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Betty A. Eipper - One of the best experts on this subject based on the ideXlab platform.
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Effects of copper occupancy on the conformational landscape of Peptidylglycine alpha-hydroxylating monooxygenase
Communications biology, 2018Co-Authors: S. Maheshwari, Betty A. Eipper, Richard E. Mains, Ninian J. Blackburn, C. Shimokawa, K. Rudzka, C.d. Kline, Sandra B. Gabelli, L.m. AmzelAbstract:The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role. Sweta Maheshwari et al. present X-ray crystal structures of the two-copper enzyme Peptidylglycine α-hydroxylating monooxygenase and three inactive mutant forms. They show that full copper occupancy is needed to maintain the catalytically competent (open) conformation of the enzyme.
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o glycosylation of a secretory granule membrane enzyme is essential for its endocytic trafficking
Journal of Biological Chemistry, 2016Co-Authors: Kurutahalli S. Vishwanatha, Nils Back, Richard E. Mains, Betty A. EipperAbstract:Abstract Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (Peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble Peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.
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Peptidylglycine α-amidating monooxygenase heterozygosity alters brain copper handling with region specificity.
Journal of neurochemistry, 2013Co-Authors: Eric D. Gaier, Richard E. Mains, Megan B. Miller, Martina Ralle, Dipendra K. Aryal, William C. Wetsel, Betty A. EipperAbstract:Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to Peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for Peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.
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Coordination of peroxide to the CuM center of Peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2012Co-Authors: Katarzyna Rudzka, Betty A. Eipper, Richard E. Mains, Diego M. Moreno, Dario A. Estrin, L. Mario AmzelAbstract:Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction—the hydroxylation of Peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-A-resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O–O interatomic distance of the copper-bound ligand is 1.5 A, characteristic of peroxide/hydroperoxide species, and the Cu–O distances are 2.0 and 2.1 A. Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)–O22− species and its isomeric, end-on structure L3CuM(I)–O2·− are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O22− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.
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Coordination of Hydrogen Peroxide by a Copper Center in Peptidylglycine Alpha-Hydroxylating Monooxygenase (PHM). Structural and Computational Study
Biophysical Journal, 2012Co-Authors: Katarzyna Rudzka, Betty A. Eipper, Richard E. Mains, Dario A. Estrin, L. Mario AmzelAbstract:Many bioactive peptides, such as hormones and growth factors require amidation of the C-terminus for their full biological activity. The enzyme Peptidylglycine α-hydroxylating monooxygenase (PHM) carries out the first step of the amidation process, the hydroxylation of Peptidylglycine substrates at the Cα position of the terminal glycine. Two non-equivalent copper sites in PHM (CuH and CuM) play distinct roles in the reaction cycle: CuM serves as an oxygen activation and hydrogen abstraction site, while CuH is involved in electron transfer. The proposed mechanism suggests that dioxygen is activated through a two-electron reduction, where each of the copper centers provides a single electron. However, there is an ambiguity regarding the characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate. To further investigate the nature of the key intermediates in the PHM cycle we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.95 A resolution structure, the (hydro)peroxide ligand coordinates solely to CuM in a slightly asymmetric side-on mode. The copper-oxygen distances are 1.9 and 2.1 A and the O-O bond 1.5 A. The interatomic O-O distance is characteristic of peroxide/hydroperoxide species, and is significantly longer than the distance typically observed in the superoxide molecule. In addition to the x-ray diffraction studies, we performed DFT calculations using the first coordination sphere of the CuM active site as a model system. Comparison of the relative energies associated with side-on Cu(II)-O22- species and its resonant, end-on structure Cu(I)-O2•- suggests that these two intermediates are significantly populated within the protein environment; this observation has important mechanistic implications.
D J Merkler - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of Peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.
Journal of enzyme inhibition and medicinal chemistry, 2015Co-Authors: Neil R. Mcintyre, Edward W. Lowe, Matthew R. Battistini, James W. Leahy, D J MerklerAbstract:AbstractPeptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance gi...
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Production of the catalytic core of human Peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli
Protein expression and purification, 2012Co-Authors: Sumit Handa, Tyler J. Spradling, Daniel R. Dempsey, D J MerklerAbstract:Abstract Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human Peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His 6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.
