Peptides play essential roles throughout the endocrine and nervous systems. The biosynthetic pathway leading from preprohormone to product peptide is fundamentally similar in human and in primitive creatures such as the sea anemone. As precursors move through the lumen of the secretory pathway, endoproteases, exopeptidases and peptidylglycine 1-amidating monooxygenase (PAM), the enzyme responsible for crucial C- terminal amidation, function sequentially. Peptidylglycine 1-hydroxylating monooxygenase (PHM;EC1.14.17.3), the first enzyme of the bifunctional PAM protein, requires copper and ascorbate;in man, both must be acquired from the diet. Cuproenzymes like PHM, rare in anaerobic species, evolved with the advent of molecular oxygen in the atmosphere and are overwhelmingly associated with its use. Our analyses of mice with one functional PAM gene (PAM mice) revealed multiple physiological and behavioral alterations with slight decreases in the amidated peptides measured. Many of the alterations were ameliorated by additional dietary copper and mimicked in copper deficient wildtype mice, leading to the conclusion that PAM plays a role in copper homeostasis. We will build on our discovery that membrane PAM yields a soluble fragment of its cytosolic domain (sfCD) that is targeted to the nucleus and alters gene expression, to determine the mechanisms through which PAM affects physiological function.
Aim 1 : The structures determined for PHM and peptidyl-1-hydroxyglycine 1-amidating lyase (PAL), the second part of the bifunctional enzyme, will be used to understand PAM function. Roles for the linker regions connecting PHM and PAL will be explored in soluble PAM proteins with the goal of crystallization. This knowledge will clarify how the linkers affect the ability of membrane PAM to signal to cytosol and nucleus. AtT-20 cells will be used to determine whether membrane tethered PAM has improved access to ascorbate and copper, facilitating peptide amidation.
Aim 2 : Focusing on two amidated peptides, vasopressin and adrenomedullin, we will evaluate the ability of PAM mice to handle a high salt diet. The mechanisms underlying the behavioral changes observed in PAM mice will be explored in electrophysiological studies focused on GABAergic signaling in the amygdala.
Aim 3 : How regulated intramembrane proteolysis generates sfCD will be evaluated in PAM-1-expressing AtT-20 cells. Effects of splice variants, luminal domain cleavage and phosphorylation will be determined. Cell permeant versions of sfCD will be used to explore the effects of PAM on gene expression, focusing on PAM targets known to play roles in the secretory pathway (aquaporin1;secretory leukocyte proteinase inhibitor) and copper metabolism (Atox1). While genetic alterations in PAM may be rare, our data strongly suggest that alterations in the availability of dietary copper and ascorbate could contribute to less than adequate functioning of PAM. It is our hope that a better understanding of the copper-reversible changes that occur in PAM mice, and are mimicked in copper deficient wildtype mice, will facilitate identification of compromised PAM function in man.

Public Health Relevance

Bioactive peptides are among the most ancient methods for intercellular communication. Peptide amidation, the modification of the ionizable COOH-terminal of a peptide to have an uncharged amide group, requires a number of cofactors and presents a set of challenges to cells using amidated peptides for intercellular signaling, performed by bifunctional peptidylglycine alpha- amidating monooxygenase, PAM. Both enzymatic activities of PAM have been extensively studied and their enzymology and biology, along with the biological consequences of inadequate ability to perform peptide amidation, form the basis of this work.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032949-31
Application #
8435472
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Malozowski, Saul N
Project Start
1998-06-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
31
Fiscal Year
2013
Total Cost
$534,718
Indirect Cost
$187,499
Name
University of Connecticut
Department
Neurosciences
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
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Kumar, Dhivya; Mains, Richard E; Eipper, Betty A (2016) 60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C. J Mol Endocrinol 56:T63-76
Kumar, Dhivya; Blaby-Haas, Crysten E; Merchant, Sabeeha S et al. (2016) Early eukaryotic origins for cilia-associated bioactive peptide-amidating activity. J Cell Sci 129:943-56
Vishwanatha, Kurutihalli S; Bäck, Nils; Lam, TuKiet T et al. (2016) O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking. J Biol Chem 291:9835-50
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Carlson, Kristina; Pomerantz, Steven C; Vafa, Omid et al. (2015) Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells. BMC Biotechnol 15:95
Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E et al. (2015) Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme. J Biol Chem 290:21264-79
Simpson, Peter D; Eipper, Betty A; Katz, Maximiliano J et al. (2015) Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells. J Biol Chem 290:24891-901
Gaier, Eric D; Eipper, Betty A; Mains, Richard E (2014) Pam heterozygous mice reveal essential role for Cu in amygdalar behavioral and synaptic function. Ann N Y Acad Sci 1314:15-23
Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E et al. (2014) A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor. J Biol Chem 289:12404-20

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