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B A Eipper - One of the best experts on this subject based on the ideXlab platform.

  • A Single Membrane Protein Required for Atrial Secretory Granule Formation
    2020
    Co-Authors: Nils Bäck, R E Mains, Raj Luxmi, Kathryn G. Powers, B A Eipper
    Abstract:

    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 Alpha-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 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the Monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in PamMyh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by PamMyh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway.

  • Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery
    MDPI AG, 2018
    Co-Authors: Raj Luxmi, R E Mains, Crysten Blaby-haas, Dhivya Kumar, Navin Rauniyar, Stephen M. King, B A Eipper
    Abstract:

    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

  • Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.
    Neurobiology of disease, 2009
    Co-Authors: Danielle Bousquet-moore, Joseph R. Prohaska, R E Mains, Eduardo A. Nillni, Traci A. Czyzyk, William C. Wetsel, B A Eipper
    Abstract:

    Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for Peptidylglycine Alpha-Amidating Monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

  • Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine α-Amidating Monooxygenase by Dietary Copper
    Endocrinology, 2008
    Co-Authors: Danielle Bousquet-moore, B A Eipper, Eduardo A. Nillni, Traci A. Czyzyk, John E. Pintar, R E Mains
    Abstract:

    Amidated peptides are critically involved in many physiological functions. Genetic deletion of Peptidylglycine Alpha-Amidating Monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

  • Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse.
    Developmental biology, 2005
    Co-Authors: Traci A. Czyzyk, R E Mains, B A Eipper, Yun Ning, Ming-sing Hsu, Bonnie Peng, John E. Pintar
    Abstract:

    Peptidylglycine Alpha-Amidating Monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.

R E Mains - One of the best experts on this subject based on the ideXlab platform.

  • A Single Membrane Protein Required for Atrial Secretory Granule Formation
    2020
    Co-Authors: Nils Bäck, R E Mains, Raj Luxmi, Kathryn G. Powers, B A Eipper
    Abstract:

    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 Alpha-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 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the Monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in PamMyh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by PamMyh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway.

  • Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery
    MDPI AG, 2018
    Co-Authors: Raj Luxmi, R E Mains, Crysten Blaby-haas, Dhivya Kumar, Navin Rauniyar, Stephen M. King, B A Eipper
    Abstract:

    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

  • Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.
    Neurobiology of disease, 2009
    Co-Authors: Danielle Bousquet-moore, Joseph R. Prohaska, R E Mains, Eduardo A. Nillni, Traci A. Czyzyk, William C. Wetsel, B A Eipper
    Abstract:

    Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for Peptidylglycine Alpha-Amidating Monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

  • Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine α-Amidating Monooxygenase by Dietary Copper
    Endocrinology, 2008
    Co-Authors: Danielle Bousquet-moore, B A Eipper, Eduardo A. Nillni, Traci A. Czyzyk, John E. Pintar, R E Mains
    Abstract:

    Amidated peptides are critically involved in many physiological functions. Genetic deletion of Peptidylglycine Alpha-Amidating Monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

  • Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse.
    Developmental biology, 2005
    Co-Authors: Traci A. Czyzyk, R E Mains, B A Eipper, Yun Ning, Ming-sing Hsu, Bonnie Peng, John E. Pintar
    Abstract:

    Peptidylglycine Alpha-Amidating Monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.

L'houcine Ouafik - One of the best experts on this subject based on the ideXlab platform.

  • Mammalian Peptidylglycine Alpha-Amidating Monooxygenase mRNA expression can be modulated by the La autoantigen.
    Molecular and Cellular Biology, 2005
    Co-Authors: Fabienne Brenet, Christine Delfino, Nadège Dussault, Jonas Borch, Géraldine Ferracci, Peter Roepstorff, Raymond Miquelis, L'houcine Ouafik
    Abstract:

    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.

  • Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo.
    American Journal of Pathology, 2002
    Co-Authors: L'houcine Ouafik, Françoise Boudouresque, Christophe Dussert, Samantha Sauze, Frederic Fina, Christine Delfino, Jacqueline Palmari, Vincent Vuaroqueaux, Olivier Chinot, Henri Dufour
    Abstract:

    Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Growth factors are potential targets for therapeutic strategies because they are essential for tumor growth and progression. Peptidylglycine Alpha-Amidating Monooxygenase is the enzyme producing alpha-amidated bioactive peptides from their inactive glycine-extended precursors. The high expression of Peptidylglycine Alpha-Amidating Monooxygenase mRNA in glioblastoma and glioma cell lines points to the involvement of alpha-amidated peptides in tumorigenic growth processes in the brain. After screening of amidated peptides, it was found that human glioblastoma cell lines express high levels of adrenomedullin (AM) mRNA, and that immunoreactive AM is released into the culture medium. AM is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities among others. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that AM mRNA was correlated to the tumor type and grade, with high expression in all glioblastomas analyzed, whereas a low expression was found in anaplastic astrocytomas and barely detectable levels in low-grade astrocytomas and oligodendrogliomas. In the present study we also demonstrate the presence of mRNA encoding the putative AM receptors, calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) in both glioma tissues and glioblastoma cell lines and further show that exogenously added AM can stimulate the growth of these glioblastoma cells in vitro. These findings suggest that AM may function as an autocrine growth factor for glioblastoma cells. One way to test the autocrine hypothesis is to interrupt the function of the endogenously produced AM. Herein, we demonstrate that a polyclonal antibody specific to AM, blocks the binding of the hormone to its cellular receptors and decreases by 33% (P < 0.001) the growth of U87 glioblastoma cells in vitro. Intratumoral administration of the anti-AM antibody resulted in a 70% (P < 0.001) reduction in subcutaneous U87 xenograft weight 21 days after treatment. Furthermore, the density of vessels was decreased in the antibody-treated tumors. These findings support that AM may function as a potent autocrine/paracrine growth factor for human glioblastomas and demonstrate that inhibition of the action of AM (produced by tumor cells) may suppress tumor growth in vivo.

  • Evidence of high expression of Peptidylglycine Alpha-Amidating Monooxygenase in the rat uterus: estrogen regulation.
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Rajaâ El Meskini, Françoise Boudouresque, Christine Delfino, Charles Oliver, Pierre-marie Martin, L'houcine Ouafik
    Abstract:

    In the present study, high levels of Peptidylglycine Alpha-Amidating Monooxygenase (PAM), which catalyzes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17beta-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

  • Identification of a novel cis-element in the 3'-untranslated region of mammalian Peptidylglycine Alpha-Amidating Monooxygenase messenger ribonucleic acid.
    Endocrinology, 1998
    Co-Authors: Sandrine Fraboulet, Françoise Boudouresque, Christine Delfino, L'houcine Ouafik
    Abstract:

    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.

  • Estrogen regulation of Peptidylglycine Alpha-Amidating Monooxygenase messenger ribonucleic acid levels by a nuclear posttranscriptional event.
    Endocrinology, 1997
    Co-Authors: Rajaa El Meskini, Françoise Boudouresque, L'houcine Ouafik
    Abstract:

    Peptidylglycine α-amidating Monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17β-estradiol was accompanied by a 4.5 ± 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland. To investigate the cellular site at which 17β-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17β-estradiol effect...

Sharon L. Milgram - One of the best experts on this subject based on the ideXlab platform.

  • Localization of integral membrane Peptidylglycine Alpha-Amidating Monooxygenase in neuroendocrine cells.
    Journal of Cell Science, 1997
    Co-Authors: Sharon L. Milgram, R E Mains, Soochuen Tricia Kho, Gregory V. Martin, B A Eipper
    Abstract:

    Peptidylglycine Alpha-Amidating Monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

  • Processing and routing of a membrane-anchored form of proneuropeptide Y.
    Molecular endocrinology (Baltimore Md.), 1996
    Co-Authors: Sharon L. Milgram, E Y Chang, R E Mains
    Abstract:

    To investigate factors governing proteolytic processing and routing of biologically active peptides in the secretory pathway, cDNAs for preproneuropeptide Y (preproNPY) and preproneuropeptide Y fused to a membrane anchor were transfected into pituitary cells. The anchor was the transmembrane and COOH-terminal cytoplasmic domain of Peptidylglycine Alpha-Amidating Monooxygenase (PAM); these domains are essential for correct routing of integral membrane forms of PAM. Like proneuropeptide Y (proNPY), the integral membrane form of proNPY was a good substrate for the endogenous prohormone convertases, yielding soluble NPY stored in regulated secretory granules. Tethering of proNPY to the membrane resulted in only a small delay in the rate of cleavage to produce mature NPY and in the arrival of NPY in regulated secretory granules. In contrast, the COOH-terminal region of proNPY remained attached to the transmembrane/COOH-terminal domain of PAM and was rerouted to the vicinity of the trans-Golgi network, where in...

  • Phosphorylation of the cytosolic domain of Peptidylglycine Alpha-Amidating Monooxygenase.
    The Journal of biological chemistry, 1995
    Co-Authors: Hye-young Yun, Sharon L. Milgram, Henry T. Keutmann, B A Eipper
    Abstract:

    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.

  • The NH2-terminal proregion of Peptidylglycine Alpha-Amidating Monooxygenase facilitates the secretion of soluble proteins.
    Molecular endocrinology (Baltimore Md.), 1995
    Co-Authors: R E Mains, Sharon L. Milgram, Henry T. Keutmann, B A Eipper
    Abstract:

    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...

  • Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins
    Journal of Cell Science, 1994
    Co-Authors: Sharon L. Milgram, R E Mains
    Abstract:

    Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble Peptidylglycine Alpha-Amidating Monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of Peptidylglycine Alpha-Amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble Peptidylglycine Alpha-Amidating Monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while Peptidylglycine Alpha-Amidating Monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.

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  • Biosynthesis of oleamide.
    Vitamins and hormones, 2009
    Co-Authors: Gregory P. Mueller, William J. Driscoll
    Abstract:

    Oleamide (cis-9-octadecenamide) is the prototype long chain primary fatty acid amide lipid messenger. The natural occurrence of oleamide was first reported in human serum in 1989. Subsequently oleamide was shown to accumulate in the cerebrospinal fluid of sleep-deprived cats and to induce sleep when administered to experimental animals. Accordingly, oleamide first became known for its potential role in the mechanisms that mediate the drive to sleep. Oleamide also has profound effects on thermoregulation and acts as an analgesic in several models of experimental pain. Although these important pharmacologic effects are well establish, the biochemical mechanism for the synthesis of oleamide has not yet been defined. This chapter reviews the biosynthetic pathways that have been proposed and highlights two mechanisms which are most supported by experimental evidence: the generation of oleamide from oleoylglycine by the neuropeptide processing enzyme, Peptidylglycine Alpha-Amidating Monooxygenase (PAM), and alternatively, the direct amidation of oleic acid via oleoyl coenzyme A by cytochrome c using ammonia as the nitrogen source. The latter mechanism is discussed in the context of apoptosis where oleamide may play a role in regulating gap junction communication. Lastly, several considerations and caveats pertinent to the future study oleamide biosynthesis are discussed.

  • In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide
    Prostaglandins & Other Lipid Mediators, 2006
    Co-Authors: Shalini Chaturvedi, William J. Driscoll, Brenda M. Elliot, Martha M. Faraday, Neil E. Grunberg, Gregory P. Mueller
    Abstract:

    Oleamide (cis-9-octadecenamide) is a member of an emerging class of lipid-signaling molecules, the primary fatty acid amides. A growing body of evidence indicates that oleamide mediates fundamental neurochemical processes including sleep, thermoregulation, and nociception. Nevertheless, the mechanism for oleamide biosynthesis remains unknown. The leading hypothesis holds that oleamide is synthesized from oleoylglycine via the actions of the peptide amidating enzyme, Peptidylglycine Alpha-Amidating Monooxygenase (PAM). The present study investigated this hypothesis using pharmacologic treatments, physiologic assessments, and measurements of serum oleamide levels using a newly developed enzyme-linked immunosorbant assay (ELISA). Oleamide and oleoylglycine both induced profound hypothermia and decreased locomotion, over equivalent dose ranges and time courses, whereas, closely related compounds, stearamide and oleic acid, were essentially without effect. While the biologic actions of oleamide and oleoylglycine were equivalent, the two compounds differed dramatically with respect to their effects on serum levels of oleamide. Oleamide administration (80mg/kg) elevated blood-borne oleamide by eight-fold, whereas, the same dose of oleoylglycine had no effect on circulating oleamide levels. In addition, pretreatment with the established PAM inhibitor, disulfiram, produced modest reductions in the hypothermic responses to both oleoylglycine and oleamide, suggesting that the effects of disulfiram were not mediated through inhibition of PAM and a resulting decrease in the formation of oleamide from oleoylglycine. Collectively, these findings raise the possibilities that: (1) oleoylglycine possesses biologic activity that is independent of its conversion to oleamide and (2) the increased availability of oleoylglycine as a potential substrate does not drive the biosynthesis of oleamide.

  • Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses.
    Biochimica et biophysica acta, 2004
    Co-Authors: Elizabeth Muth, William J. Driscoll, Alexi Smalstig, Gertrud Goping, Gregory P. Mueller
    Abstract:

    The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being Peptidylglycine Alpha-Amidating Monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.

  • Peptide alpha-amidation and Peptidylglycine alpha-hydroxylating Monooxygenase: control by disulfiram.
    Molecular pharmacology, 1993
    Co-Authors: Gregory P. Mueller, R E Mains, E J Husten, B A Eipper
    Abstract:

    The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by Peptidylglycine alpha-hydroxylating Monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine Alpha-Amidating lyase (PAL; EC 4.3.2.5). These enzymes are derived from the bifunctional precursor protein, Peptidylglycine Alpha-Amidating Monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL. Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low. Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of Peptidylglycine Alpha-Amidating Monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme.