Inhibitory action of polyunsaturated fatty acids on Cdt1-geminin interaction

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme-linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural compounds, and found that a fatty acid, linoleic acid (C18:2), from a basidiomycete, inhibited Cdt1-geminin interaction in vitro. Of the commercially purchased linear-chain fatty acids tested, the inhibitory effect of oleic acid (C18:1) was the strongest, with 50% inhibition observed at concentrations of 9.6 μM. Since trans-configuration, the ester form, and the addition of the hydroxyl group of oleic acid had no influence on C18:1 fatty acid derivatives, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Surface plasmon resonance analysis demonstrated that oleic acid was able to bind selectively to Cdt1, but did not interact with geminin. Using a three-dimensional computer modeling analysis, oleic acid was conjectured to interact with the geminin interaction interface on Cdt1, and the carboxyl group of oleic acid was assumed to form hydrogen bonds with the residue of Arg342 of Cdt1. These results suggested that, at least in vitro, oleic acid-containing cell membranes of the lipid bilayer inhibit Cdt1-geminin complex formation by binding to Cdt1 and thereby liberating Cdt1 from inhibition by geminin.