Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

PubMed

Aktories, K; Wegner, A

1992-10-01

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaslow, H.R.; Groppi, V.E.; Abood, M.E.

1981-11-01

Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila.

PubMed

Ji, Yingbiao; Tulin, Alexei V

2016-10-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3' untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3' UTR, increasing the translation in vivo and in vitro hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

PubMed Central

Ji, Yingbiao

2016-01-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3′ untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3′ UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3′ UTR, increasing the translation in vivo and in vitro. hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. PMID:27402862

Hierarchical and Helical Self-assembly of ADP-ribosyl Cyclase into Large-scale Protein Microtubes

PubMed Central

Liu, Qun; Kriksunov, Irina A.; Wang, Zhongwu; Graeff, Richard; Lee, Hon Cheung; Hao, Quan

2013-01-01

Proteins are macromolecules with characteristic structures and biological functions. It is extremely challenging to obtain protein microtube structures through self-assembly as proteins are very complex and flexible. Here we present a strategy showing how a specific protein, ADP-ribosyl cyclase, helically self-assembles from monomers into hexagonal nanochains and further to highly ordered crystalline microtubes. The structures of protein nanochains and consequently self-assembled superlattice were determined by X-ray crystallography at 4.5 Å resolution and imaged by Scanning Electron Microscopy. The protein initially forms into dimers that have a fixed size of 5.6 nm, and then, helically self-assembles into 35.6 nm long hexagonal nanochains. One such nanochain consists of six dimers (12 monomers) that stack in order by a pseudo P61 screw axis. Seven nanochains produce a series of largescale assemblies, nanorods, forming the building blocks for microrods. A proposed aging process of microrods results in the formation of hollow microstructures. Synthesis and characterization of large scale self-assembled protein microtubes may pave a new pathway, capable of not only understanding the self-assembly dynamics of biological materials, but also directing design and fabrication of multifunctional nanobuilding blocks with particular applications in biomedical engineering. PMID:18956900

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2.

PubMed

Kowieski, Terri M; Lee, Susan; Denu, John M

2008-02-29

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, J.; Schultz, R.M.

1990-06-01

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alphamore » 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.« less

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins.

PubMed

Aktories, K

1994-09-01

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years. Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization. Clostridium botulinum exoenzyme C3 and Clostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.

The role of ADP-ribosylation in regulating DNA interstrand crosslink repair

PubMed Central

Gunn, Alasdair R.; Banos-Pinero, Benito; Paschke, Peggy; Sanchez-Pulido, Luis; Ariza, Antonio; Day, Joseph; Emrich, Mehera; Leys, David; Ponting, Chris P.

2016-01-01

ABSTRACT ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2. PMID:27587838

PARPs and ADP-Ribosylation: 50 Years … and Counting.

PubMed

Kraus, W Lee

2015-06-18

Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion-the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed. Copyright © 2015 Elsevier Inc. All rights reserved.

Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

PubMed Central

Hassa, Paul O.; Haenni, Sandra S.; Elser, Michael; Hottiger, Michael O.

2006-01-01

Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD+-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as “programmed necrosis” (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., “histone code”), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

PubMed Central

Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

2003-01-01

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38− cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38− cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave. PMID:14623867

Quantification of diphtheria toxin mediated ADP-ribosylation in a solid-phase assay.

PubMed

Bachran, Christopher; Sutherland, Mark; Bachran, Diana; Fuchs, Hendrik

2007-09-01

Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT. Solid phase-bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD(+) as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

PubMed Central

Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

2013-01-01

Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+-bound form (NAD+-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD+-Ia-actin complex. Here we report high-resolution structures of NAD+-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD+ under different conditions. The structures of NAD+-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the βTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases. PMID:23382240

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

PubMed Central

Yamamoto-Katayama, S; Sato, A; Ariyoshi, M; Suyama, M; Ishihara, K; Hirano, T; Nakamura, H; Morikawa, K; Jingami, H

2001-01-01

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies. PMID:11439087

Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

1988-01-01

The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated /sup 32/P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingestedmore » ethanol in a liquid diet. /sup 32/P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.« less

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scaife, R.M.; Wilson, L.; Purich, D.L.

1992-01-14

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extentmore » of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.« less

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages*

PubMed Central

Kang, John; Park, Kwang-Hyun; Kim, Jwa-Jin; Jo, Eun-Kyeong; Han, Myung-Kwan; Kim, Uh-Hyun

2012-01-01

Phagocytosis is a crucial event in the immune system that allows cells to engulf and eliminate pathogens. This is mediated through the action of immunoglobulin (IgG)-opsonized microbes acting on Fcγ receptors (FcγR) on macrophages, which results in sustained levels of intracellular Ca2+ through the mobilization of Ca2+ second messengers. It is known that the ADP-ribosyl cyclase is responsible for the rise in Ca2+ levels after FcγR activation. However, it is unclear whether and how CD38 is involved in FcγR-mediated phagocytosis. Here we show that CD38 is recruited to the forming phagosomes during phagocytosis of IgG-opsonized particles and produces cyclic-ADP-ribose, which acts on ER Ca2+ stores, thus allowing an increase in FcγR activation-mediated phagocytosis. Ca2+ data show that pretreatment of J774A.1 macrophages with 8-bromo-cADPR, ryanodine, blebbistatin, and various store-operated Ca2+ inhibitors prevented the long-lasting Ca2+ signal, which significantly reduced the number of ingested opsonized particles. Ex vivo data with macrophages extracted from CD38−/− mice also shows a reduced Ca2+ signaling and phagocytic index. Furthermore, a significantly reduced phagocytic index of Mycobacterium bovis BCG was shown in macrophages from CD38−/− mice in vivo. This study suggests a crucial role of CD38 in FcγR-mediated phagocytosis through its recruitment to the phagosome and mobilization of cADPR-induced intracellular Ca2+ and store-operated extracellular Ca2+ influx. PMID:22396532

Hydrofluoric Acid-Based Derivatization Strategy To Profile PARP-1 ADP-Ribosylation by LC-MS/MS.

PubMed

Gagné, Jean-Philippe; Langelier, Marie-France; Pascal, John M; Poirier, Guy G

2018-06-11

Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

Inhibiting poly(ADP-ribosylation) improves axon regeneration.

PubMed

Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

2016-10-04

The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration.

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin.

PubMed

Popoff, M R; Boquet, P

1988-05-16

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

PubMed Central

Wykes, Michelle N; Beattie, Lynette; MacPherson, Gordon G; Hart, Derek N

2004-01-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b− DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs. PMID:15500618

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses.

PubMed

Wykes, Michelle N; Beattie, Lynette; Macpherson, Gordon G; Hart, Derek N

2004-11-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.

Inhibiting poly(ADP-ribosylation) improves axon regeneration

PubMed Central

Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

2016-01-01

The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.12734.001 PMID:27697151

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

PubMed Central

Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

2015-01-01

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

PubMed Central

Matsumoto, Yasuko; Nakano, Tsuyoshi; Yamamoto, Masafumi; Matsushima-Hibiya, Yuko; Odagiri, Ken-Ichi; Yata, Osamu; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

2008-01-01

Cabbage butterflies, Pieris rapae and Pieris brassicae, contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), and Appiadina (Appias nero and Appias paulina). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ≈100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly. PMID:18256183

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

PubMed Central

Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

2014-01-01

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as

Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking.

