How histatin enhances wound closure: structural and cellular aspects

We have identified histatin as the factor in human saliva which stimulate migration of epithelial cells and fibroblasts and enhance wound closure in a human skin model. The aim of this proposal is to provide fundamental insights into (i) the structural and conformational properties that endow histatin with the wound healing activity, (ii) the identity of the cognate receptor of histatin, and (iii) histatin?s intracellular effects. EXPERIMENTAL APPROACH (i) Structure-activity analysis of histatin In this part the minimal domain(s) on histatin responsible for binding and cell-activation will be mapped. The impact of conformation will be investigated using cyclic variants, synthesized by a novel enzyme-based method, in comparison with the linear counterparts. Structural properties will be investigated with high resolution spectroscopic techniques. (ii) Cognate receptor The histatin receptor will be purified by immunoprecipitation from epithelial cells using biotinylated cyclic histatin as high-affinity bait, followed by mass-spectrometrical identification. The histatin-binding domains of the receptor will be mapped by label transfer techniques, in which a biotin label is transferred by photoactivation from histatin to an approximate residue of the receptor. After enzymatic digestion the biotin-containing fragments will be identified. (iii)Intracellular effects Cells treated with histatin or control peptides will be subjected to phosphoproteomic analysis of the signal transduction machinery. To obtain a fingerprint of the cellular functions that are influenced by histatins, effects at the transcriptional level will be analyzed using an oligonucleotide microarray.