Grant: $202,670 - National Institutes of Health - Jul. 16, 2009
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Award Description: This proposal is based on the overall hypothesis that non-collagenous extracellular matrix (ECM) proteins play vital roles in the formation of dentin by odontoblasts and in the homeostatic mechanisms of formation and breakdown of bone by osteoblasts, osteocytes and osteoclasts. Specifically, we are focusing upon the biological functions of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). The importance of these two proteins in mineralized tissue formation is supported by a number of experiments showing that mice lacking genes coding for DMP1 and DSPP display defects in bone and/or dentin mineralization. It is well known that DSPP is proteolytically processed into DSP and DPP, found in the ECM of dentin. Recent data from our laboratory indicate that DPM1 is processed into 37K and 57K fragments, present in the ECM of bone and dentin. Our detailed sequence analyses show that four cleavage sites in DMP1 and two in DSPP are X-Asp bonds, strongly suggesting that these two proteins are processed by the same proteinase. We hypothesize that this proteinase is PHEX protein, that is predominantly expressed in bone and tooth and that has a strong preference for cleavage at the NH2-terminus of aspartyl residue. This idea is supported by observations that the bony and dental defects of Dmp1 and Dspp knock-out mice are similar to those of X-linked hypophosphatemic rickets caused by mutations of the PHEX gene. Based on these findings, we propose the following hypotheses: DMPI and DSPP are inactive precursors that are proteolytically processed by cleavage of selected X-Asp bonds during or after secretion. These proteolytic reactions, catalyzed by PHEX proteinase, release functional components, 37K and/or 57K fragments (in the case of DMP1), and DSP and/or DPP (from DSPP). These processing events play significant, crucial roles in the conversion of osteoid to bone and predentin to dentin. Failure to process these two proteins during PHEX deficiency gives rise to abnormal phenotypes similar to those when Dmp1 or Dspp genes are mutated or knocked out. To test these hypotheses, we propose the following Specific Aims: 1. To determine if PHEX protein is the enzyme responsible for catalyzing the proteolytic processing of DMP1 and DSPP. 2. To study the effects of 37K and 57K fragments on mineralization. 3. To study the biosynthesis and secretion of DMP1, 37K and 57K fragments.
Project Description: The recently awarded ARRA funds were used to create a Research Assistant position, which was filled by a recent college graduate. The RA has begun working on a couple of projects related to the original grant proposal and has been learning several basic science techniques including measures of vascular reactivity and flow cytometry for immunophenotyping. Expectations for the RA include performing the experiments necessary to generate publications and presentations, of which he will assist in the preparation of.
Jobs Summary: Research Assistant-Texas A&M Health Science Center-Temple, TX (Mitchell Lab) Perform scientific experiments and data analyses as directed by the Principal Investigator (PI) or project leader. Assist Laboratory Manager with ordering of supplies necessary to perform experiments as well as other laboratory personnel with cleaning of supplies/equipment. Assist PI with the preparation of grant proposals, presentations, and manuscripts. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 16, 2009. Help expand these official descriptions using the wiki below.