A wide variety of proteins in neural, endocrine and immune tissues undergo proteolytic processing. Many of these proteins and peptides are important intercellular messenger. Although proteolytic cleavages are often essential for the activation and/or inactivation of these molecules, most of the enzymes responsible for the proteolytic processing of proteins and peptides are poorly understood. The focus of this grant application is to study a proteases involved in processing of neuropeptide precursors such as prodynorphin. We have purified and characterized a neuropeptide processing endoprotease designated """"""""dynorphin converting enzyme"""""""" capable of producing dynorphin B- 13 from leumorphin. The enzyme exhibits a broad tissue-distribution suggesting involvement in the generation of a number of neuropeptides. In addition, this enzyme is secreted from cells and thus could act extracellularly on proteins such as amyloid precursor protein. In this grant application, we propose to examine the substrate specificity to the specificity of the recently identified prohormone convertases. In addition, the ability of the enzyme to process a variety of proteins including, prodynorphin, proenkephalin and amyloid precursor protein will be examined. We also propose to isolate a cDNA clone encoding this enzyme (Specific Aim 2). polymerase chain reaction with oligonucleotides based on cysteine protease consensus sequences has led to the identification of four novels cDNAs; one of these shows tissue-specific expression which generally matched dynorphin converting enzyme. We propose to examine the distribution of the mRNAs by in situ hybridization analysis, to isolate a full length cDNA and to express and characterize the protein using the various substrates proposed in Specific Aim 1. These studies will provide important information on the substrate specificity, primary structure and distribution of a peptide-processing enzyme. This information will also form a basis for further investigations of the involvement of proteases in normal cell function and in disease processes such as Alzheimer's disease.
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