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Evidence for substrate preorganization in the Peptidylglycine α-amidating monooxygenase reaction describing the contribution of ground state structure to hydrogen tunneling.
Journal of the American Chemical Society, 2010Co-Authors: Neil R. Mcintyre, Edward W. Lowe, Jonathan L. Belof, Milena Ivkovic, Jacob Shafer, Brian Space, D J MerklerAbstract:Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O2-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by Peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by Peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM-dependent Cα-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy pert...
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thiorphan tiopronin and related analogs as substrates and inhibitors of Peptidylglycine α amidating monooxygenase pam
FEBS Letters, 2006Co-Authors: Neil R. Mcintyre, Edward W. Lowe, Geoffrey H. Chew, Terrence C Owen, D J MerklerAbstract:Abstract Peptidyglycine α-amidating monooxygenase is a copper- and zinc-dependent, bifunctional enzyme that catalyzes the cleavage of glycine-extended peptides or N-acylglycines to the corresponding amides and glyoxylate. This reaction is a key step in the biosynthesis of bioactive α-amidated peptides and, perhaps, the primary fatty acids amides also. Two clinically useful N-acylglycines are thiorphan and tiopronin, each with a thiol moiety attached to the acyl group. We report here that thiorphan and tiopronin are substrates for PAM, exhibiting relatively low KM,app and VMAX,app values. The low VMAX,app values result, most likely, from a decrease in active PAM · 2Cu(II) as the enzyme competes ineffectively with thiorphan and tiopronin for free copper.
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Ubiquitin and ubiquitin-derived peptides as substrates for Peptidylglycine α-amidating monooxygenase
FEBS letters, 2005Co-Authors: Geoffrey H. Chew, Neil R. Mcintyre, Lamar Galloway, Laura Aaron Schroder, Karla M. Richards, Scott A. Miller, David W. Wright, D J MerklerAbstract:Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding α-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is Peptidylglycine α-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)amidation that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.
B A Eipper - One of the best experts on this subject based on the ideXlab platform.
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Coordination of peroxide to the Cu_M center of Peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study
JBIC Journal of Biological Inorganic Chemistry, 2013Co-Authors: Katarzyna Rudzka, B A Eipper, Richard Mains, Diego M. Moreno, Dario A. Estrin, L. Mario AmzelAbstract:Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction—the hydroxylation of Peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O_2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O_2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (Cu_H and Cu_M). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-Å-resolution structure (hydro)peroxide binds solely to Cu_M in a slightly asymmetric side-on mode. The O–O interatomic distance of the copper-bound ligand is 1.5 Å, characteristic of peroxide/hydroperoxide species, and the Cu–O distances are 2.0 and 2.1 Å. Density functional theory calculations using the first coordination sphere of the Cu_M active site as a model system show that the computed energies of the side-on L_3Cu_M(II)–O_2 ^2− species and its isomeric, end-on structure L_3Cu_M(I)–O_2 ^·− are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L_3Cu_M(II)O_2 ^2− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.
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Peptidylglycine alpha-amidating monooxygenase: an ascorbate-requiring enzyme.
Methods in enzymology, 1997Co-Authors: Aparna S. Kolhekar, Richard E. Mains, B A EipperAbstract:Publisher Summary Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyterminal amidation of glycine-extended peptides. Amidation is a two-step reaction, with the first step being hydroxylation at the α-carbon of the carboxy-terminal glycine, catalyzed by Peptidylglycine α-hydroxylating monooxygenase (PHM), a copper, ascorbate, and molecular oxygen-dependent enzyme. The second step of the reaction is dealkylation of the peptidyl α-hydroxyglycine intermediate, catalyzed by peptidyl α-hydroxyglycine α-amidating lyase (PAL), a divalent metal ion-dependent enzyme. Peptidylglycine α-amidating monooxygenase is the only enzyme known to catalyze the α-amidation of peptides. Although PAM is encoded by a single gene, soluble and membrane-bound monofunctional and bifunctional forms are generated by tissue-specific alternative splicing and endoproteolytic cleavage. The PHM-catalyzed reaction requires a reducing cofactor and ascorbate is the most likely physiological reductant, although the cofactor requirement in vivo can be cell-type specific. Overall, the enzyme converts 2 mol of reduced ascorbate into 2 mol of semidehydroascorbate, consuming 1 mol of ascorbate for each mole of α-amidated product peptide. The optimal ascorbate concentration is typically about 1 mM. Dopamine β-monooxygenase performs a similar reaction, using the same cofactors.
