Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.
"AQP1 has been found in DCVs in pituitary, chromaffin granules from adrenal medulla and pancreatic zymogen granules, while AQP5 has been found in parotid gland DCVs (Cho et al., 2002; Arnaoutova et al., 2008; Francone et al., 2010). AQP1 has also been found in the TGN (Francone et al., 2010). Its function in TGN and granules is to remove water to facilitate condensation of aggregated DCV proteins at the TGN and during DCV maturation. "
[Show abstract][Hide abstract] ABSTRACT: The release of intercellular messengers from synaptic (SVs) and dense-core vesicles (DCVs) constitutes the primary mechanism for communication between neighboring or distant cells and organs in response to stimuli. Here we review the life span of SVs and DCVs found in the brain, neuroendocrine and exocrine tissues. These vesicles must be formed, trafficked, and their contents secreted; processes which require orchestrated actions of a great repertoire of lipids, proteins, and enzymes. For biogenesis and vesicular budding, lipids that influence curvature and aggregation of cargo are necessary for pinching off of vesicles. Vesicles travel on cytoskeletal filaments powered by motors that control the dynamics: location, speed, and directionality of movement. Regardless of mechanisms of traffic, vesicles arrive at sites of release and are docked for exocytosis, followed by membrane fusion, and release of vesicular content to exert physiological responses. Neurological disorders with pathology involving abnormal vesicular budding, trafficking, or secretion are discussed.
International review of cell and molecular biology 09/2012; 299:27-115. DOI:10.1016/B978-0-12-394310-1.00002-3 · 3.42 Impact Factor
"In vertebrates, PAM is a Type I integral membrane protein; its luminal catalytic domains and unstructured cytosolic domain are highly conserved. A γ-secretase-mediated cleavage within the PAM transmembrane domain generates a cytosolic fragment that accumulates in the nucleus and is thought to affect gene expression , , , . "
[Show abstract][Hide abstract] ABSTRACT: Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.
PLoS ONE 12/2011; 6(12):e28679. DOI:10.1371/journal.pone.0028679 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptidylgycine alpha-amidating monooxygenase (PAM), a highly conserved copper-dependent enzyme, is essential for the synthesis of all amidated neuropeptides. Biophysical studies revealed that the binding of copper to PAM affects its structure, and cell biological studies demonstrated that the endocytic trafficking of PAM was sensitive to copper. We review data indicating that genetic reduction of PAM expression and mild copper deficiency in mice cause similar alterations in several physiological functions known to be regulated by neuropeptides: thermal regulation, seizure sensitivity, and anxiety-like behavior.
Journal of Neuroscience Research 09/2010; 88(12):2535-45. DOI:10.1002/jnr.22404 · 2.59 Impact Factor
Anupama Singh, Sunderasha Siddappa, Vinay Bhardwaj, Brajesh Singh, Devendra Kumar, Bir Pal Singh
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