PAM: diverse roles in neuroendocrine cells, cardiomyocytes, and green algae

Abstract

Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin and insulin, each of which is stored in electron dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the endoplasmic reticulum lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by Peptidylglycine -Amidating Monooxygenase (PAM), an ascorbate-and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell-types.This article is protected by copyright. All rights reserved encoding their precursors revealed the importance of post-translational processing in the production of bioactive peptides [5,6]. With the identification of COOH-terminal amidation as a critical step in the synthesis of peptides like vasopressin, oxytocin, neuropeptide Y, cholecystokinin and -melanocyte stimulating hormone, searches for an amidating enzyme began [7][8][9]. The sequencing of cDNAs encoding the prepropeptides for amidated peptides indicated that each amidated peptide was produced from a precursor with a COOH-terminal glycine.Using bovine and rodent pituitaries, a soluble monooxygenase that converted peptidylglycine substrates into amidated products, peptidylglycine -hydroxylating monooxygenase (PHM; E.C. 1.14.17.3), was identified and shown to require copper and ascorbic acid (Fig. 1A) [9]. The purification of soluble, ~40 kDa PHM led to the expression cloning of a cDNA that encoded a 110 kDa Type I integral membrane protein that included an NH 2terminal signal sequence followed by PHM (Fig. 1A) [10]. With a cDNA in hand, it quickly became apparent that the luminal region of this membrane protein included two catalytic activities, PHM and peptidyl-hydroxyglycine -amidating lyase (PAL; E.C. 4.3.2.5) (Fig. 1A) [7,11]. Under the slightly acidic conditions encountere...

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion

“…In the absence of PAM, proANP storage granules failed to form; the reintroduction of PAM facilitated proANP storage. Crucially, enzymatically inactive PAM also supported proANP storage ( 49 , 50 ), leading to the suggestion that PAM acts as a cargo receptor that directly binds to and protects newly synthesized proANP, facilitating its delivery to granules ( 9 , 49 ). A potential role for PAM as a cargo receptor in pituitary cells remains to be explored.…”

“…In a large, international cohort of individuals with PAs, we identified germline loss-of-function (LOF) variants of PAM (peptidylglycine α-amidating monooxygenase, MIM: 170270) in subjects with familial and sporadic GH and ACTH hypersecretion. PAM is a highly conserved, multifunctional protein that is increasingly recognized as an important regulator of peptide amidation and secretion, among many other functions, in health and disease ( 9 ). We functionally evaluated 36 SNVs, demonstrating a deleterious effect on PAM function/expression in eight variants.…”

An Update on the Genetic Drivers of Corticotroph Tumorigenesis

“…Another molecular defect possibly contributing to FIPA was recently proposed, after the finding of germline missense PAM variants in three first-degree relatives with gigantism and in sporadic patients with functioning pituitary tumours [232]. This gene encodes an enzyme catalysing the C-terminal amidation of secreted peptides; LOF was demonstrated in vitro for multiple variants [268,269]. Among these cases, two patients with isolated paediatric CD carried a frameshift and a 5'UTR variant that displayed deleterious effects.…”

Germline loss-of-functionPAMvariants are enriched in subjects with pituitary hypersecretion

“…Interestingly, the depletion of proANP in the absence of PAM was unaccompanied by decreased proANP mRNA or protein biosynthesis, but rather occurred due to 3-fold increased basal ANP secretion. Bäck, Mains & Eipper recently suggested that PAM may also have a role as a cargo receptor that directly binds to and protects proANP soon after its synthesis in the rough endoplasmic reticulum through to newly formed granules [9,51]. A potential role for PAM as a cargo receptor in pituitary cells remains to be for use under a CC0 license.…”