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Phosphorylation of the cytosolic domain of Peptidylglycine alpha-amidating monooxygenase.
The Journal of biological chemistry, 1995Co-Authors: Hye-young Yun, Sharon L. Milgram, Henry T. Keutmann, B A EipperAbstract:Abstract Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the COOH-terminal α-amidation of neural and endocrine peptides through a two-step reaction carried out sequentially by its monooxygenase and lyase domains. PAM occurs in soluble and integral membrane forms. Metabolic labeling of stably transfected hEK-293 and AtT-20 cells showed that [32P]PO4 was efficiently incorporated into Ser and Thr residues of membrane PAM but not into soluble PAM. Truncation of integral membrane PAM proteins (which terminate with Ser) at Tyr eliminated their phosphorylation, suggesting that the COOH-terminal region of the protein was the site of phosphorylation. Recombinant PAM COOH-terminal domain was phosphorylated on Ser and Ser by protein kinase C (PKC). PAM-1 protein recovered from different subcellular fractions of stably transfected AtT-20 cells was differentially susceptible to calcium-dependent, staurosporine-inhibitable phosphorylation catalyzed by endogenous cytosolic protein kinase(s). Although phorbol ester treatment of hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a bifunctional 105-kDa PAM protein, the effect was an indirect one since it was also observed in hEK-293 cells expressing a truncated PAM-1 protein that was not phosphorylated. AtT-20 cells expressing PAM-1 lacking one of the PKC sites (PAM-1/Ser Ala) exhibited an altered pattern of PAM•PAM antibody internalization, with the mutant protein targeted to lysosomes upon internalization. Thus, phosphorylation of Ser in the COOH-terminal cytosolic domain of membrane PAM plays a role in a specific step in the targeting of this protein.
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The NH2-terminal proregion of Peptidylglycine alpha-amidating monooxygenase facilitates the secretion of soluble proteins.
Molecular endocrinology (Baltimore Md.), 1995Co-Authors: Richard E. Mains, Sharon L. Milgram, Henry T. Keutmann, B A EipperAbstract:A highly conserved ten amino acid proregion separates the Peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain of the bifunctional Peptidylglycine alpha-amidating monooxygenase (PAM) protein from the NH2-terminal signal peptide; propeptides with amino acid sequences similar to the PAM proregion have been identified in other secreted proteins. In AtT-20 cells, but not in human embryonic kidney (hEK)-293 cells, an endogenous endoprotease acting at a site distal to the trans-Golgi network efficiently removes the propeptide from stably transfected monofunctional PHM (PHMs). We constructed a mutant PHM protein (delta ProPHMs) in which the proregion was deleted and the signal peptide joined directly to the monooxygenase domain. Newly synthesized, enzymatically active delta ProPHMs was secreted from both AtT-20 cells and hEK-293 cells more slowly than PHMs. In endocrine cells, the proregion was not required for storage in regulated secretory granules. We transferred the PAM proregion to prohormone conv...
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Localization of the Gene Encoding Peptidylglycine α-Amidating Monooxygenase (PAM) to Human Chromosome 5q14-5q21
Genomics, 1993Co-Authors: L Ouafik, B A Eipper, Charles Oliver, Marie Geneviève Mattei, Pierre Giraud, Richard MainsAbstract:Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).
Richard E. Mains - One of the best experts on this subject based on the ideXlab platform.
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Effects of copper occupancy on the conformational landscape of Peptidylglycine alpha-hydroxylating monooxygenase
Communications biology, 2018Co-Authors: S. Maheshwari, Betty A. Eipper, Richard E. Mains, Ninian J. Blackburn, C. Shimokawa, K. Rudzka, C.d. Kline, Sandra B. Gabelli, L.m. AmzelAbstract:The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role. Sweta Maheshwari et al. present X-ray crystal structures of the two-copper enzyme Peptidylglycine α-hydroxylating monooxygenase and three inactive mutant forms. They show that full copper occupancy is needed to maintain the catalytically competent (open) conformation of the enzyme.
