Histone removal in sperm protects paternal chromosomes from premature division at fertilization.

The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)2 tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.

PAM variants were associated with type 2 diabetes mellitus risk in the Chinese population.

This study aimed to assess the association between PAM single-nucleotide polymorphisms (SNPs) and T2DM risk in the Chinese population. We performed the genotype of PAM SNPs using Agena MassARRAY in 1002 subjects. The effect of PAM polymorphisms on T2DM occurrence was evaluated by logistic regression analysis. False-positive report probability (FPRP) was utilized to assess the noteworthiness of the significant results. This study showed that PAM rs406761, rs17154889, and rs6889592 were related to an increased risk of T2DM. The similar results were also in subjects with ≤ 60 years. Rs2431320 and rs406761 were related to an increased risk of T2DM in males, and rs6889592 was only found to be associated with T2DM risk in females. Rs2431320 and rs406761 increased T2DM risk in people with BMI > 24, and rs6889592 and rs26431 significantly correlated with T2DM risk in people with BMI ≤ 24. By comparing patients with no retinopathy with controls, the correlation between PAM rs406761 and rs17154889 and T2DM risk was observed. The significant association between T2DM risk and PAM SNPs was remarkable by FPRP values. PAM SNPs were correlated with T2DM risk in the Chinese population, illustrating the importance of PAM SNPs in the pathogenesis of T2DM.

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.

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex.

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

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 (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.

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

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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-1ß, 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 24 weeks 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. CONCLUSIONS/INTERPRETATION: Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

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.

PAM50 for prediction of response to neoadjuvant chemotherapy for ER-positive breast cancer.

PURPOSE: There is an urgent need for the development of a predictor of response to chemotherapy for ER-positive breast cancer which is less chemosensitive than for ER-negative breast cancer in order to avoid unnecessary chemotherapy. In the present study, intrinsic subtyping by PAM50 was evaluated for its ability to predict a response to chemotherapy. PATIENTS AND METHODS: For this study, 124 patients with ER-positive breast cancer treated with neoadjuvant sequential paclitaxel and FEC (NAC) were evaluated. Tumor biopsy specimens obtained before NAC were subjected to intrinsic subtyping (IS) by gene expression (GE) using PAM50 (PAM50-IS) or immunohistochemistry (IHC-IS). RESULTS: Of the PAM50-ISs (Luminal A, Luminal B, HER2-enriched, and Basal-like), GE-Luminal A showed the lowest pCR rate (1.9%), and multivariate analysis revealed that GE-Luminal A was a significant (P = 0.031) predictor of non-pCR independently of other clinicopathological parameters, including Ki67, and tumor-infiltrating lymphocytes. Of the IHC-ISs, on the other hand, IHC-Luminal A was not significantly associated with pCR. We also found that breast tumors with low ER levels (1-9%), like ER-negative tumors, were mostly GE-HER2-enriched and GE-Basal-like, and more sensitive to NAC than those with high ER levels (≥ 10%). CONCLUSIONS: GE-Luminal A intrinsically subtyped by PAM50 was the least sensitive to NAC and very unlikely to attain pCR. IHC-Luminal A identified by IHC, on the other hand, was not significantly predictive of pCR. In addition, PAM50 revealed that tumors with low ER (1-9%) were more like ER-negative tumors than ER-positive tumors, and most such cases should therefore would better be treated with chemotherapy.

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic ß-cells.

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human ß-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in ß-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans.

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

The peptidylglycine-α-amidating monooxygenase (PAM) gene rs13175330 A>G polymorphism is associated with hypertension in a Korean population.

BACKGROUND: Peptidylglycine-α-amidating monooxygenase (PAM) may play a role in the secretion of atrial natriuretic peptide (ANP), which is a hormone involved in the maintenance of blood pressure (BP). The objective of the present study was to determine whether PAM is a novel candidate gene for hypertension (HTN). RESULTS: A total of 2153 Korean participants with normotension and HTN were included. Genotype data were obtained using the Korean Chip. The rs13175330 polymorphism of the PAM gene was selected from the ten single nucleotide polymorphisms (SNPs) most strongly associated with BP. The presence of the G allele of the PAM rs13175330 A>G SNP was associated with a higher risk of HTN after adjustments for age, sex, BMI, smoking, and drinking [OR 1.607 (95% CI 1.220-2.116), p = 0.001]. The rs13175330 G allele carriers in the HTN group treated without antihypertensive therapy (HTN w/o therapy) had significantly higher systolic and diastolic BP than the AA carriers, whereas the G allele carriers in the HTN group treated with antihypertensive therapy (HTN w/ therapy) showed significantly higher diastolic BP. Furthermore, rs13175330 G allele carriers in the HTN w/o therapy group had significantly increased levels of insulin, insulin resistance, and oxidized low-density lipoprotein (LDL) and significantly decreased LDL-cholesterol levels and LDL particle sizes compared to the AA carriers. CONCLUSION: These results suggest that the PAM rs13175330 A>G SNP is a novel candidate gene for HTN in the Korean population. Additionally, the PAM rs13175330 G allele might be associated with insulin resistance and LDL atherogenicity in patients with HTN.

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C.

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

Identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type 2 diabetes.

Through whole-genome sequencing of 2,630 Icelanders and imputation into 11,114 Icelandic cases and 267,140 controls followed by testing in Danish and Iranian samples, we discovered 4 previously unreported variants affecting risk of type 2 diabetes (T2D). A low-frequency (1.47%) variant in intron 1 of CCND2, rs76895963[G], reduces risk of T2D by half (odds ratio (OR) = 0.53, P = 5.0 × 10(-21)) and is correlated with increased CCND2 expression. Notably, this variant is also associated with both greater height and higher body mass index (1.17 cm per allele, P = 5.5 × 10(-12) and 0.56 kg/m(2) per allele, P = 6.5 × 10(-7), respectively). In addition, two missense variants in PAM, encoding p.Asp563Gly (frequency of 4.98%) and p.Ser539Trp (frequency of 0.65%), confer moderately higher risk of T2D (OR = 1.23, P = 3.9 × 10(-10) and OR = 1.47, P = 1.7 × 10(-5), respectively), and a rare (0.20%) frameshift variant in PDX1, encoding p.Gly218Alafs*12, associates with high risk of T2D (OR = 2.27, P = 7.3 × 10(-7)).

Extracellular ammonia at sites of pulmonary infection with Coccidioides posadasii contributes to severity of the respiratory disease.

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen.

Exome array analysis identifies new loci and low-frequency variants influencing insulin processing and secretion.

Insulin secretion has a crucial role in glucose homeostasis, and failure to secrete sufficient insulin is a hallmark of type 2 diabetes. Genome-wide association studies (GWAS) have identified loci contributing to insulin processing and secretion; however, a substantial fraction of the genetic contribution remains undefined. To examine low-frequency (minor allele frequency (MAF) 0.5-5%) and rare (MAF < 0.5%) nonsynonymous variants, we analyzed exome array data in 8,229 nondiabetic Finnish males using the Illumina HumanExome Beadchip. We identified low-frequency coding variants associated with fasting proinsulin concentrations at the SGSM2 and MADD GWAS loci and three new genes with low-frequency variants associated with fasting proinsulin or insulinogenic index: TBC1D30, KANK1 and PAM. We also show that the interpretation of single-variant and gene-based tests needs to consider the effects of noncoding SNPs both nearby and megabases away. This study demonstrates that exome array genotyping is a valuable approach to identify low-frequency variants that contribute to complex traits.

An alternative pathway for ureide usage in legumes: enzymatic formation of a ureidoglycolate adduct in Cicer arietinum and Phaseolus vulgaris.

Ureidoglycolate is an intermediate in the degradation of the ureides, allantoin and allantoate, found in many organisms. In some leguminous plant species these compounds are used to transport recently fixed nitrogen in the root nodules to the aerial parts of the plant. In the present study, it was demonstrated that purified ureidoglycolases from chickpea (Cicer arietinum) and French bean (Phaseolus vulgaris) do not produce glyoxylate, and can use phenylhydrazine as a substrate with K(m) values of 4.0 mM and 8.5 mM, respectively. Furthermore, these enzymes catalyse the transfer of the ureidoglycolyl group to phenylhydrazine to produce ureidoglycolyl phenylhydrazide, which degrades non-enzymatically to glyoxylate phenylhydrazone and urea. This supports their former classification as ureidoglycolate urea-lyases. The enzymatic reaction catalysed by the characterized ureidoglycolases uncovered here can be viewed as a novel type of phenylhydrazine ureidoglycolyl transferase. The implications of these findings for ureide metabolism in legume nitrogen metabolism are discussed.

Amidation of bioactive peptides: the structure of the lyase domain of the amidating enzyme.

Many neuropeptides and peptide hormones require amidation of their carboxy terminal for full biological activity. The enzyme peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate. Here we report the X-ray crystal structure of the PAL catalytic core (PALcc) alone and in complex with the nonpeptidic substrate alpha-hydroxyhippuric acid. The structures show that PAL folds as a six-bladed beta-propeller. The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its alpha-hydroxyl group coordinated to the Zn(II) ion. The structures also reveal a tyrosine residue (Tyr(654)) at the active site as the catalytic base for hydroxyl deprotonation, an unusual role for tyrosine. A reaction mechanism is proposed based on this structural data and validated by biochemical analysis of site-directed PALcc mutants.

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2.

Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase.

We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.

Essential features of the catalytic core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.

Bioactive peptides frequently terminate with an essential alpha-amide that is generated from a COOH-terminal Gly in a two-step enzymatic process occurring within the lumen of the secretory pathway. The first enzyme, peptidylglycine alpha-hydroxylating monooxygenase, is a member of the copper- and ascorbate-dependent monooxygenase family. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL, EC 4.3.2.5), has no known homologues. Examination of the catalytic core of PAL (PALcc) using trypsin, BNPS skatole, and COOH-terminally truncated proteins failed to identify stable subdomains. Treatment of PALcc with divalent metal ion chelators inactivated the enzyme and increased its protease and thermal sensitivity, suggesting a structural role for bound metal. Purified PALcc contained 0.7 +/- 0.4 mol of zinc/mol of enzyme. Since the four Cys residues in PALcc form two disulfide bonds, potential Zn ligands include conserved Asp, Glu, and His residues. The secretion and activity of PALcc bearing mutations in each conserved Asp, Glu, and His residue were evaluated. Mutation of three conserved Asp residues and two conserved His residues yielded a protein that could not be secreted, suggesting that these residues play a structural role. Analysis of mutants that were efficiently secreted identified three His residues along with single Asp residue that may play a role in catalysis. These essential residues occur in a pattern unique to PAL.