Peptidylglycine Α-amidating Monooxygenase Research Articles

PAM variants in patients with thyrotrophinomas, cyclical Cushing's disease and prolactinomas.

Germline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC). We performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing's disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%. We identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing's disease (2/7, 29%), compared to prolactinomas (1/12, 8%). This is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing's disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol.

The adrenomedullin system and risk of cardiogenic shock or death complicating acute coronary syndromes

Abstract Introduction Cardiogenic shock (CS) is the leading cause of death due to acute coronary syndromes (ACS) and mortality remained nearly unchanged over the past two decades. Immediate revascularization of the infarct-related artery is the treatment strategy of choice to reduce CS-related mortality, but early identification of patients at high risk of CS and death is challenging. The adrenomedullin (ADM) system modulates vascular tone and endothelial barrier function and monitoring its activity may enhance early risk assessment of ACS patients prone to experience CS and ultimately death. Methods In 4293 ACS patients, prospectively recruited at 4 Swiss university hospitals, the 3 major ADM system components were assessed in plasma at the time of acute presentation by blinded study personnel: ADM-glycine (its inactive substrate), peptidylglycine α-amidating monooxygenase (PAM; the ADM-amidating enzyme), and the biologically active ADM-amide (bio-ADM). Patients were monitored for in-hospital development of CS and followed at 30 days and 1 year with external event adjudication. The relationship of each ADM system component with features of CS pathophysiology (such as proxies for the decrease in cardiac contractile mass, increase in cardiac stress or systemic inflammation) was estimated by means of partial correlation coefficients. Multivariable-adjusted regression models were fit to assess their independent association with CS development and death. Results Baseline ADM-glycine, PAM and bio-ADM levels were tightly linked to features of CS (Fig. 1), with each ADM system component associating with in-hospital CS (odds ratio, 95% confidence interval [CI] per doubling in each, 2.04, 1.79-2.33, P&amp;lt;.001; 1.91, 1.63-2.23, P&amp;lt;.001; 1.72, 1.11-2.66, P=.016). The association of each marker with CS remained robust after adjusting for established risk factors for in-hospital development of CS, including all hemodynamic-, patient-, and procedure-related variables informing the ORBI risk score (1.65, 1.35-2.00, P&amp;lt;.001; 1.50, 1.26-1.77, P&amp;lt;.001; and 1.87, 1.13-3.11, P=.015). Notably, integration of ADM-glycine, PAM, and bio-ADM into the ORBI risk prediction model conferred increased discriminative performance for the prediction of in-hospital CS (AUC, 0.76 vs. 0.81, P&amp;lt;.001), ranking the ADM system second among all variables informing the score (Fig. 2). While both ADM-glycine and bio-ADM independently associated with mortality risk at 30-days (multivariable-adjusted hazard ratio per doubling, 95% CI, 1.93, 1.54-2.42, P&amp;lt;.001; and 1.81, 1.34-2.44, P&amp;lt;.001) and 1-year (1.41, 1.18-1.68, P&amp;lt;.001; and 1.52, 1.24-1.87, P&amp;lt;.001), PAM showed no association with these outcomes (P&amp;gt;.05). Conclusions Monitoring key regulators of the ADM system improves early risk assessment of ACS patients at high risk of CS, with admission ADM-glycine and bio-ADM representing independent risk factors for 30-day and 1-year mortality post-ACS.

Immunoassay-based quantification of full-length peptidylglycine alpha-amidating monooxygenase in human plasma

A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and intra-assay (2.2%) variabilities. It exhibited linearity when accessed by gradual dilution or random mixing of plasma samples. The accuracy of the PAM-LIA was determined to be 94.7% through spiking recovery experiments, and the signal recovery after substance interference was 94–96%. The analyte showed 96% stability after six freeze–thaw cycles. The assay showed strong correlation with matched EDTA and serum samples, as well as matched EDTA and Li-Heparin samples. Additionally, a high correlation was observed between α-amidating activity and PAM-LIA. Finally, the PAM-LIA assay was successfully applied to a sub-cohort of a Swedish population-based study, comprising 4850 individuals, confirming its suitability for routine high throughput screening.

Peptidylglycine α-amidating monooxygenase and adrenomedullin measurement in patients with hepatic cirrhosis.

