Recent studies have sought to connect Alzheimer's disease (AD), a severe progressive neurodegenerative disorder leading to senile dementia and death, to aberrant brain proteinase function in the mis-processing of amyloid precursor protein (APP). In addition, preliminary evidence indicates elevated levels of truncated nerve growth factor receptor (NGFr) are observed with CNS trauma, including AD. Isolation and characterization of proteinases associated with APP processing and NGFr truncation will lead to an understanding of the role these enzymes play in Alzheimers and potentially other neurodegenerative disorders.
The specific aims of this project are: to develop isolation procedures for these proteinases; to characterize the proteinases with respect to primary/secondary structure and confirmation and stability; to characterize the enzymology and substrate specificity of these proteins; to obtain information on structural properties useful for tertiary structure determination; and to develop procedures for characterizing enzyme interactions with inhibitors. The proteinases will be isolated from normal human and AD-diagnosed brain tissues, and brain tissues from other dementias using combinations of standard analytical and preparative separation techniques. Amino terminal sequences and sequences from peptide mapping of the proteins will be established to permit cloning and expression in E. coli or mammalian cell lines. The recombinant proteinases will be isolated and will undergo structural and physical characterization and comparison to the natural proteinases. The recombinant-derived enzymes will be used for subsequent three-dimensional structure determination in support of structure-based drug (inhibitor) design. Substrate specificities will be defined using novel fluorogenic and recombinant protein substrates and inhibitors will be tested to determine how the active center specificity and catalytic mechanism of the proteinases is related to other proteinases. These studies will help elucidate the role of normal/aberrant proteolysis in AD, and possibly other proteinase-related neurodegenerative disorders. In conjunction with structure-based drug design efforts these studies will lay the foundation for development of enzyme inhibitors capable of acting selectively to halt disease progress.
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