PubMed

Funaro, Ada; Ortolan, Erika; Bovino, Paola; Lo Buono, Nicola; Nacci, Giulia; Parrotta, Rossella; Ferrero, Enza; Malavasi, Fabio

2009-01-01

CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes

PubMed Central

Gupte, Rebecca; Liu, Ziying; Kraus, W. Lee

2017-01-01

The discovery of poly(ADP-ribose) >50 years ago opened a new field, leading the way for the discovery of the poly(ADP-ribose) polymerase (PARP) family of enzymes and the ADP-ribosylation reactions that they catalyze. Although the field was initially focused primarily on the biochemistry and molecular biology of PARP-1 in DNA damage detection and repair, the mechanistic and functional understanding of the role of PARPs in different biological processes has grown considerably of late. This has been accompanied by a shift of focus from enzymology to a search for substrates as well as the first attempts to determine the functional consequences of site-specific ADP-ribosylation on those substrates. Supporting these advances is a host of methodological approaches from chemical biology, proteomics, genomics, cell biology, and genetics that have propelled new discoveries in the field. New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer. These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field. PMID:28202539

Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies. PMID:24911624

Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fendrick, J.L.; Iglewski, W.J.

1989-01-01

Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of trypticmore » peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.« less

Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

PubMed Central

Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

2010-01-01

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

Novel bacterial ADP-ribosylating toxins: structure and function

PubMed Central

Simon, Nathan C.; Aktories, Klaus; Barbieri, Joseph T.

2018-01-01

Preface Bacterial ADP-ribosyltransferase toxins (bARTTs) transfer ADP-ribose to eukaryotic proteins to promote bacterial pathogenesis. In this review we use prototype bARTTs, such as diphtheria and pertussis toxins, as references for the characterization of several new bARTTs from human, insect, and plant pathogens, which were identified recently through bioinformatic analyses. Several of these toxins, including Cholix toxin from Vibrio cholerae, SpyA from Streptococcus pyogenes, HopU1 from Pseudomonas syringae, and the Tcc toxins from Photorhabdus luminescens, ADP-ribosylate novel substrates and possess unique organizations, which distinguish them from the reference toxins. The characterization of these toxins extends our appreciation for the variety of structure-function properties possessed by bARTTs and their roles in bacterial pathogenesis. PMID:25023120

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A/sub 1/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gill, D.M.; Coburn, J.

1987-10-06

The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTP..gamma..S), forms over a period of 10-15 min at 37/sup 0/C. Both guanosine 5'-O-(2-thiodiphosphate)more » and GTP block this quasi-permanent activation. Some S x GTP..gamma..S forms in membranes that are exposed to CF alone and then to GTP..gamma..S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTP..gamma..S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45/sup 0/C is decreased by GTP..gamma..S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A/sub 1/ fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified.« less

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

Serine is the major residue for ADP-ribosylation upon DNA damage

PubMed Central

Dauben, Helen

2018-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling. PMID:29480802

Structure of Plasmodium falciparum ADP-ribosylation factor 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J.; Smith, Craig D.; Senkovich, Olga

Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in Rhodobacter capsulatus Is Correlated with Cellular Nitrogen Status

PubMed Central

Yakunin, Alexander F.; Laurinavichene, Tatyana V.; Tsygankov, Anatoly A.; Hallenbeck, Patrick C.

1999-01-01

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions. PMID:10094674

The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, S.; Tainer, J.A.

2001-08-01

ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core hasmore » been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within

Bacillus cereus Certhrax ADP-ribosylates Vinculin to Disrupt Focal Adhesion Complexes and Cell Adhesion*

PubMed Central

Simon, Nathan C.; Barbieri, Joseph T.

2014-01-01

Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton. PMID:24573681

A Cell-Line-Specific Atlas of PARP-Mediated Protein Asp/Glu-ADP-Ribosylation in Breast Cancer.

PubMed

Zhen, Yuanli; Zhang, Yajie; Yu, Yonghao

2017-11-21

PARP1 plays a critical role in regulating many biological processes linked to cellular stress responses. Although DNA strand breaks are potent stimuli of PARP1 enzymatic activity, the context-dependent mechanism regulating PARP1 activation and signaling is poorly understood. We performed global characterization of the PARP1-dependent, Asp/Glu-ADP-ribosylated proteome in a panel of cell lines originating from benign breast epithelial cells, as well as common subtypes of breast cancer. From these analyses, we identified 503 specific ADP-ribosylation sites on 322 proteins. Despite similar expression levels, PARP1 is differentially activated in these cell lines under genotoxic conditions, which generates signaling outputs with substantial heterogeneity. By comparing protein abundances and ADP-ribosylation levels, we could dissect cell-specific PARP1 targets that are driven by unique expression patterns versus cell-specific regulatory mechanisms of PARylation. Intriguingly, PARP1 modifies many proteins in a cell-specific manner, including those involved in transcriptional regulation, mRNA metabolism, and protein translation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription.

PubMed

Bütepage, Mareike; Preisinger, Christian; von Kriegsheim, Alexander; Scheufen, Anja; Lausberg, Eva; Li, Jinyu; Kappes, Ferdinand; Feederle, Regina; Ernst, Sabrina; Eckei, Laura; Krieg, Sarah; Müller-Newen, Gerhard; Rossetti, Giulia; Feijs, Karla L H; Verheugd, Patricia; Lüscher, Bernhard

2018-04-30

Macrodomains are conserved protein folds associated with ADP-ribose binding and turnover. ADP-ribosylation is a posttranslational modification catalyzed primarily by ARTD (aka PARP) enzymes in cells. ARTDs transfer either single or multiple ADP-ribose units to substrates, resulting in mono- or poly-ADP-ribosylation. TARG1/C6orf130 is a macrodomain protein that hydrolyzes mono-ADP-ribosylation and interacts with poly-ADP-ribose chains. Interactome analyses revealed that TARG1 binds strongly to ribosomes and proteins associated with rRNA processing and ribosomal assembly factors. TARG1 localized to transcriptionally active nucleoli, which occurred independently of ADP-ribose binding. TARG1 shuttled continuously between nucleoli and nucleoplasm. In response to DNA damage, which activates ARTD1/2 (PARP1/2) and promotes synthesis of poly-ADP-ribose chains, TARG1 re-localized to the nucleoplasm. This was dependent on the ability of TARG1 to bind to poly-ADP-ribose. These findings are consistent with the observed ability of TARG1 to competitively interact with RNA and PAR chains. We propose a nucleolar role of TARG1 in ribosome assembly or quality control that is stalled when TARG1 is re-located to sites of DNA damage.

Involvement of cytosolic NAD+ glycohydrolase in cyclic ADP-ribose metabolism.

PubMed

Matsumura, N; Tanuma, S

1998-12-18

The NAD+ glycohydrolase homogeneously purified from bovine brain cytosol was found to catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). Although the formation of cADPR from NAD+ does not exceed about 2% of the reaction products, the cyclase activity is clearly evidenced by its conversion of NGD+ to cyclic GDP-ribose (cGDPR), which cannot be hydrolyzed to GDPR. Importantly, a steep increase in cADPR hydrolytic activity was observed at cADPR concentrations above 60 microM, which could be reproduced on a Hill curve with a Hill coefficient of 2. Thus, the allosteric binding of cADPR to the NAD+ glycohydrolase (E) molecule promotes the hydrolysis of cADPR. These results suggest that NAD+ hydrolysis to ADPR and nicotinamide catalyzed by the NAD+ glycohydrolase occurs through the formation of a cADPR. E. cADP-ribosyl complex. The low production of cADPR by NAD+ glycohydrolase compared with invertebrate ADP-ribosyl cyclase is believed to be attributable to the fast hydrolysis of cADPR by the allosteric effect of cADPR bound to the same enzyme that produces it. Copyright 1998 Academic Press.

Site of ADP-ribosylation and the RNA-binding site are situated in different domains of the elongation factor EF-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davydova, E.K.

1987-01-01

One of the proteins participating in the process of elongation of polypeptide chains - elongation factor 2 (EF-2) - can be ADP-ribosylated at a unique amino acid residue - diphthamide. Since the ADP-ribosylation of EF-2 at dipthamide leads to a loss of affinity of the factor for RNA while the presence of RNA inhibits the ADP-ribosylation reaction, it seemed probable to the authors that diphthamide participated directly in the binding of EF-2 to DNA. The experiments presented in this article showed that this was not the case: diphthamide and the RNA-binding site are situated on different domains of EF-2. Thus,more » ADP-ribosylation of factor EF-2 in one domain leads to a loss of the ability to bind to RNA in the other. The authors investigated the mutual arrangement of diphthamide and the RNA-binding site on the EF-2 molecule by preparing a factor from rabbit reticulocytes and subjecting it to proteolytic digestion with elastase. The factor was incubated with elastase for 15 min at 37/sup 0/C at an enzyme:substrate ratio of 1:100 in buffer solution containing 20 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM MgCl/sub 2/, and 2 mM dithiothreitol. The reaction was stopped by adding para-methylsulfonyl fluoride to 50 micro-M. The authors obtained a preparation as a result of proteolysis and applied it on a column with RNA-Sepharose and separated into two fractions: RNA-binding and without affinity for RNA. The initial preparation and its fractions were subjected to exhaustive ADP-ribosylation in the presence of diphtheria toxin and (U-/sup 14/C) nicotinaide adenine dinucleotide ((/sup 14/C)NAD) (296 mCi/mmole). The samples were analyzed electrophoretically in a polyacrylamide gel gradient in the presence of sodium dodecyl sulfate. For the detection of (/sup 14/C) ADP-ribosylated components, the gels were dried and exposed with RM-V x-ray film.« less

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

PubMed

Maehama, T; Takahashi, K; Ohoka, Y; Ohtsuka, T; Ui, M; Katada, T

1991-06-05

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.

Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

PubMed

Li, Mo; Bian, Chunjing; Yu, Xiaochun

2014-01-01

Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

Rapid evolution of PARP genes suggests a broad role for ADP-ribosylation in host-virus conflicts.

PubMed

Daugherty, Matthew D; Young, Janet M; Kerns, Julie A; Malik, Harmit S

2014-01-01

Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic 'arms races' with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in 'housekeeping' functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our

Rapid Evolution of PARP Genes Suggests a Broad Role for ADP-Ribosylation in Host-Virus Conflicts

PubMed Central

Daugherty, Matthew D.; Young, Janet M.; Kerns, Julie A.; Malik, Harmit S.

2014-01-01

Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic ‘arms races’ with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in ‘housekeeping’ functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our

Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

PubMed Central

Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V. V.; Intorne, Aline C.; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A.; Shan, Libo; He, Ping

2015-01-01

Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks. PMID:25569773

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

PubMed Central

Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

2017-01-01

Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

Social memory, amnesia, and autism: brain oxytocin secretion is regulated by NAD+ metabolites and single nucleotide polymorphisms of CD38.

PubMed

Higashida, Haruhiro; Yokoyama, Shigeru; Huang, Jian-Jun; Liu, Li; Ma, Wen-Jie; Akther, Shirin; Higashida, Chiharu; Kikuchi, Mitsuru; Minabe, Yoshio; Munesue, Toshio

2012-11-01

Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C>A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C>T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by

Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin.

PubMed

Aktories, Klaus; Barth, Holger

2004-04-01

Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

ADP-Ribosylation Factor 6 Regulates a Novel Plasma Membrane Recycling Pathway

PubMed Central

Radhakrishna, Harish; Donaldson, Julie G.

1997-01-01

ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor α subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton. PMID:9314528

Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

PubMed Central

Belov, George A.; Fogg, Mark H.; Ehrenfeld, Ellie

2005-01-01

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication. PMID:15890959

Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komissarova, Elena V.; Rossman, Toby G., E-mail: toby.rossman@nyumc.or

2010-03-15

Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage,more » is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.« less

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

PubMed

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed Central

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins. Images PMID:2521214

Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

PubMed Central

Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

2013-01-01

The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 ·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 ·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 ·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 ·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 ·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, G.P.

1987-01-01

Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted inmore » a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.« less

Toward a unified nomenclature for mammalian ADP-ribosyltransferases.

PubMed

Hottiger, Michael O; Hassa, Paul O; Lüscher, Bernhard; Schüler, Herwig; Koch-Nolte, Friedrich

2010-04-01

ADP-ribosylation is a post-translational modification of proteins catalyzed by ADP-ribosyltransferases. It comprises the transfer of the ADP-ribose moiety from NAD+ to specific amino acid residues on substrate proteins or to ADP-ribose itself. Currently, 22 human genes encoding proteins that possess an ADP-ribosyltransferase catalytic domain are known. Recent structural and enzymological evidence of poly(ADP-ribose)polymerase (PARP) family members demonstrate that earlier proposed names and classifications of these proteins are no longer accurate. Here we summarize these new findings and propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. A unified nomenclature would facilitate communication between researchers both inside and outside the ADP-ribosylation field. 2009 Elsevier Ltd. All rights reserved.

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE PAGES

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.; ...

2015-04-06

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Autophagy requires poly(adp-ribosyl)ation-dependent AMPK nuclear export

PubMed Central

Rodríguez-Vargas, José M; Rodríguez, María I; Majuelos-Melguizo, Jara; García-Diaz, Ángel; González-Flores, Ariannys; López-Rivas, Abelardo; Virág, László; Illuzzi, Giuditta; Schreiber, Valerie; Dantzer, Françoise; Oliver, F Javier

2016-01-01

AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation. PMID:27689873

ADP-Ribosylation Factor 6 and a Functional PIX/p95-APP1 Complex Are Required for Rac1B-mediated Neurite Outgrowth

PubMed Central

Albertinazzi, Chiara; Za, Lorena; Paris, Simona; de Curtis, Ivan

2003-01-01

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1–derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis. PMID:12686588

Functions of the poly(ADP-ribose) polymerase superfamily in plants.

PubMed

Lamb, Rebecca S; Citarelli, Matteo; Teotia, Sachin

2012-01-01

Poly(ADP-ribosyl)ation is the covalent attachment of ADP-ribose subunits from NAD(+) to target proteins and was first described in plants in the 1970s. This post-translational modification is mediated by poly(ADP-ribose) polymerases (PARPs) and removed by poly(ADP-ribose) glycohydrolases (PARGs). PARPs have important functions in many biological processes including DNA repair, epigenetic regulation and transcription. However, these roles are not always associated with enzymatic activity. The PARP superfamily has been well studied in animals, but remains under-investigated in plants. Although plants lack the variety of PARP superfamily members found in mammals, they do encode three different types of PARP superfamily proteins, including a group of PARP-like proteins, the SRO family, that are plant specific. In plants, members of the PARP family and/or poly(ADP-ribosyl)ation have been linked to DNA repair, mitosis, innate immunity and stress responses. In addition, members of the SRO family have been shown to be necessary for normal sporophytic development. In this review, we summarize the current state of plant research into poly(ADP-ribosyl)ation and the PARP superfamily in plants.

His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II.

PubMed Central

Wozniak, D J; Hsu, L Y; Galloway, D R

1988-01-01

Exotoxin A (ETA) is recognized as the most toxic product associated with the opportunistic pathogen Pseudomonas aeruginosa. Identification of the amino acids in the polypeptide sequence that are required for toxin activity is critical for vaccine development. By defining the nucleotide sequence of the structural gene of a mutant that encodes an enzymatically inactive ETA (CRM 66), we identified an essential amino acid (His-426), which is involved in the ADP-ribosyltransferase activity associated with functional ETA. A monoclonal antibody that inhibits ETA enzymatic activity in vitro fails to react with ETA variants that have a His 426----Tyr substitution. Several mono-ADP-ribosylating toxins, including diphtheria and pertussis toxins, within the primary amino acid sequences carry a histidine residue that is conserved in spacing and in location with respect to other critical residues. Analysis of the three-dimensional structure of ETA revealed that His-426 is not associated with the proposed NAD+ binding site. These findings should be useful for the design and construction of toxin vaccines. Images PMID:3143111

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis.

PubMed

Yoo, Cheol-Min; Wen, Jiangqi; Motes, Christy M; Sparks, J Alan; Blancaflor, Elison B

2008-08-01

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases.

PubMed

Hassa, Paul O; Hottiger, Michael O

2008-01-01

Poly-ADP-ribose metabolism plays a mayor role in a wide range of biological processes, such as maintenance of genomic stability, transcriptional regulation, energy metabolism and cell death. Poly-ADP-ribose polymerases (PARPs) are an ancient family of enzymes, as evidenced by the poly-ADP-ribosylating activities reported in dinoflagellates and archaebacteria and by the identification of Parp-like genes in eubacterial and archaeabacterial genomes. Six genes encoding "bona fide" PARP enzymes have been identified in mammalians: PARP1, PARP2, PARP3, PARP4/vPARP, PARP5/Tankyrases-1 and PARP6/Tankyrases-2. The best studied of these enzymes PARP1 plays a primary role in the process of poly-ADP-ribosylation. PARP1-mediated poly-ADP-ribosylation has been implicated in the pathogenesis of cancer, inflammatory and neurodegenerative disorders. This review will summarize the novel findings and concepts for PARP enzymes and their poly-ADP-ribosylation activity in the regulation of physiological and pathophysiological processes. A special focus is placed on the proposed molecular mechanisms involved in these processes, such as signaling, regulation of telomere dynamics, remodeling of chromatin structure and transcriptional regulation. A potential functional cross talk between PARP family members and other NAD+-consuming enzymes is discussed.

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

PubMed

Piberger, Ann Liza; Keil, Claudia; Platz, Stefanie; Rohn, Sascha; Hartwig, Andrea

2015-11-01

The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 μM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 μM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genes Related to Oxytocin and Arginine-Vasopressin Pathways: Associations with Autism Spectrum Disorders.

PubMed

Zhang, Rong; Zhang, Hong-Feng; Han, Ji-Sheng; Han, Song-Ping

2017-04-01

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorders characterized by impaired social interactions, communication deficits, and repetitive behavior. Although the mechanisms underlying its etiology and manifestations are poorly understood, several lines of evidence from rodent and human studies suggest involvement of the evolutionarily highly-conserved oxytocin (OXT) and arginine-vasopressin (AVP), as these neuropeptides modulate various aspects of mammalian social behavior. As far as we know, there is no comprehensive review of the roles of the OXT and AVP systems in the development of ASD from the genetic aspect. In this review, we summarize the current knowledge regarding associations between ASD and single-nucleotide variants of the human OXT-AVP pathway genes OXT, AVP, AVP receptor 1a (AVPR1a), OXT receptor (OXTR), the oxytocinase/vasopressinase (LNPEP), and ADP-ribosyl cyclase (CD38).

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

PubMed Central

Talhaoui, Ibtissam; Lebedeva, Natalia A.; Zarkovic, Gabriella; Saint-Pierre, Christine; Kutuzov, Mikhail M.; Sukhanova, Maria V.; Matkarimov, Bakhyt T.; Gasparutto, Didier; Saparbaev, Murat K.; Lavrik, Olga I.; Ishchenko, Alexander A.

2016-01-01

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs. PMID:27471034

Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

PubMed

Skuta, G; Fischer, G M; Janaky, T; Kele, Z; Szabo, P; Tozser, J; Sumegi, B

1999-12-15

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short

Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

PubMed

De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

2013-09-01

Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity. © 2013 Elsevier Inc. All rights reserved.

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

PubMed Central

Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

2016-01-01

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis1[W][OA

PubMed Central

Yoo, Cheol-Min; Wen, Jiangqi; Motes, Christy M.; Sparks, J. Alan; Blancaflor, Elison B.

2008-01-01

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs. PMID:18539780

Expression of ADP-ribosylation factor (ARF)-like protein 6 during mouse embryonic development.

PubMed

Takada, Tatsuyuki; Iida, Keiko; Sasaki, Hiroshi; Taira, Masanori; Kimura, Hiroshi

2005-01-01

ADP-ribosylation factor (ARF)-like protein 6 (ARL6) is a member of the ARF-like protein (ARL) subfamily of small GTPases (Moss, 1995; Chavrier, 1999). ARLs are highly conserved through evolution and most of them possess the consensus sequence required for GTP binding and hydrolysis (Pasquallato, 2002). Among ARLs, ARL6 which was initially isolated from a J2E erythroleukemic cell line is divergent in its consensus sequences and its expression has been shown to be limited to the brain and kidney in adult mouse (Ingley, 1999). Recently, it was reported that mutations of the ARL6 gene cause type 3 Bardet-Biedl syndrome in humans and that ARL6 is involved in ciliary transport in C. elegans (Chiang, 2004; Fan, 2004). Here, we investigated the expression pattern of ARL6 during early mouse development by whole-mount in situ hybridization and found that interestingly, ARL6 mRNA was localized around the node at 7.0-7.5 days post coitum (dpc) embryos, while weak expression was also found in the ectoderm. At the later stage (8.5 dpc) ARL6 was expressed in the neural plate and probably in the somites. Based on these results, a possible role of ARL6 in early development is discussed in relation to the findings in human and C. elegans (Chiang, 2004; Fan, 2004).

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

PubMed Central

Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

2013-01-01

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

PubMed

Svedova, Martina; Masin, Jiri; Fiser, Radovan; Cerny, Ondrej; Tomala, Jakub; Freudenberg, Marina; Tuckova, Ludmila; Kovar, Marek; Dadaglio, Gilles; Adkins, Irena; Sebo, Peter

2016-04-01

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

ADP-ribosylation Factor 6 (ARF6) Bidirectionally Regulates Dendritic Spine Formation Depending on Neuronal Maturation and Activity*

PubMed Central

Kim, Yoonju; Lee, Sang-Eun; Park, Joohyun; Kim, Minhyung; Lee, Boyoon; Hwang, Daehee; Chang, Sunghoe

2015-01-01

Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons. PMID:25605715

Suppression of breast cancer metastasis through the inactivation of ADP-ribosylation factor 1.

PubMed

Xie, Xiayang; Tang, Shou-Ching; Cai, Yafei; Pi, Wenhu; Deng, Libin; Wu, Guangyu; Chavanieu, Alain; Teng, Yong

2016-09-06

Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

PubMed

Gariani, Karim; Ryu, Dongryeol; Menzies, Keir J; Yi, Hyon-Seung; Stein, Sokrates; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Katsyuba, Elena; Jha, Pooja; Vijgen, Sandrine; Rubbia-Brandt, Laura; Kim, Yong Kyung; Kim, Jung Tae; Kim, Koon Soon; Shong, Minho; Schoonjans, Kristina; Auwerx, Johan

2017-01-01

To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD + ), we hypothesized that overactivation of PARPs drives NAD + depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD + and activate NAD + -dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD + levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD + , increasing mitochondrial biogenesis and β-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1 hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse

Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

PubMed

Purdy, Alexandra E; Balch, Deborah; Lizárraga-Partida, Marcial Leonardo; Islam, Mohammad Sirajul; Martinez-Urtaza, Jaime; Huq, Anwar; Colwell, Rita R; Bartlett, Douglas H

2010-02-01

Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rybin, V.O.; Gureeva, A.A.

1986-05-10

The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature ofmore » the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.« less

Mucosal adjuvants and long-term memory development with special focus on CTA1-DD and other ADP-ribosylating toxins.

PubMed

Lycke, N; Bemark, M

2010-11-01

The ultimate goal for vaccination is to stimulate protective immunological memory. Protection against infectious diseases not only relies on the magnitude of the humoral immune response, but more importantly on the quality and longevity of it. Adjuvants are critical components of most non-living vaccines. Although little attention has been given to qualitative aspects of the choice of vaccine adjuvant, emerging data demonstrate that this function may be central to vaccine efficacy. In this review we describe efforts to understand more about how adjuvants influence qualitative aspects of memory development. We describe recent advances in understanding how vaccines induce long-lived plasma and memory B cells, and focus our presentation on the germinal center reaction. As mucosal vaccination requires powerful adjuvants, we have devoted much attention to the adenosine diphosphate (ADP)-ribosylating cholera toxin and the CTA1-DD adjuvants as examples of how mucosal adjuvants can influence induction of long-term memory.

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality*

PubMed Central

Ahmed, Shaimaa; Bott, Debbie; Gomez, Alvin; Tamblyn, Laura; Rasheed, Adil; Cho, Tiffany; MacPherson, Laura; Sugamori, Kim S.; Yang, Yang; Grant, Denis M.; Cummins, Carolyn L.; Matthews, Jason

2015-01-01

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp−/− mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp+/+ mice survived the 30-day treatment. Dioxin-treated Tiparp−/− mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin. PMID:25975270

ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujita, Atsushi

2008-02-01

Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p,more » for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.« less

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

CD38 exacerbates focal cytokine production, postischemic inflammation and brain injury after focal cerebral ischemia.

PubMed

Choe, Chi-un; Lardong, Kerstin; Gelderblom, Mathias; Ludewig, Peter; Leypoldt, Frank; Koch-Nolte, Friedrich; Gerloff, Christian; Magnus, Tim

2011-01-01

Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion. We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8(+) cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model. CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.

Poly ADP-Ribose Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes

PubMed Central

Long, Chandler A.; Boloum, Valy; Albadawi, Hassan; Tsai, Shirling; Yoo, Hyung-Jin; Oklu, Rahmi; Goldman, Mitchell H.; Watkins, Michael T.

2013-01-01

Introduction Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes; ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods db/db mice underwent 1.5hrs of hind limb ischemia followed by 1, 7, or 24hrs reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24hrs period; the untreated group received Lactated ringer’s (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity /intracellular localization and poly-ADP-ribosylation of GAPDH. Results PARP activity was significantly lower in the PJ34 treated groups compared to the LR group at 7 and 24 hours reperfusion. There was significantly less muscle fiber injury in the PJ34 treated group compared to LR treated mice at 24 hrs reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7hrs and 24hrs IR. There were significant increases in metabolic activity only at 24 hours IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly ADP-ribosylation and nuclear translocation of GAPDH. Conclusions PJ34 reduced PARP activity, GAPDH ribosylation, GAPDH translocation, ameliorated muscle fiber injury, and increased metabolic activity following hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy following IR in diabetic humans. PMID:23549425

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.

2008-07-15

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity againstmore » ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.« less

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.

PubMed

Chiang, Shian-Huey; Harrington, W Wallace; Luo, Guizhen; Milliken, Naphtali O; Ulrich, John C; Chen, Jing; Rajpal, Deepak K; Qian, Ying; Carpenter, Tiffany; Murray, Rusty; Geske, Robert S; Stimpson, Stephen A; Kramer, Henning F; Haffner, Curt D; Becherer, J David; Preugschat, Frank; Billin, Andrew N

2015-01-01

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

Tankyrase1-mediated poly(ADP-ribosyl)ation of TRF1 maintains cell survival after telomeric DNA damage

PubMed Central

Yang, Lu; Sun, Luxi; Teng, Yaqun; Chen, Hao; Gao, Ying; Levine, Arthur S.; Nakajima, Satoshi

2017-01-01

Abstract Oxidative DNA damage triggers telomere erosion and cellular senescence. However, how repair is initiated at telomeres is largely unknown. Here, we found unlike PARP1-mediated Poly-ADP-Ribosylation (PARylation) at genomic damage sites, PARylation at telomeres is mainly dependent on tankyrase1 (TNKS1). TNKS1 is recruited to damaged telomeres via its interaction with TRF1, which subsequently facilitates the PARylation of TRF1 after damage. TNKS inhibition abolishes the recruitment of the repair proteins XRCC1 and polymerase β at damaged telomeres, while the PARP1/2 inhibitor only has such an effect at non-telomeric damage sites. The ANK domain of TNKS1 is essential for the telomeric damage response and TRF1 interaction. Mutation of the tankyrase-binding motif (TBM) on TRF1 (13R/18G to AA) disrupts its interaction with TNKS1 concomitant recruitment of TNKS1 and repair proteins after damage. Either TNKS1 inhibition or TBM mutated TRF1 expression markedly sensitizes cells to telomere oxidative damage as well as XRCC1 inhibition. Together, our data reveal a novel role of TNKS1 in facilitating SSBR at damaged telomeres through PARylation of TRF1, thereby protecting genome stability and cell viability. PMID:28160604

Characterization of the Enzymatic Component of the ADP-Ribosyltransferase Toxin CDTa from Clostridium difficile

PubMed Central

Gülke, Irene; Pfeifer, Gunther; Liese, Jan; Fritz, Michaela; Hofmann, Fred; Aktories, Klaus; Barth, Holger

2001-01-01

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1–240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244–263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208–413 but loss of activity of several N-terminally deleted C2I proteins including C2I103–431, C2I190–431, and C2I30–431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity. PMID:11553537

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

PubMed

Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

2017-10-27

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Family-wide analysis of poly(ADP-ribose) polymerase activity

PubMed Central

Uchima, Lilen; Rood, Jenny; Zaja, Roko; Hay, Ronald T.; Ahel, Ivan; Chang, Paul

2014-01-01

The poly(ADP-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as substrate. Based on the composition of three NAD+ coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino acid targets. In addition, we identify cysteine as a novel amino acid target for ADP-ribosylation on PARPs. PMID:25043379

CD38 enhances the proliferation and inhibits the apoptosis of cervical cancer cells by affecting the mitochondria functions.

PubMed

Liao, Shan; Xiao, Songshu; Chen, Hongxiang; Zhang, Manying; Chen, Zhifang; Long, Yuehua; Gao, Lu; Zhu, Guangchao; He, Junyu; Peng, Shuping; Xiong, Wei; Zeng, Zhaoyang; Li, Zheng; Zhou, Ming; Li, Xiaoling; Ma, Jian; Wu, Minghua; Xiang, Juanjuan; Li, Guiyuan; Zhou, Yanhong

2017-10-01

Cervical cancer is one of the most common malignant tumors in women all over the world. The exact mechanism of occurrence and development of cervical cancer has not been fully elucidated. CD38 is a type II transmembrane glycoprotein, which was found to mediate diverse activities, including signal transduction, cell adhesion, and cyclic ADP-ribose synthesis. Here, we reported that CD38 promoted cell proliferation and inhibited cell apoptosis in cervical cancer cells by affecting the mitochondria functions. We established stable cervical cancer cell lines with CD38 over-expressed. CCK8 assay and colony formation assay indicated that CD38 promoted cervical cancer cell proliferation. Nude mouse tumorigenicity assay showed that CD38 significantly promotes tumor growth in vivo. CD38 also induced S phase accumulation in cell cycle analysis and suppressed cell apoptosis in cervical cancer cells. Meanwhile, flow cytometry analysis of mitochondria functions suggested that CD38 decreased intracellular Ca 2+ levels in cervical cancer cells and CD38 was involved in down-regulation of ROS levels and prevented mitochondrial apoptosis in cervical cancer cells. The percentage of cells with loss of mitochondrial membrane potential (Δψm) in CD38-overexpressed cervical cancer cells was less than control groups. Furthermore, we found an up-regulation of MDM2, cyclinA1, CDK4, cyclinD1, NF-kB P65, c-rel, and a downregulation of P53, P21, and P38 by Western blot analysis. These results indicated that CD38 enhanced the proliferation and inhibited the apoptosis of cervical cancer cells by affecting the mitochondria functions. © 2017 Wiley Periodicals, Inc.

Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase

PubMed Central

Baum, Paul D.; Sullam, Paul M.; Stoddart, Cheryl A.; McCune, Joseph M.

2011-01-01

Objective To provide a molecular mechanism that explains the association of the antiretroviral guanosine analogue, abacavir, with an increased risk of myocardial infarction. Design Drug effects were studied with biochemical and cellular assays. Methods Human platelets were incubated with nucleoside analogue drugs ex vivo. Platelet activation stimulated by ADP was studied by measuring surface P-selectin with flow cytometry. Inhibition of purified soluble guanylyl cyclase was quantified using an ELISA to measure cGMP production. Results Pre-incubation of platelets in abacavir significantly increased activation in response to ADP in a time and dose-dependent manner. The active anabolite of abacavir, carbovir triphosphate, competitively inhibited soluble guanylyl cyclase activity with a Ki of 55 μmol/l. Conclusion Abacavir competitively inhibits guanylyl cyclase, leading to platelet hyper-reactivity. This may explain the observed increased risk of myocardial infarction in HIV patients taking abacavir. PMID:21941165

The Sound of Silence: RNAi in Poly (ADP-Ribose) Research

PubMed Central

Blenn, Christian; Wyrsch, Philippe; Althaus, Felix R.

2012-01-01

Poly(ADP-ribosyl)-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose) (PAR) is regulated by its synthesis by poly(ADP-ribose) polymerases (PARPs) and on the catabolic side by poly(ADP-ribose) glycohydrolase (PARG). PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR) moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption. PMID:24705085

The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

PubMed Central

Kickhoefer, Valerie A.; Siva, Amara C.; Kedersha, Nancy L.; Inman, Elisabeth M.; Ruland, Cristina; Streuli, Michel; Rome, Leonard H.

1999-01-01

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle. PMID:10477748

ADP-ribosylation factor6 regulates both [3H]-noradrenaline and [14C]-glutamate exocytosis through phosphatidylinositol 4,5-bisphosphate.

PubMed

Zheng, Qian; Bobich, Joseph A

2004-10-01

GTP phosphohydrolase (cell regulating) (EC 3.6.1.47, ADP-ribosylation factor6, ARF6) has been shown to play an important role in different steps of membrane trafficking. It also regulates chromaffin granule exocytosis through phosphatidylcholine phosphatidohydrolase (EC 3.1.4.14, PLD) activation. In this study, the role of ARF6 in neurotransmitter release from both dense-core granules (DCGs) and synaptic vesicles (SVs) in rat brain cortex nerve endings was investigated. We observed that synaptosomal ARF6 is largely particulate but moves to a less easily pelleted compartment upon nerve ending stimulation. We also found that direct inhibition of ARF6 by a specific antibody or interference with ARF6 downstream effects by a myristoylated N-terminal ARF6 peptide both significantly decreased both [3H]-noradrenaline and [14C]-glutamate exocytosis. Addition of phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2) partially or completely restored exocytosis. These findings suggest that ARF6 plays important regulatory roles for both DCG and SV exocytosis by activating PLD and ATP:1-phosphatidyl-1D-myo-inositol 4-phosphate 5-phosphotransferase (EC 2.7.1.68, PI4P-5K) to enhance PIP2 synthesis and nerve ending membrane trafficking.

