Enamel, the outermost covering of teeth forms extracellularly through the ordered assembly of a protein scaffolding that regulates crystallite formation. The underlying mechanism for protein assembly within the enamel extracellular matrix and the regulatory roles for protein to protein and crystallite to protein interactions are not known. Amelogenesis imperfecta, genetic defects of enamel, include several forma with mutations in the human amelogenin locus that affect amelogenin expression, resulting in alterations in protein stoichiometry and/or the loss of protein domains required to regulate enamel organic matrix biogenesis. An emerging view of enamel biogenesis includes protein to protein interactions essential for assembly of the enamel organic matrix since protein to protein interaction will affect the spatial organization of the matrix that provides protein to crystallite regulatory interactions. THE HYPOTHESIS UNDERLYING THIS APPLICATION IS THAT SUPRAMOLECULAR ASSEMBLY OF THE ENAMEL ORGANIC MATRIX REQUIRES SPECIFIC PROTEIN DOMAINS AND IT IS THIS PROTEIN ASSEMBLY THAT ULTIMATELY DIRECTS BIOMINERALIZATION. We will use the yeast two-hybrid system to identify and define domains essential to protein to protein interactions used during supra molecular assembly of the enamel organic matrix. We will search for protein(s) other than amelogenin and tuftelin that interact with amelogenin and participate during protein assembly of the enamel organic matrix. To test our hypothesis we propose five interrelated specific aims: 1) To use the yeast two hybrid system to screen for specific enamel protein interactions; 2) To identify and map minimal domains required for specific enamel protein interactions; 3) To identify unknown enamel protein(s) associated with enamel organic matrix assembly; 4) To map minimal protein domain(s) required for interactions among newly identified enamel matrix proteins; 5) To perturb assembly functions of the identified domains by site-directed mutagenesis. Successful completion of these aims will permit critical insights into the assembly and disassembly of the enamel extracellular matrix during biomineralization. Such insights can be used to model the molecular basis for enamel defects resulting from environmental insults, such as fluoride, or from genetic disorders, such as in amelogenesis imperfecta. Identification of the minimal protein domains and their structure will provide enhanced understanding of protein structures which are required for enamel matrix self-assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE011704-02
Application #
2414704
Study Section
Special Emphasis Panel (ZRG4-OBM-2 (02))
Project Start
1996-05-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Southern California
Department
Dentistry
Type
Schools of Dentistry
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Fong, Hanson; White, Shane N; Paine, Michael L et al. (2003) Enamel structure properties controlled by engineered proteins in transgenic mice. J Bone Miner Res 18:2052-9
Paine, M L; White, S N; Luo, W et al. (2001) Regulated gene expression dictates enamel structure and tooth function. Matrix Biol 20:273-92
Paine, C T; Paine, M L; Luo, W et al. (2000) A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts. J Biol Chem 275:22284-92
Paine, M L; Krebsbach, P H; Chen, L S et al. (1998) Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix. J Dent Res 77:496-502
Snead, M L; Paine, M L; Luo, W et al. (1998) Transgene animal model for protein expression and accumulation into forming enamel. Connect Tissue Res 38:279-86;discussion 295-303
Paine, C T; Paine, M L; Snead, M L (1998) Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system. Connect Tissue Res 38:257-67;discussion 295-303
Paine, M L; Snead, M L (1997) Protein interactions during assembly of the enamel organic extracellular matrix. J Bone Miner Res 12:221-7