The focus of this application is to define how enamelysin (MMP-20) processes enamel proteins in vitro and in vivo, and to determine how such processing allows for proper enamel development. The proposed research takes advantage of a unique development in the enamel field: the generation of an enamelysin knockout mouse. The enamelysin (-/-) mouse displays a severe enamel phenotype consisting of hypoplastic enamel and an obliterated rod pattern with no other detectable tissue malformations. The knockout mouse proves that proteolysis of enamel matrix proteins is a critical part of Dental enamel formation and allows us to test specific hypotheses concerning the functional effects of enamelysin cleavages in vivo. These hypotheses are: 1) enamelysin is the only protease that cleaves enamel proteins during the secretory stage of mouse amelogenesis, 2) enamel matrix proteins characterized from the enamelysin knockout mouse will be uncleaved, and therefore still in their secreted forms, 3) amelogenin and/or ameloblastin cleavage products are necessary for enamel rod organization, and 4) assemblies of full-length enamel proteins catalyze an increment of crystal elongation that, once completed, is followed by proteolysis to disassemble the structure and make way for another round of crystal extension. By performing a proteomic characterization of the enamel matrix of developing teeth obtained from the enamelysin knockout and from wild-type mice (Aim 1), important new information will be gained on the relative abundance of various enamel protein cleavage and alternative splice products. By determining the substrate specificity of enamelysin (Aim 2) we will understand the relative contributions of enamelysin cleavage site preferences and the accessibility of preferred cleavage sites (i.e., does protein folding play a role?) in determining the make-up of the enamel matrix. To test the validity of our hypotheses, we propose to utilize the knockout mice for transgenic experiments that determine if amelogenin and/or ameloblastin cleavage products are necessary for enamel rod organization (Aim 3) and to determine if proteolysis of full-length enamel proteins is necessary for enamel crystallite elongation (Aim 4). This application utilizes a unique tool (enamelysin knockout mouse) and recent, highly advanced technologies including, the ProteoSep system for protein isolation and mixture based oriented peptide libraries for substrate specificity analysis, to help determine how protein processing allows for proper enamel development. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE016276-02
Application #
7267081
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Scholnick, Steven
Project Start
2006-08-01
Project End
2011-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
2
Fiscal Year
2007
Total Cost
$417,594
Indirect Cost
Name
Forsyth Institute
Department
Type
DUNS #
062190616
City
Cambridge
State
MA
Country
United States
Zip Code
02142
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