Simmer Lab - research
current research
ENAMEL MATRIX PROTEINASE-1
Enamel matrix serine proteinase 1 (EMSPl) is proposed to process enamel proteins during the secretory stage, and degrade enamel proteins during the early maturation stage of amelogenesis. EMSP1 is believed to be critical for proper dental enamel formation and defects in the human ESP1 gene are suspected of causing amelogenesis imperfecta. Two hypotheses are tested:
- EMSP1 function is essential for the correct processing of enamel matrix proteins during the secretory stage of enamel formation, and that a loss of function can result in hypoplastic enamel.
- EMSP1 function is essential for the degradation of enamel matrix proteins during the transition/early maturation stages of enamel fonnation, and that a loss of function can result in hypomineralized enamel.
The following 4 Specific Aims are proposed to test these hypotheses:
- 1. To determine the temporal and spatial expression of EMSP1 during the secretory, transition, and maturationstages of enamel biomineralization. In situ hybridization ans immunohistochemistry will be perfonned on mouse maxillae and mandibles, The first and second molars and incisors will be analyzed from newborn, postnatal (PN) days 2, 4, 6, 8 and 14 and adult.
- 2. To characterize the enzymatic activity of EMSP1 on amelogenin.Recombinant pig amelogenin will be digested by recombinant pig EMSP1 in vitro. The digestion products will be characterized by Edman degradation and mass spectrometry and compared to amelogenin cleavage sites that occur in vivo.
- 3.To characterize the enamel formation in the absence of mouse EMSP1 expression. EMSP1 knock-out mice will be generated and characterized.
- 4. To determine the genetic linkage of human EMSP1 gene wit inherited defects of dental enamel formation. Incollaboration with 3 laboratories that have assembled large numbers of kindreds suffering for amelogenesis imperfecta (AI), a candidate gene approach will be used to establish linkage between the human EMSPl gene and AI.
Accomplishing these aims will gain important insights into the action of EMSP1 in the organization processing, turnover, and degradation of enamel matrix proteins during enamel biomineralization.
PROTEOMICS AND GENETICS OF ENAMEL AND DENTIN
Proteomics and Genetics of Enamel and Dentin: Our goals are:
- to isolate and characterize proteins in the enamel and dentin matrices of developing teeth.
- to discover mutations in defective genes that cause dental disorders
A proteomics approach is proposed to isolate and characterize proteins normally expressed during tooth formation. By discovering novel dentin and enamel matrix proteins, we will identify new candidate genes in the etiologies of inherited dental disorders, such as amelogenesis impelfecta (AI) and dentinogenesis imperfecta (DGI).
Two Specific Aims are proposed:
- SA 1: Isolate and characterize molecules in the extracellular matrices of developing enamel and dentin.
- SA 2: Identify genes and mutations that cause amelogenesis impelfecta and dentinogenesis imperfecta.
In Specific Aim 1 we perform a comprehensive isolation and characterization of the protein components in developing enamel and dentin. Complete resolution of virtually every soluble matrix protein is achieved using a two-dimensional method that separates proteins first by isoelectric point and second by hydrophobicity. Isolated proteins are characterized by on-line electrospray ionization (ESI) time-of-flight mass spectrometry, by N-terminal sequencing, peptide mapping and characterization of posttranslational modifications.
In Specific Aim 2 genetic studies are performed on kindreds having AI or DGI, using a candidate gene approach. The five candidate genes for autosomal AI (enamelin & ameloblastin on 4q11-q21, MMP-20 on 11 q22, tuftelin on 1 q21-31, and kallikrein-4 on 19q13.3-q13.4), the one candidate gene for X-linked AI (amelogenin on Xp22.3-p22.1), the two candidate genes for DGI (DSPP and DMP1 on 4q21), and candidate genes identified discovered in the Proteomics study will be tested for linkage to dental disease. This study will provide gene-based diagnostic criteria, improved genetic counseling, and lead to the development of novel prevention and therapeutic strategies for families suffering from inherited disease.