Prohormone convertases PC1, PC2 and PC5 are good candidates for the enzymes responsible for the post-translational processing of CCK in rodent brain. PC2 is essential for normal pro CCK processing in some mouse brain regions, although there is clearly another enzyme(s) that processes pro CCK in place of PC2. Knowing the extent of colocalization of PC1 and PC5 with CCK will indicate whether they are candidates for the enzyme(s) that work in concert with PC2. We have made significant progress toward understanding how the sequence of pro CCK determines where it is cleaved and what cleavages are required for production of amidated CCK in an endocrine cell line. Proposed site-directed mutagenesis and expression studies would extend our understanding of this process. Tumor cells have been very useful to study pro CCK processing, but they are not good models for pro CCK processing in rodent brain. The use of antisense strategies to inhibit the expression of these enzymes in organotypic rat brain slices will allow us to learn more about their role in CCK processing. Together these experiments represent an integrated and novel approach to understanding the biosynthesis and processing of pro CCK in endocrine tumor cells and in rodent brain. The experiments test the following hypotheses: 1). PC1, PC2 and PC5 comprise a redundant system to insure production of active CCK in rodent brain. 2). Pro CCK has 3 dimensional structure that influences where it is cleaved. This structure changes as it is processed, making other sites more accessible to subsequent cleavage. The overall goal of this work is to understand the mechanism and enzymology of post-translational processing of pro CCK. CCK is known to be an important element in the neurochemical balance that is essential for normal nervous system function. The following Specific Aims are proposed: 1. The distribution and co-localization of PCI, PC2 and PC5 mRNA with CCK mRNA will be determined using in situ hybridization histochemistry in rat brain. In parallel, the colocalization of PCI, PC2 and PC5 enzyme protein with CCK immunoreactivity will be determined using immunohistochemistry. 2. Ongoing studies on the effect of altering the sequence of rat pro CCK on its processing in pituitary At-T20 cells will be continued. 3. The importance of PCI, PC2 and PC5 for pro CCK processing will be determined using organotypic rat brain slices in culture. Inhibition of enzyme expression will be achieved by incubation with peptide nucleic acid compounds, specific antisense oligonucleotides, double stranded RNA, DNA transfection and/or treatment with viral vectors expressing antisense cDNAs. The effect of this inhibition on the processing of pro CCK will be determined.
Showing the most recent 10 out of 30 publications
