Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains.

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.

Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisometry modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4' position is not the sole determinant for antioxidant activity. In contrast, the presence of 4'-OH together with stereoisometry in the trans-conformation (4'-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases alpha and delta.