Understanding the physiologic relationship between genomics and proteomics is a common theme and focus for investigation. The use of cell models and organ systems remains a fundamental approach to achieving such an understanding. Enamel is a complex bioceramic tissue that is a reflection of its unique molecular and cellular formation. From their ectodermal origins, ameloblasts exist for the sole purpose of producing a mineralized shell. The shell is almost entirely devoid of protein and is deposited over the """"""""bone-like"""""""" dentine. We have recently discovered TIP39kDa, a protein that favors the secretory surface of ameloblasts (Tomes' processes). Antibodies to TIP39kDa react to secretory vesicles within Tomes' processes, and also the immature enamel matrix. TIP39kDa expression is not unique to ameloblasts, but preliminary data suggest that it may be unique to cells with a significant secretory function. This project will pursue an experimental strategy to identify a definitive function for TIP39kDa, and also explain the nature of the multiple messenger RNA isoforms observed for TIP39kDa.
The first aim will test the hypothesis that TIP39kDa is involved with the cells secretory activity by down-regulating TIP39kDa expression within cell and organ cultures, and look at the subsequent levels of a secreted protein (amelogenin). For this I will employ both an antisense RNA and a ribozyme approach.
The second aim will be to use the TIP39kDa gene promoter in an appropriate reporter system and produce transgenic animals to define organs for which TIP39kDa plays a role.
The third aim will identify the subcellular localization of TIP39kDa. For this I will employ available TIP39kDa antibodies and green fluorescent protein tags. Regular database searches will be performed to assist in the identification of functional domains.
The fourth aim will be to screen a yeast two-hybrid mouse embryonic library to look for TIP39kDa protein partners. This data will be used to help identify a physiological role for TIP39kDa. The fifth and final specific aim will seek to answer the question of how multiple messenger RNA isoforms of TIP39kDa are generated as evident from previously published data. These first four specific aims will assign a subcellular location and function to a TIP39kDa that, in the case of ameloblasts, has a preference for Tomes' processes, secretory vacuoles and the immature enamel matrix. The fifth specific aim will answer a very basic question related to how TIP39kDa is processed post-transcriptionally, and the potential significance of this. ? ? ? ?
