Molecular Mechanisms of GI Peptide Hormone Processing

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DESCRIPTION (provided by applicant): Gastrointestinal peptide hormones undergo extensive post-translational modification before they achieve their biologically relevant forms. It is important to elucidate these processing mechanisms since they result in the tissue-specific generation of distinct bioactive peptides. During the past funding period we characterized two important processing reactions-endoproteolysis and c-terminal amidation. In preliminary data we noted that prosomatostatin (proSS) is cleaved at single and double basic amino acid residues are performed by distinct enzymes leading to the formation of 2 distinct peptides (SS-28 and SS-14, respectively). Although the enzymes and molecular determinants for dibasic processing have been extensively characterized, little is known of monobasic prohormone processing. In other studies, we noted that the immediate processing precursor of amidated gastrin (glycine-extended gastrin, G-Gly) is a distinct end product of gastrin biosynthesis with trophic effects mediated by a receptor distinct from the gastrin/CCK8 receptors that mediate the effects of amidated gastrin. The conversion of G-Gly to amidated gastrin is mediated by a cooper-dependent enzyme, peptidyl-glycine alpha-amidating monooxygenase (PAM). Recently, our collaborators have isolated an important gene encoding a copper transporter (Menkes protein). In the present proposal we will extend our previous observations and examine the role of the Menkes protein in peptide amidation, gastrin biosynthesis and gastrointestinal function. Thus, toward the goal of elucidating the molecular determinants of peptide processing and its relationships to gastrointestinal physiology we will expand upon our previous work and focus on the following specific aims: 1) Characterize the molecular determinants of prohormone endoproteolysis single basic amino acids found in prosomatostatin; and 2) define the role of the Menkes protein in peptide amidation and gastrointestinal function utilizing endocrine cell lines, a well-characterized mouse model of disordered copper transport, and in human sera from patients with Menkes syndrome and related disorders. Since these processing reactions are common to a wide variety of neuroendocrine precursors, the results of these investigations will also aid in our understanding of the molecular mechanisms that regulate prohormone processing in other gut and neuroendocrine tissues.