OBJECTIVES:Carriers of PAM (peptidylglycine alpha-amidating monooxygenase) coding variant alleles have reduced insulinogenic index, higher risk of developing type 2 diabetes (T2D), and islets from heterozygous carriers of the PAM p.Asp563Gly variant display reduced insulin secretion. Exactly how global PAM deficiency contributes to hyperglycemia remains unclear. PAM is the only enzyme capable of converting glycine-extended peptide hormones into amidated products. Like neuropeptide Y (NPY), α-melanocyte stimulating hormone (αMSH), and glucagon-like peptide 1 (GLP-1), islet amyloid polypeptide (IAPP), a beta cell peptide that forms islet amyloid in type 2 diabetes, is a PAM substrate. We hypothesized that Pam deficiency limited to beta cells would lead to reduced insulin secretion, prevent the production of amidated IAPP, and reveal the extent to which loss of Pam in β-cells could accelerate the onset of hyperglycemia in mice. METHODS:PAM activity was assessed in human islets from donors based on their PAM genotype. We generated beta cell-specific Pam knockout (Ins1Cre/+, Pamfl/fl; βPamKO) mice and performed islet culture, histological, and metabolic assays to evaluate the physiological roles of Pam in beta cells. We analyzed human IAPP (hIAPP) amyloid fibril forming kinetics using synthetic amidated and non-amidated hIAPP peptides, and generated hIAPP knock-in beta cell-specific Pam knockout (hIAPPw/w βPamKO) mice to determine the impact of hIAPP amidation on islet amyloid burden, islet graft survival, and glucose tolerance. RESULTS:PAM enzyme activity was significantly reduced in islets from donors with the PAM p. Asp563Gly T2D-risk allele. Islets from βPamKO mice had impaired second-phase glucose- and KCl-induced insulin secretion. Beta cells from βPamKO mice had larger dense-core granules and fewer and shorter cilia. Interestingly, non-amidated hIAPP was less fibrillogenic in vitro, and high glucose-treated hIAPPw/w βPamKO islets had reduced amyloid burden. Despite these changes in beta cell function, βPamKO mice were not more susceptible to diet-induced hyperglycemia. In vitro beta cell death and in vivo islet graft survival remained comparable between hIAPPw/w βPamKO and hIAPPw/w islets. Surprisingly, aged hIAPPw/w βPamKO mice had improved insulin secretion and glucose tolerance. CONCLUSIONS:Eliminating Pam expression only in beta cells leads to morphological changes in insulin granules, reduced insulin secretion, reduced hIAPP amyloid burden and altered ciliogenesis. However, in mice beta-cell Pam deficiency has no impact on the development of diet- or hIAPP-induced hyperglycemia. Our data are consistent with current studies revealing ancient, highly conserved roles for peptidergic signaling in the coordination of the diverse signals needed to regulate fundamental processes such as glucose homeostasis.

This record contains raw data related to article "Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion" Abstract Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Gated entry of lipophilic ligands into the enclosed hydrophobic pocket in stand-alone Sec14 domain proteins often links lipid metabolism to membrane trafficking. Similar domains occur in multidomain mammalian proteins that activate small GTPases and regulate actin dynamics. The neuronal RhoGEF Kalirin, a central regulator of cytoskeletal dynamics, contains a Sec14 domain (Kal bSec14 ) followed by multiple spectrin-like repeats and catalytic domains. Previous studies demonstrated that Kalirin lacking its Sec14 domain fails to maintain cell morphology or dendritic spine length, yet whether and how Kal bSec14 interacts with lipids remain unknown. Here, we report the structural and biochemical characterization of Kal bSec14 . Kal bSec14 adopts a closed conformation, sealing off the canonical ligand entry site, and instead employs a surface groove to bind a limited set of lysophospholipids. The low-affinity interactions of Kal bSec14 with lysolipids are expected to serve as a general model for the regulation of Rho signaling by other Sec14-containing Rho activators.

Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia-derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Peptidergic intercellular communication controls a wide range of physiological and behavioral responses and occurs throughout eukaryotes. The Chlamydomonas reinhardtii genome encodes what appear to be numerous prepropeptides and enzymes homologous to those used to convert metazoan prepropeptides into bioactive peptide products. Since C. reinhardtii, a green alga, lack the dense core vesicles in which metazoan peptides are processed and stored, we explored the hypothesis that propeptide processing and secretion occur through the regulated release of ciliary ectosomes. A synthetic peptide (GATI-amide) that could be generated from a 91-kDa peptide precursor (proGATI) serves as a chemotactic modulator, attracting minus gametes while repelling plus gametes. Here we dissect the processing pathway that leads to formation of an amidated peptidergic sexual signal specifically on the ciliary ectosomes of plus gametes. Unlike metazoan propeptides, modeling studies identified stable domains in proGATI. Mass spectrometric analysis of a potential prohormone convertase and the amidated proGATI-derived products found in cilia and mating ectosomes link endoproteolytic cleavage to ectosome entry. Extensive posttranslational modification of proGATI confers stability to its amidated product. Analysis of this pathway affords insight into the evolution of peptidergic signaling; this will facilitate studies of the secretory functions of metazoan cilia.

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

Peptidergic intercellular communication occurs throughout the eukaryotes, and regulates a wide range of physiological and behavioral responses. Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Our previous study revealed the presence of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), in cilia and its key role in ciliogenesis. Furthermore, PAM and its amidated products are released in ciliary ectosomes from the green alga Chlamydomonas reinhardtii. One amidated product (GATI-amide) serves as a chemotactic modulator for C. reinhardtii gametes, attracting minus gametes while repelling plus gametes. Here we dissect the complex processing pathway that leads to formation of this amidated peptidergic sexual signal specifically on the ectosomes of plus gametes. We also identify a potential prohormone convertase that undergoes domain rearrangement during ectosomal secretion as a substrate for PAM. Analysis of this pathway affords insight into how single-celled organisms lacking dense core vesicles engage in regulated secretion, and provides a paradigm for understanding how amidated peptides that transmit sexual and other signals through cilia are generated.

Peptidylglycine α-Amidating Monooxygenase (PAM) Tami C. Steveson, Tami C. Steveson University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorJoseph Bell, Joseph Bell University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorRichard E. Mains, Richard E. Mains University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorBetty A. Eipper, Betty A. Eipper University of Connecticut, Farmington, ConnecticutSearch for more papers by this author Tami C. Steveson, Tami C. Steveson University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorJoseph Bell, Joseph Bell University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorRichard E. Mains, Richard E. Mains University of Connecticut, Farmington, ConnecticutSearch for more papers by this authorBetty A. Eipper, Betty A. Eipper University of Connecticut, Farmington, ConnecticutSearch for more papers by this author First published: 15 January 2002 https://doi.org/10.1002/0471203076.emm0240 Read the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Wiley Encyclopedia of Molecular MedicineBrowse other articles of this reference work:BROWSE BY TOPICBROWSE A-Z RelatedInformation

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 ER 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 α-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.

The electron-dense spherical granules found in the perinuclear region of atrial myocytes store and release both proatrial and probrain natriuretic peptides (proANP and proBNP, respectively). Mature ANP and BNP produce vasodilation and natriuresis and inhibit the renin-angiotensin and sympathetic nervous systems. Although neither ANP nor BNP is a-amidated, Peptidylglycine a-Amidating Monooxygenase (PAM), an integral membrane enzyme known to catalyze the a-amidation of peptidylglycine precursors, is the major atrial granule membrane protein. Selective deletion of PAM from cardiomyocytes impairs their ability to store proANP, resulting in an increase in proANP secretion. Exogenous expression of active or inactive PAM protein restores the ability of atrial myocytes to store proANP, leading to the suggestion that PAM functions as a cargo receptor for newly synthesized proANP.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine alpha-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (K-m = 99 +/- 19 mu M) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2 [Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.

Nontransgenic and 3xTG transgenic mice, which express mutant transgenes encoding human amyloid precursor protein (hAPP) along with Alzheimer's disease-associated versions of hTau and a presenilin mutation, acquired the Barnes Maze escape task equivalently at 3-9 months of age. Although nontransgenics retested at 6 and 9 months acquired the escape task more quickly than naive mice, 3xTG mice did not. Deficits in Kalirin, a multidomain protein scaffold and guanine nucleotide exchange factor that regulates dendritic spines, has been proposed as a contributor to the cognitive decline observed in Alzheimer's disease. To test whether deficits in Kalirin might amplify deficits in 3xTG mice, mice heterozygous/hemizygous for Kalirin and the 3xTG transgenes were generated. Mouse strain, age and sex affected cortical expression of key proteins. hAPP levels in 3xTG mice increased total APP levels at all ages. Kalirin expression showed strong sex-dependent expression in C57 but not B6129 mice. Decreasing Kalirin levels to half had no effect on Barnes Maze task acquisition or retraining in 3xTG hemizygous mice. (C) 2020 Elsevier Inc. All rights reserved.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM ( Pam Myh6-cKO/cKO ) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam 0-Cre -cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in Pam Myh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by Pam Myh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis -Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway. Significance Transmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

Unfortunately, the human islet checklist was omitted from the electronic supplementary material (ESM) linked to this paper.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Background: Kalirin-7 (Kal7) is a multidomain scaffold and guanine nucleotide exchange factor localized to the postsynaptic density, where Kal7 is crucial for synaptic plasticity. Kal7 knockout mice exhibit marked suppression of long-term potentiation and long-term depression in hippocampus, cerebral cortex and spinal cord, with depressed surface expression of GluN2B receptor subunits and dramatically blunted perception of pain. Kal7 knockout animals show exaggerated locomotor responses to psychostimulants and self-administer cocaine more enthusiastically than wildtype mice. Results: To explore the underlying cellular and molecular mechanisms which are deranged by loss of Kal7, we infused candidate intracellular interfering peptides to acutely challenge the synaptic function(s) of Kal7 with potential protein binding partners, to determine if plasticity deficits in Kal7-/- mice are the product of developmental processes since conception, or could be produced on a much shorter time scale. We demonstrated that these small intracellular peptides disrupted normal long-term potentiation and long-term depression, strongly suggesting that maintenance of established interactions of Kal7 with PSD-95 and/or GluN2B is crucial to synaptic plasticity. Conclusions: Blockade of the Kal7-GluN2B interaction was most effective at blocking long-term potentiation, but had no effect on long-term depression. Biochemical approaches indicated that Kal7 interacted with PSD-95 at multiple sites within Kal7.

Neuropeptides and peptide hormones play a crucial role in integrating the many factors that affect physiologic and cognitive processes. The potency of many of these peptides requires an amidated amino acid at the C-terminus; a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), catalyzes this modification. Anxiety-like behavior is known to be altered in mice with a single functional Pam allele (Pam+/- ) and in mice unable to express Pam in excitatory forebrain neurons (PamEmx1-cKO/cKO ) or in cardiomyocytes (PamMyh6-cKO/cKO ). Examination of PAM-positive and glutamic acid decarboxylase 67 (GAD)-positive cells in the amygdala of PamEmx1-cKO/cKO mice demonstrated the absence of PAM in pyramidal neurons and its continued presence in GAD-positive interneurons, suggestive of altered excitatory/inhibitory balance. Additional behavioral tests were used to search for functional alterations in these cell-type specific knockout mice. PamEmx1-cKO/cKO mice exhibited a less focused search pattern for the Barnes Maze escape hole than control or PamMyh6-cKO/cKO mice. While wildtype mice favor interacting with novel objects as opposed to familiar objects, both PamEmx1-cKO/cKO and PamMyh6-cKO/cKO mice exhibited significantly less interest in the novel object. Since PAM levels in the central nervous system of PamMyh6-cKO/cKO mice are unaltered, the behavioral effect observed in these mice may reflect their inability to produce atrial granules and the resulting reduction in serum levels of atrial natriuretic peptide. In the sociability test, male mice of all three genotypes spent more time with same-sex stranger mice; while control females showed no preference for stranger mice, female PamEmx1-cKO/cKO mice showed preference for same-sex stranger mice in all trials.

Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology.