A vast array of polyproteins, peptide hormones and neuropeptides are synthesized as part of larger precursor molecules that must be proteolytically cleaved to release bioactive products. Over the past 24 years, numerous candidate enzymes have been proposed as potential eukaryotic prohormone converting endopeptidases (PCEs), but none has yet been demonstrated to function in vivo as an authentic PCE. Recently, several putative PCEs having characteristics similar to the yeast Kex2 pro- alpha-mating factor endoprotease have been identified by using the polymerase chain reaction (PCR) to amplify cDNA derived from mammalian cells. These enzymes include human, mouse and rat furins, as well as two mouse and two human prohormone convertases (PCs). Results from co- transfection studies have demonstrated a potential role for these proteases in pro-polypeptide processing. However, very little is currently known regarding tissue distribution or breadth of specificity of any of these putative PCEs, and essentially nothing is known about regulation of their expression and activity. The work proposed in this application is designed to generate information in these areas. Cultured cell lines that are known to synthesize and process prosomatostatin (pro-SS) and/or pro-neuropeptide Y (pro-NPY) will be used. Antisense DNAs having sequences complementary to specified regions of the mRNAS encoding identified PCEs will be introduced into cells previously determined to express PCE mRNA by Northern blot analysis and in situ hybridization. The antisense DNA should hybridize with the PCE MRNA for which it is specific and inhibit or block its translation. If the blocked PCE is involved in the processing of pro-NPY or pro-SS, then processing should be inhibited, thereby establishing a role for that specific PCE in cleaving that precursor to release its product hormone. Once a specific PCE has been shown to process pro-NPY and/or pro- SS, the regulation of expression and activity of the PCE will be examined by incubating the cells with physiologic regulators of the prohormone substrate for that PCE. Regulation of PCE expression and activity will be assessed by monitoring PCE MRNA levels, PCE enzyme levels (by PCE-specific RIA), and prohormone processing activity. The results from these studies will provide new data on PCE characteristics, specificity and regulators of PCE function. There are direct health-related implications of this research. Prohormone processing defects have been demonstrated to be components of, or associated with, both Type II Diabetes mellitus and Alzheimer's disease. A thorough characterization of the processing enzymes and their regulation in normal tissues must precede any attempt to elucidate PCE-related defects which may be involved in the etiology of disease processes.
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