and compared with NMR titration data. The analysis of theoretical and experimental results suggests that (1) there may be more than one binding sites for negatively charged ligands; (2) at low pH the core of the molecule is more compact than observed in the structures obtained via restrained molecular dynamics from NMR data, but loop and terminal regions must be disordered.

We have determined a crude structure of the apo form of bovine L L-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear I H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a L L-barrel fold, wellsuperimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.z 1998 Federation of European Biochemical Societies.

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.