Protein-protein interactions are intrinsic to virtually every cellular process, including DNA replication, transcription, translation, splicing, secretion, cell cycle control, signal transduction, and intermediary metabolism. We hypothesize that protein-protein interactions are integral to enamel biomineralization and that knowledge of these interactions is essential for understanding the molecular mechanisms of enamel formation. In this Sub-program, protein-protein interactions among enamel matrix proteins are investigated using the yeast two hybrid system and immuno-protein methods. The yeast two-hybrid system is a molecular biology tool for identify potential interactions between proteins. It is ideally suited for cloning cDNAs encoding unknown proteins that interact with a chosen protein, or for identifying interactions between two chosen proteins.
Two Specific Aims are propose. 1) To isolate and characterize cDNAs encoding enamel matrix proteins, with a focus on proteinase inhibitors.
This aim will be accomplished by identifying enamel matrix proteins by their protein-protein interactions with previously isolated constituents of the developing enamel matrix. Proteins interacting with a chosen, or """"""""bait"""""""" protein are identified by the yeast two-hybrid system and affinity chromatography. The enamel proteins used as bait include enamelysin (MMP-20), enamel matrix serine proteinase 1 (EMSP1), enamelin, sheathlin, and amelogenin. Protein- protein interactions identified by these methods are confirmed by affinity blotting, and/or immunoprecipitation. Priority is given to identifying enamel proteinase inhibitors that regulate the activities of enamelysin and EMSP1. 2) To characterize protein-protein interactions during amelogenesis.
This aim will be accomplished using affinity methods and the yeast two- hybrid system. In contrast to SA1 where a """"""""bait"""""""" protein is crossed with a library of unknown proteins, in SA2 specific enamel protein is crossed with itself, or with a second enamel protein. Five potential protein-protein interactions are investigated: 1) enamelin and enamelin, 2) sheathlin and sheathlin, 3) enamelin and sheathlin, 4) amelogenin and enamelin, and 5) amelogenin and sheathlin.
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