Fusicoccin; molecular mimicry and novel 14-3-3 targets in animal cells

The study described in this project seeks to explore novel aspects related to the action of the phytotoxin fusicoccin (FC): a diterpene glycoside produced by the fungus Fusicoccum amygdali Del. FC was the first ligand reported to interact with members of the 14-3-3 protein family. Intriguingly, high affinity interaction with 14-3-3 proteins only occurs when a second protein, the plant plasma membrane H+-ATPase, participates. The dogma, that FC works in plants only, where it targets the dual complex formed by the H+-ATPase and a 14-3-3 dimer, is no longer valid since we published the dramatic effect of FC on the left/right asymmetry development of Xenopus embryos. Besides the effect on embryo development, other observations and articles demand a re-opening of the case: i) FC in synergy with interferon-&; strongly inhibits the proliferation of human non-small lung carcinoma cells (De Boer et al. unpubl. res.), ii) outward K+ channels in the plasma membrane of cells of the embryonic barley root are inhibited by FC independent from H+-ATPase activation, iii) in sycamore cells FC stimulates H2O2 production, 14-3-3 accumulation and cytochrome c leakage from mitochondria and iv) cDNA microarray analysis revealed a massive FC-induced change in gene expression in tomato plants. Another intriguing question is whether the production of FC by the fungus Fusicoccum amygdali is a case of molecular mimicry: i.e. the pathogen mimics a molecule of the host. Thus, the question is whether FC mimics a plant or animal molecule that acts as 14-3-3 ligand with a thus far unknown physiological function. A precedent for molecular mimicry in plant/fungus interaction is for example the production of the plant hormone gibberellin by the fungus Gibberella fujikuroi, causing the so-called ?foolish seedling? disease. In an earlier study we confirmed literature reports that plants contain a ligand of small molecular mass that competes with FC for binding, but due to the low concentration the structural information could not be obtained. Now, that FC has been shown to act on animal cells and in view of the widespread functions of 14-3-3 proteins in animal and plant physiology, this question is ever so relevant. Considering the role of 14-3-3 proteins in cell cycle control, apoptosis, cell signaling and transport processes a novel 14-3-3 ligand might have great pharmacological potential. This project seeks to identify and characterize novel protein targets for fusicoccin in animal cells. Simultaneously, we want to identify novel endogenous ligands that form ternary complexes with 14-3-3 and these target proteins, as does FC. We aim to unravel the biosynthetic pathway, reveal the structure and eventually show the physiological function.