Amelogenin undergoes self-assembly into nanospheres and contains a self-assembly domain located at the amino-terminus. We will pursue hypothesis driven experimental strategy into critical issues impacting upon assembly of the enamel organic matrix into a structure competent to direct biomineralization. We will test the hypothesis that amelogenin self- assembly into nanospheres requires an unaltered amino-terminal self- assembly domain. We predict that alteration in the assembly domain will alter the size of the nanospheres compared to controls. To test this hypothesis we propose two specific aims: 1) To engineer transgenic animals compared to controls. To test this hypothesis we propose two specific aims: 1) To engineer transgenic animals expressing amelogenin that contains alterations or deletions to the self-assembly amino-terminal """"""""A""""""""-domain and to characterize the resulting enamel; 2) to directly measure the strength of amelogenin interactions. Next, we screened for previously unrecognized protein partner(s) that interact with amelogenin interactions. Next, we screened for previously unrecognized protein partner(s) that interact with amelogenin or with tuftelin. We identified an amelogenin interacting protein (AIP) and several tuftelin interacting proteins (TIPs). We will test the hypothesis that the physiologic function for an AIP or TIP is to participate in the assembly of an enamel organic matrix that is competent to direct biomineralization.
In specific aim 3 we propose to reduce the availability of the AIP or each TIP target protein using hammerhead ribozymes. We predict pathogenomic alterations in the resulting enamel specific for each reduced target protein. Lastly, we predict that additional proteins will play essential roles during assembly of the enamel organic extracellular matrix. We will focus attention on the critical dentine to enamel junction (DEJ) which serves to anchor the brittle enamel bioceramic onto the underlying softer dentine. We hypothesize that the unique physical properties of the DEJ form as the result of an admixture of proteins synthesized by ameloblasts and dentinoblasts. We will search for critical protein partners expressed during the formation of the DEJ. We will use dentine sialophosphoprotein (DSPP) as bait to screen a cDNA library of tooth specific transcripts for those proteins that interact with DSPP.
In specific aim 4, we propose to search for novel proteins that interact with the dentine matrix protein DSPP.
The specific aims are a logical extension of data derived from our own accomplishments and from others in the field.
Each specific aim complements and builds upon the others. Completing these experiments will extend our understanding of normal and abnormal enamel and DEJ formation. This information will allow us to pursue the creation of an enamel and DEJ biomimetic.
|Wen, Xin; Lacruz, Rodrigo S; Smith, Charles E et al. (2014) Gene-expression profile and localization of Na+/K(+)-ATPase in rat enamel organ cells. Eur J Oral Sci 122:21-6|
|Lacruz, R S; Smith, C E; Kurtz, I et al. (2013) New paradigms on the transport functions of maturation-stage ameloblasts. J Dent Res 92:122-9|
|Wen, Xin; Paine, Michael L (2013) Iron deposition and ferritin heavy chain (Fth) localization in rodent teeth. BMC Res Notes 6:1|
|Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin et al. (2013) Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis. J Bone Miner Res 28:672-87|
|Lees, James D; Robinson, Colin; Shore, Roger C et al. (2013) Cellular uptake and processing of enamel matrix derivative by human periodontal ligament fibroblasts. Arch Oral Biol 58:348-54|
|Lacruz, Rodrigo S; Smith, Charles E; Moffatt, Pierre et al. (2012) Requirements for ion and solute transport, and pH regulation during enamel maturation. J Cell Physiol 227:1776-85|
|Lacruz, Rodrigo S; Hacia, Joseph G; Bromage, Timothy G et al. (2012) The circadian clock modulates enamel development. J Biol Rhythms 27:237-45|
|Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James et al. (2012) Targeted overexpression of amelotin disrupts the microstructure of dental enamel. PLoS One 7:e35200|
|Lacruz, Rodrigo S; Smith, Charles E; Bringas Jr, Pablo et al. (2012) Identification of novel candidate genes involved in mineralization of dental enamel by genome-wide transcript profiling. J Cell Physiol 227:2264-75|
|Lacruz, Rodrigo S; Smith, Charles E; Chen, Yi-Bu et al. (2011) Gene-expression analysis of early- and late-maturation-stage rat enamel organ. Eur J Oral Sci 119 Suppl 1:149-57|
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