Background: Peptidylglycine α-amidating monooxygenase (PAM) is an enzyme involved in the maturation of regulatory peptides. Here we examined PAM activity and adrenomedullin (bio-ADM) concentrations in patients with hepatic cirrhosis and determined net changes across the liver, kidneys and leg. Materials&methods: A total of 48 patients with hepatic cirrhosis and 16 control subjects were included. All patients and controls underwent an invasive procedure with blood collected across organs. Results: PAM activity was increased in cirrhotic patients but without a net change across the liver, leg or kidney. In contrast, bio-ADM concentrations were associated with severity of disease and found to be higher in venous blood from the liver. Conclusion: Increased PAM activity in patients with hepatic cirrhosis may reflect other organs involved in cirrhotic disease.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Copper Deficiency Alters Rat Peptidylglycine α-Amidating Monooxygenase Activity

Perinatal copper deficiency was studied in 1-mo-old female and male Sprague-Dawley rat offspring to investigate changes in cuproenzymes. Offspring of dams given the low Cu treatment beginning at d 7 of gestation exhibited signs characteristic of Cu deficiency, including a 90% reduction in liver Cu levels compared with Cu-adequate controls. Compared with Cu-adequate rats, Cu-deficient rats had lower activities of the cuproenzymes peptidylglycine α-amidating monooxygenase (PAM), cytochrome c oxidase (CCO), and Cu,Zn-superoxide dismutase (SOD) in heart and midbrain samples. Activity of dopamine-β-monooxygenase (DBM) was higher in midbrain and lower in heart samples from Cu-deficient compared with Cu-adequate rats. Following 1 mo of Cu repletion, PAM and CCO activity were still lower in heart of Cu-replete rats. Midbrain DBM activity was still elevated in the former Cu-deficient males. A second study was conducted using weanling male Holtzman rats. After 5.5 wk of treatment, Cu-deficient rats had signs characteristic of Cu deficiency and lower PAM, CCO and DBM activities in heart but not midbrain as compared with Cu-adequate rats. The PAM activity was lower following Cu deficiency. Perhaps neuropeptide maturation is compromised by Cu deficiency.

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker for human diseases.

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 catalysing 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 tumours. 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.

Cognitive stimulation in the workplace, plasma proteins, and risk of dementia: three analyses of population cohort studies

ObjectivesTo examine the association between cognitively stimulating work and subsequent risk of dementia and to identify protein pathways for this association.DesignMulticohort study with three sets of analyses.SettingUnited Kingdom, Europe, and...

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

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 thebioactive 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.

Multiple roles for peptidylglycine α-amidating monooxygenase in the response to hypoxia.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-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 (Km = 99 ± 19 μM) 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.

Cell‐type specific knockout of peptidylglycine α‐amidating monooxygenase reveals specific behavioral roles in excitatory forebrain neurons and cardiomyocytes

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.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation

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 13-fold drop in the number of secretory granules. When primary cultures of Pam 0-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 Pam Myh6-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 Pam Myh6-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 Pam Myh6-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.

PERK-mediated expression of peptidylglycine \u03b1-amidating monooxygenase supports angiogenesis in glioblastoma

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.

PAM haploinsufficiency does not accelerate the development of diet- and human IAPP-induced diabetes in mice.

Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam+/- and Pam+/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1β, IFN-γ and TFN-α. Despite Pam+/- only having 50% PAM expression and enzyme activity as compared with Pam+/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24weeks of HFD, both Pam+/- and Pam+/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

Cilia-based peptidergic signaling.

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.

Identifying roles for peptidergic signaling in mice

Despite accumulating evidence demonstrating the essential roles played by neuropeptides, it has proven challenging to use this information to develop therapeutic strategies. Peptidergic signaling can involve juxtacrine, paracrine, endocrine, and neuronal signaling, making it difficult to define physiologically important pathways. One of the final steps in the biosynthesis of many neuropeptides requires a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), and lack of amidation renders most of these peptides biologically inert. PAM, an ancient integral membrane enzyme that traverses the biosynthetic and endocytic pathways, also affects cytoskeletal organization and gene expression. While mice, zebrafish, and flies lacking Pam (PamKO/KO ) are not viable, we reasoned that cell type-specific elimination of Pam expression would generate mice that could be screened for physiologically important and tissue-specific deficits. Conditional PamcKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global PamKO/KO mouse, were indistinguishable from wild-type mice. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine, and improved thermoregulation in the cold. A number of amidated peptides play essential roles in each of these behaviors. Although atrial natriuretic peptide (ANP) is not amidated, Pam expression in the atrium exceeds levels in any other tissue. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior and improved thermoregulation. Atrial and serum levels of ANP fell sharply in PAM myosin heavy chain 6 conditional knockout mice, and RNA sequencing analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of therapeutic approaches targeted to peptidergic pathways.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

25-OR: Investigating a Candidate Causal Allele in Type 2 Diabetes Susceptibility Gene PAM as a Cause of Neonatal Diabetes Mellitus

