Peptides; membrane protein trafficking; cuproenzymes; neurons; pituitary peptides seem to have preceded the 'classical' transmitters as the nervous system developed - creatures like Hydra and Drosophila utilize peptides to control key developmental decisions. Beginning with our work on proopiomelanocortin and the coordinate biosynthesis of ACTH and the opioid peptide beta-endorphin, I have been fascinated with the effort that neurons and endocrine cells devote to the biosynthesis, storage and regulated secretion of peptides. As we have learned more about the specific enzymes involved in the biosynthesis of peptides, we have learned important questions to ask about how these enzymes function in cells and in the whole animal.We have focused a great deal of our effort on the process of peptide amidation. This seemingly trivial modification to the COOH-terminus of peptides often turns out to be essential for their biological activity. Hypothalamic peptides like oxytocin and vasopressin along with neuropeptides like substance P and gastrointestinal mediators like gastrin must be amidated in order to affect their target tissues. By purifying an enzyme capable of converting peptidylglycine precursors into amidated products, we were then able to clone a cDNA encoding this enzyme.To our surprise, this modification requires the sequential action of two enzymes, a monooxygenase and a lyase. The enzyme has been named PAM, short for peptidylglycine alpha-amidating monooxygenase. The monooxygenase itself is called PHM, short for peptidylglycine alpha-hydroxylating monooxygenase, and the lyase is called PAL, short for peptidyl-alpha-hydroxylglycine alpha-amidating lyase. PHM uses ascorbic acid (vitamin C) to reduce the two copper atoms that are bound to its catalytic core and molecular oxygen is the final component of the reaction. PAL also requires a metal ion, zinc, for activity. With its need for copper, zinc and ascorbate, PAM function is sensitive to genetic and environmental factors.We have expressed the bifunctional PAM protein in soluble and membrane forms and have purified milligram amounts of the two separate catalytic domains, PHM and PAL. With Dr. Mario Amzel, we were able to deduce the crystal structures for PHM and for PAL. One copper binds to the N-terminal domain of the PHM catalytic core and the other to the C-terminal domain; how both sites contribute to the reaction is not yet clear. The beta-propeller structure of PAL constrains PHM to a location near the granule membrane, with important functional implications.Along with structure function studies, our current efforts are aimed at understanding what the cells that use PAM have to do in order to provide copper to the enzyme. Copper is an extremely toxic metal and specific pumps and chaperones are used to deliver it to the proteins that need it. PAM knockout mice do not survive beyond mid-gestation; PAM heterozygous mice are viable, but exhibit increased anxiety-like behavior, an inability to thermoregulate and increased seizure sensitivity. Many of these deficits are mimicked in mildly copper-deficient wildtype mice and ameliorated by providing supplementary dietary copper to PAM heterozygous mice. We are using these animal models to understand the role of PAM in coping with copper availability and are collaborating with our clinical colleagues to see if mild copper deficiency occurs in patient populations.In addition to its catalytic domains, PAM has non-catalytic regions. In particular, the transmembrane domain and cytosolic domain of PAM need not be present for the enzyme to function. The role of these non-catalytic domains seems to be in getting PAM to the right place in the cell so that it can do its job. In particular, the cytosolic domain is essential for targeting PAM to the secretory granules of pituitary endocrine cells and for guiding PAM protein that has reached the cell surface back into secretory granules following internalization. We recently found that a gamma-secretase-like cleavage releases a soluble cytosolic domain fragment of PAM that enters the nucleus and alters gene expression. This adds a new dimension to our studies of PAM and we are in seach of the underlying mechanism.A PAM cytosolic domain interactor protein of great interest is kalirin, a member of the Dbl family of GDP/GTP exchange factors for small GTP binding proteins of the Rho sub-family. The cytosolic domain of PAM binds to the spectrin-like repeat region of kalirin. This region of Kalirin is followed by a Dbl homology or DH domain, and a PH domain. Kalirin occurs naturally in a variety of isoforms and this first DH/PH domain can be followed by a PDZ-binding motif (Kalirin-7), an SH3 motif (Kalirin-8), another DH/PH domain (Kalirin-9) or another DH/PH domain and a putative serine/threonine protein kinase (Kalirin-12). The various isoforms of kalirin are expressed at different times during development and are localized to different regions of the cell. Mice engineered to lack expression of Kalirin globally or only in pituitary corticotropes are being used to identify the major roles of Kalirin in the pituitary and in the nervous system.

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Peptide Amidation and the Control of Ciliogenesis (King and Eipper Laboratories)

Cilia are essential signaling and motile organelles that play key roles in development and homeostasis.  Defects in these microtubule-based structures lead to a broad array of phenotypes (including infertility, obesity, neurological disorders, polycystic kidneys, retinal degeneration, skeletal abnormalities and many others) collectively known as ciliopathies.  The enzyme peptidylglycine alpha-amidating monoxygenase (PAM) converts glycine-extended peptides into bioactive amidated products (e.g. oxytocin and vasopressin).  Although originally thought to have co-evolved with the nervous system, we recently found that PAM has deeper evolutionary roots and has been conserved in all organisms (even unicellular ones) that build sensory cilia.  Furthermore, using amiRNA knockdown strains of the green alga Chlamydomonas (the premier model system for analysis of cilia assembly and motility) we have now demonstrated that active PAM is not only present in cilia, but is actually essential for ciliogenesis, potentially through its effects on post-Golgi trafficking.  This project will focus on using biochemical, cell biological and molecular genetic approaches to further elucidate the mechanism(s) by which PAM activity impacts on cilia assembly and function. 

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