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RESEARCH ARTICLE Open Access Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells
2016Co-Authors: Kristina Carlson, Steven C. Pomerantz, Omid Vafa, Michael Naso, William Strohl, Richard E. MainsAbstract:Background: Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is Peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein. Methods: To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway. Results: PAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing Peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate. Conclusions: Peptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM
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o glycosylation of a secretory granule membrane enzyme is essential for its endocytic trafficking
Journal of Biological Chemistry, 2016Co-Authors: Kurutahalli S. Vishwanatha, Nils Back, Richard E. Mains, Betty A. EipperAbstract:Abstract Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (Peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble Peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.
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Biochemical Characterization of Peptide a-Amidation Enzyme Activities of Human Neuroendocrine Lung
2015Co-Authors: Cancer Cell Lines, Richard E. Mains, Anthony M. Treston, Frank Cuttitta, Frank M. Scott, Michele Vos, Naomichi Lwai, James I. MulshineAbstract:Peptide a-amidation is a posttranslational modification of approximately half of all endocrine and neuroendocrine peptide hormones, including several hormones with mitogenic effeds for tumor cells, and is typically essential for complete hormonal bioadivity. a-Amidated peptide hormones have been reported to be autocrine growth fadors for small cell lung cancer cells. We report here that a variety of human lung tumor cell lines express both enzymes required for the two-step conversion of inadive glycine-extended peptides into their adive COOH-terminal a-amide analogues. Human tumor cell Peptidylglycine a-amidation enzymes are present in multiple molecular forms. Both proteins are metalloenzymes which are present at highest concentrations in secretory granules in neuroendocrine cell lines. The expression of these enzymes is positively correlated with expression of other markers of the neuroendocrine phenotype, such as DOPA decarboxylase. Peptidylglycine a-amidating enzyme-specific adivities are approximately 50-fold higher in extracts of endocrine cell lines (lung small cell and carcinoid) than of nonendocrine lines. Biochemical charaderization of the Peptidylglycine a-amidating enzymes will enable development of tools for detedion of endocrine processes in the early stages of neoplasia and for interruption of autocrine stimulation pathways in tumor cells
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Peptidylglycine α-amidating monooxygenase heterozygosity alters brain copper handling with region specificity.
Journal of neurochemistry, 2013Co-Authors: Eric D. Gaier, Richard E. Mains, Megan B. Miller, Martina Ralle, Dipendra K. Aryal, William C. Wetsel, Betty A. EipperAbstract:Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to Peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for Peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.
L Ouafik - One of the best experts on this subject based on the ideXlab platform.
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Mammalian Peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen.
Molecular and Cellular Biology, 2005Co-Authors: Fabienne Brenet, Christine Delfino, Nadège Dussault, Jonas Borch, Géraldine Ferracci, Peter Roepstorff, Raymond Miquelis, L OuafikAbstract:Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of Peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.
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Identification of a novel cis-element in the 3'-untranslated region of mammalian Peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid.
Endocrinology, 1998Co-Authors: Sandrine Fraboulet, Christine Delfino, F. Boudouresque, L OuafikAbstract:Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of Peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3'-untranslated region (3'-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3'-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.
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Estrogen Regulation of Peptidylglycine α-Amidating Monooxygenase Expression in Anterior Pituitary Gland
Endocrinology, 1997Co-Authors: Rajaâ El Meskini, Christine Delfino, F. Boudouresque, Charles Oliver, Micheline Hery, L OuafikAbstract:The pituitary is a rich source of Peptidylglycine α-amidating monooxygenase (PAM). This bifunctional protein contains Peptidylglycineα -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycineα -amidating lyase catalytic domains necessary for the two-step formation of α-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 β-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 β-estradiol treatment induced an overall decrease of the ...
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Localization of the Gene Encoding Peptidylglycine α-Amidating Monooxygenase (PAM) to Human Chromosome 5q14-5q21
Genomics, 1993Co-Authors: L Ouafik, B A Eipper, Charles Oliver, Marie Geneviève Mattei, Pierre Giraud, Richard MainsAbstract:Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).
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The multifunctional Peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.
Molecular Endocrinology, 1992Co-Authors: L Ouafik, Richard E. Mains, Doris A. Stoffers, T A Campbell, Richard C. Johnson, Brian T. Bloomquist, B A EipperAbstract:Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM r...