Peptidylglycine α-amidating monooxygenase (PAM) catalyses the amidation of glycine-extended peptides, thereby maximising their biological potency. We recently showed that coding alleles in PAM mediate type 2 diabetes (T2D) risk through alterations to PAM protein activity and expression in the β cell. Identifying alleles along a continuum of functional severity informs on the relationship between protein perturbation and clinical effect. We hypothesised that rare, loss of function PAM alleles could cause severe β cell dysfunction presenting as neonatal diabetes. Sequencing of 15 probands presenting with diabetes at ≤ 6 months of age and their unaffected immediate family, identified a novel de novo nonsynonymous coding variant (p.R36S) in PAM in a single proband. We investigated the impact of the variant allele on amidating activity, protein stability and localisation as a measure of PAM function and the consequences on β cell viability. Recombinant R36S-PAM exhibited normal amidating activity in vitro which was consistent with PAM activity measured in patient serum (WT: 391pmol/μl/hr vs. R36S: 305pmol/μl/hr). In human β cells (EndoC-βh1) the secreted luminal R36S-PAM isoform displayed reduced protein expression (-78%, p=0.004) compared to WT-PAM, which could be rescued by inhibiting the proteosomal pathway (+120% of WT); whilst the non-secreted membrane-integral isoform was retained in the endoplasmic reticulum (ER). We observed that overexpression of R36S-PAM elevated levels of ER stress (p-IRE1: +196%, p=0.02; CHOP: +114%, p=0.051) and pro-apoptotic markers (ASK1: +145%, caspase-3: +154%) compared with WT-PAM. We have identified a novel, de novo coding variant, in a gene with a proven role in T2D risk, in a single patient with neonatal diabetes. Functional studies provide evidence for protein mislocalisation and instability and support a role for this allele in ER stress and β cell dysfunction consistent with neonatal diabetes pathogenesis. Disclosure S. Sengupta: None. L.L. Bonnycastle: None. B. Hastoy: None. A. Grotz: None. M.M. Umapathysivam: None. A. Raimondo: None. A. Swift: None. P.S. Chines: None. A. Clark: None. H. Huopio: None. F.S. Collins: None. M. Laakso: None. A.L. Gloyn: Consultant; Spouse/Partner; Eli Lilly and Company, Merck &amp; Co., Inc. Consultant; Self; Merck &amp; Co., Inc. Consultant; Spouse/Partner; Novo Nordisk A/S, Pfizer Inc. Research Support; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Other Relationship; Self; Diabetes UK, European Foundation for the Study of Diabetes. Funding UK Wellcome Trust

Long Term Follow up of Cerebellar Ataxia CA) and MRI Lesions Which Responded to Copper (Cu) Supplementation (P12-055-19)

ObjectivesCu deficiency (CuD) produces anemia and leukopenia. CuD neurologic dysfunction can also occur. We follow a patient with CuD with CA and MRI lesions that responded to Cu replacement. MethodsA 64-year-old male with progressive ataxia and neuropathy was found to have a significant CuD (copper level 38 mmol/L, normal range 70–175) 7 years ago. The etiology of CuD was unknown. There was no previous gastric surgery, malabsorption, ingestion of zinc containing products or medications associated with Cu loss.He had an ataxic gait with a steppage component. Muscle stretch reflexes were brisk at the knees and absent at the ankles. He had length dependent loss to all modalities. Other etiologies for CA were not found. The blood smear showed anisocytes and poikilocytes, but anemia and leukopenia were absent. He became wheelchair bound after several weeks.Brain MRI without contrast revealed non-enhancing symmetric T2 signal prolongation of the medial cerebellar white matter extending into left middle cerebral peduncle. Neuroconductive studies showed moderate-to-severe axonal sensorimotor polyneuropathy. ResultsCu supplementation with using 5 mg cupric oxide daily raised serum Cu to 97 mmol/L. Symptoms improved after 1 month. Brain MRI 3 months later showed no cerebellar lesions with serum Cu of 123 mmol/L. At this point he was able to walk with a cane. His gait disturbance further improved to the extent that he was able to walk without assistance or support. His follow up Cu levels remain in the normal range (last value = 128 mmol/L). ConclusionsCu is a component of the neurologic metalloenzymes cytochrome-c oxidase (CcO), Cu-zinc superoxide dismutase, tyrosinase, dopamine β-hydroxylase, peptidylglycine α-amidating monooxygenase and ceruloplasmin. Cu and iron pass electrons through CcO to oxygen, forming water. CuD impacts the electron transport chain, impairing ATP production which injures susceptible tissues.This case demonstrates the reversibility of neurologic dysfunction and MRI lesions by means of correcting CuD. Our 7-year follow up period confirms the validity of the pathogenesis and management. Further study is needed to identify the specific roles of Cu metabolism in such cases and the individual factors allowing for the dramatic response we observed. Funding Sourcesnone.

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.