REGULATION OF NEUROPEPTIDE BIOSYNTHESIS

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The long range objective of this project is to apply the techniques of biochemistry to investigate how genes of the nervous system are expressed and regulated. The nervous system is composed of many neurons which interconnect with one another in a precis manner. The diversity of neurons and the nature of their interactions suggest that specific genes or sets of genes are activated in some neurons and not in others. One of the specific aims of this proposal is to develop a better understanding of the molecular events associated with the expression and regulation of the most abundant and widely distributed neuropeptide the mammalian nervous system, neuropeptide Y. The specific regions of the NPY gene responsible for expression, tissue-specific expression, and response to nerve growth factor will be dissected using molecular and biochemical approaches. Most neuropeptides and numerous proteins undergo post-translational processing which requires limited proteolysis. At least two (and often more) enzymes appear to function in their processing: 1) a trypsin-like protease, which recognizes precursors to both proteins and neuropeptides where two adjacent basic residues are present; and 2) a carboxypeptidase B-like enzyme, which removes the C-terminal basic residues form the C- terminus of intermediates which are formed by limited proteolysis. These second aim of this proposal is to better define the molecular events associated with the limited proteolysis of precursors to proteins and neuropeptides. Our laboratory has identified and sequenced a full-length clone of the carboxypeptidase B-like processing enzyme (referred to as carboxypeptidase H [CPH]). We have shown that carboxypeptidase H is synthesized as a precursor, which is the first reported example of a proteolytic-processing enzyme being synthesized as a precursor. The identification and characterization of the intermediates produced during the processing of this precursor is also a goal of this work. In addition, the gene encoding carboxypeptidase H should contain sequences which result in its selective expression in hormone producing cells. Therefore, we would like to identify and characterize these regions of the gene.