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

PubMed

Slama, J T; Simmons, A M

1989-09-19

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity.

PubMed

Considine, R V; Simpson, L L

1991-01-01

Clostridial organisms produce a number of binary toxins. Thus far, three complete toxins (botulinum, perfringens and spiroforme) and one incomplete toxin (difficile) have been identified. In the case of complete toxins, there is a heavy chain component (Mr approximately 100,000) that binds to target cells and helps create a docking site for the light chain component (Mr approximately 50,000). The latter is an enzyme that possesses mono(ADP-ribosyl)transferase activity. The toxins appear to proceed through a three step sequence to exert their effects, including a binding step, an internalization step and an intracellular poisoning step. The substrate for the toxins is G-actin. By virtue of ADP-ribosylating monomeric actin, the toxins prevent polymerization as well as promoting depolymerization. The most characteristic cellular effect of the toxins is alteration of the cytoskeleton, which leads directly to changes in cellular morphology and indirectly to changes in cell function (e.g. release of chemical mediators). Binary toxins capable of modifying actin are likely to be useful tools in the study of cell biology.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PubMed

Ida, Chieri; Yamashita, Sachiko; Tsukada, Masaki; Sato, Teruaki; Eguchi, Takayuki; Tanaka, Masakazu; Ogata, Shin; Fujii, Takahiro; Nishi, Yoshisuke; Ikegami, Susumu; Moss, Joel; Miwa, Masanao

2016-02-01

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.

1988-05-01

Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/submore » z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.« less

CD38 Mediates Angiotensin II–Induced Intracellular Ca2+ Release in Rat Pulmonary Arterial Smooth Muscle Cells

PubMed Central

Lee, Suengwon; Paudel, Omkar; Jiang, Yongliang; Yang, Xiao-Ru

2015-01-01

CD38 is a multifunctional enzyme that catalyzes the formation of the endogenous Ca2+-mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors (RyRs) of sarcoplasmic reticulum and NAADP-sensitive Ca2+ release channels in endolysosomes, respectively. It plays important roles in systemic vascular functions, but there is little information on CD38 in pulmonary arterial smooth muscle cells (PASMCs). Earlier studies suggested a redox-sensing role of CD38 in hypoxic pulmonary vasoconstriction. This study sought to characterize its roles in angiotensin II (Ang II)–induced Ca2+ release (AICR) in PASMCs. Examination of CD38 expression in various rat arteries found high levels of CD38 mRNA and protein in pulmonary arteries. The Ang II–elicited Ca2+ response consisted of extracellular Ca2+ influx and intracellular Ca2+ release in PASMCs. AICR activated in the absence of extracellular Ca2+ was reduced by pharmacological or siRNA inhibition of CD38, by the cADPR antagonist 8-bromo-cADPR or ryanodine, and by the NAADP antagonist Ned-19 or disruption of endolysosomal Ca2+ stores with the vacuolar H+-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibitions of cADPR- and NAADP-dependent pathways were nonadditive, indicating interdependence of RyR- and NAADP-gated Ca2+ release. Furthermore, AICR was inhibited by the protein kinase C inhibitor staurosporine, the nonspecific NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium, the NOX2-specific inhibitor gp91ds-tat, and the scavenger of reactive oxygen species (ROS) tempol. These results provide the first evidence that Ang II activates CD38-dependent Ca2+ release via the NOX2-ROS pathway in PASMCs. PMID:25078456

The contributions of oxytocin and vasopressin pathway genes to human behavior.

PubMed

Ebstein, Richard P; Knafo, Ariel; Mankuta, David; Chew, Soo Hong; Lai, Poh San

2012-03-01

Arginine vasopressin (AVP) and oxytocin (OXT) are social hormones and mediate affiliative behaviors in mammals and as recently demonstrated, also in humans. There is intense interest in how these simple nonapeptides mediate normal and abnormal behavior, especially regarding disorders of the social brain such as autism that are characterized by deficits in social communication and social skills. The current review examines in detail the behavioral genetics of the first level of human AVP-OXT pathway genes including arginine vasopressin 1a receptor (AVPR1a), oxytocin receptor (OXTR), AVP (AVP-neurophysin II [NPII]) and OXT (OXT neurophysin I [NPI]), oxytocinase/vasopressinase (LNPEP), ADP-ribosyl cyclase (CD38) and arginine vasopressin 1b receptor (AVPR1b). Wherever possible we discuss evidence from a variety of research tracks including molecular genetics, imaging genomics, pharmacology and endocrinology that support the conclusions drawn from association studies of social phenotypes and detail how common polymorphisms in AVP-OXT pathway genes contribute to the behavioral hard wiring that enables individual Homo sapiens to interact successfully with conspecifics. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior. Copyright © 2011 Elsevier Inc. All rights reserved.

PARP-1 may be involved in hydroquinone-induced apoptosis by poly ADP-ribosylation of ZO-2

PubMed Central

Liu, Jiaxian; Yuan, Qian; Ling, Xiaoxuan; Tan, Qiang; Liang, Hairong; Chen, Jialong; Lin, Lianzai; Xiao, Yongmei; Chen, Wen; Liu, Linhua; Tang, Huanwen

2017-01-01

Hydroquinone (HQ), a major reactive metabolite of benzene, contributes to benzene-induced leukemia. The molecular mechanisms that underlie this activity remain to be elucidated. Poly ADP-ribosylation (PARylation) is a type of reversible posttranslational modification that is performed by enzymes in the PAR polymerase (PARP) family and mediates different biological processes, including apoptosis. Zona occludens 2 (ZO-2) is a tight junction scaffold protein, which is involved in cell proliferation and apoptosis. The present study investigated the activity and mechanisms regulated by PARP-1 during HQ-induced apoptosis using TK6 lymphoblastoid cells and PARP-1-silenced TK6 cells. The results revealed that exposure to 10 µM HQ for 72 h induced apoptosis in TK6 cells and that apoptosis was attenuated in PARP-1-silenced TK6 cells. In cells treated with HQ, inhibition of PARP-1 increased the expression of B cell leukemia/lymphoma 2 (Bcl-2), increased ATP production and reduced reactive oxygen species (ROS) production relative to the levels observed in cells treated with HQ alone. Co-localization of ZO-2 and PAR (or PARP-1 protein) was determined using immunofluorescence confocal microscopy. The findings of the present study revealed that ZO-2 was PARylated via an interaction with PARP-1, which was consistent with an analysis of protein expression that was performed using western blot analysis, which determined that ZO-2 protein expression was upregulated in HQ-treated control cells and downregulated in HQ-treated PARP-1-silenced TK6 cells. These findings indicated that prolonged exposure to a low dose of HQ induced TK6 cells to undergo apoptosis, whereas inhibiting PARP-1 attenuates cellular apoptosis by activating Bcl-2 and energy-saving processes and reducing ROS. The present study determined that PARP-1 was involved in HQ-induced apoptosis by PARylation of ZO-2. PMID:28983606

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis*

PubMed Central

Mushtaq, Mazhar; Nam, Tae-Sik; Kim, Uh-Hyun

2011-01-01

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca2+ signal, resulting from a coordinated interplay of Ca2+-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca2+ signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38+/+ platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38−/− platelets. Similarly, PS exposure and Ca2+ signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca2+ signaling mediated by its products, cADPR and NAADP. PMID:21339289

Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

PubMed

Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

2010-08-15

A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. Copyright 2010 Elsevier GmbH. All rights reserved.

Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.

1989-08-01

ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A){sup +} RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A){sup +} RNA are consistent with the presence of at least two,more » and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.« less

Functional role and prognostic significance of CD157 in ovarian carcinoma.

PubMed

Ortolan, Erika; Arisio, Riccardo; Morone, Simona; Bovino, Paola; Lo-Buono, Nicola; Nacci, Giulia; Parrotta, Rossella; Katsaros, Dionyssios; Rapa, Ida; Migliaretti, Giuseppe; Ferrero, Enza; Volante, Marco; Funaro, Ada

2010-08-04

CD157, an ADP-ribosyl cyclase-related cell surface molecule, regulates leukocyte diapedesis during inflammation. Because CD157 is expressed in mesothelial cells and diapedesis resembles tumor cell migration, we investigated the role of CD157 in ovarian carcinoma. We assayed surgically obtained ovarian cancer and mesothelial cells and both native and engineered ovarian cancer cell lines for CD157 expression using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and for adhesion to extracellular matrices, migration, and invasion using cell-based assays. We investigated invasion of human peritoneal mesothelial cells by serous ovarian cancer cells with a three-dimensional coculture model. Experiments were performed with or without CD157-blocking antibodies. CD157 expression in tissue sections from ovarian cancer patients (n = 88) was examined by immunohistochemistry, quantified by histological score (H score), and categorized as at or above or below the median value of 60, and compared with clinical parameters. Statistical tests were two-sided. CD157 was expressed by ovarian cancer cells and mesothelium, and it potentiated the adhesion, migration, and invasion of serous ovarian cancer cells through different extracellular matrices. CD157-transfected ovarian cancer cells migrated twice as much as CD157-negative control cells (P = .001). Blockage of CD157 inhibited mesothelial invasion by serous ovarian cancer cells in a three-dimensional model. CD157 was expressed in 82 (93%) of the 88 epithelial ovarian cancer tissue specimens. In serous ovarian cancer, patients with CD157 H scores of 60 or greater had statistically significantly shorter disease-free survival and overall survival than patients with lower CD157 H scores (CD157 H score > or =60 vs <60: median disease-free survival = 18 months, 95% confidence interval [CI] = 5.92 to 30.07 vs unreached, P = .005; CD157 H score > or =60 vs <60: median overall survival = 45 months, 95% CI = 21

Signaling Mechanism of Poly(ADP-Ribose) Polymerase-1 (PARP-1) in Inflammatory Diseases

PubMed Central

Ba, Xueqing; Garg, Nisha Jain

2011-01-01

Poly(ADP-ribosyl)ation, attaching the ADP-ribose polymer chain to the receptor protein, is a unique posttranslational modification. Poly(ADP-ribose) polymerase-1 (PARP-1) is a well-characterized member of the PARP family. In this review, we provide a general update on molecular structure and structure-based activity of this enzyme. However, we mainly focus on the roles of PARP-1 in inflammatory diseases. Specifically, we discuss the signaling pathway context that PARP-1 is involved in to regulate the pathogenesis of inflammation. PARP-1 facilitates diverse inflammatory responses by promoting inflammation-relevant gene expression, such as cytokines, oxidation-reduction–related enzymes, and adhesion molecules. Excessive activation of PARP-1 induces mitochondria-associated cell death in injured tissues and constitutes another mechanism for exacerbating inflammation. PMID:21356345

Early life stage trimethyltin exposure induces ADP-ribosylation factor expression and perturbs the vascular system in zebrafish.

PubMed

Chen, Jiangfei; Huang, Changjiang; Truong, Lisa; La Du, Jane; Tilton, Susan C; Waters, Katrina M; Lin, Kuanfei; Tanguay, Robert L; Dong, Qiaoxiang

2012-12-16

Trimethyltin chloride (TMT) is an organotin contaminant, widely detected in aqueous environments, posing potential human and environmental risks. In this study, we utilized the zebrafish model to investigate the impact of transient TMT exposure on developmental progression, angiogenesis, and cardiovascular development. Embryos were waterborne exposed to a wide TMT concentration range from 8 to 96 h post fertilization (hpf). The TMT concentration that led to mortality in 50% of the embryos (LC(50)) at 96 hpf was 8.2 μM; malformations in 50% of the embryos (EC(50)) was 2.8 μM. The predominant response observed in surviving embryos was pericardial edema. Additionally, using the Tg (fli1a: EGFP) y1 transgenic zebrafish line to non-invasively monitor vascular development, TMT exposure led to distinct disarrangements in the vascular system. The most susceptible developmental stage to TMT exposure was between 48 and 72 hpf. High density whole genome microarrays were used to identify the early transcriptional changes following TMT exposure from 48 to 60 hpf or 72 hpf. In total, 459 transcripts were differentially expressed at least 2-fold (P<0.05) by TMT compared to control. Using Ingenuity Pathway Analysis (IPA) tools, it was revealed that the transcripts misregulated by TMT exposure were clustered in numerous categories including metabolic and cardiovascular disease, cellular function, cell death, molecular transport, and physiological development. In situ localization of highly elevated transcripts revealed intense staining of ADP-ribosylation factors arf3 and arf5 in the head, trunk, and tail regions. When arf5 expression was blocked by morpholinos, the zebrafish did not display the prototypical TMT-induced vascular deficits, indicating that the induction of arf5 was necessary for TMT-induced vascular toxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)*

PubMed Central

Harlan, Benjamin A.; Pehar, Mariana; Sharma, Deep R.; Beeson, Gyda; Beeson, Craig C.; Vargas, Marcelo R.

2016-01-01

Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD+-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1–7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD+ salvage pathway capable of resynthesizing NAD+ from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD+ levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD+ salvage pathway in astrocytes. Supplementation with the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD+ levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1–2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD+ content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD+ salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, T.; Ui, M.

1985-06-25

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

PubMed

Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

2015-09-15

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

PubMed Central

Keith, G; Glasser, A L; Desgrès, J; Kuo, K C; Gehrke, C W

1990-01-01

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65. PMID:2235481

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

PubMed

Keith, G; Glasser, A L; Desgrès, J; Kuo, K C; Gehrke, C W

1990-10-25

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

PubMed

Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

2016-05-13

Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. © 2016 by The American Society for Biochemistry and Molecular

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

PubMed Central

Pulliainen, Arto T.; Pieles, Kathrin; Brand, Cameron S.; Hauert, Barbara; Böhm, Alex; Quebatte, Maxime; Wepf, Alexander; Gstaiger, Matthias; Aebersold, Ruedi; Dessauer, Carmen W.; Dehio, Christoph

2012-01-01

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium. PMID:22635269

The Structure of RalF, an ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amor,J.; Swails, J.; Zhu, X.

2005-01-01

The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms amore » cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the 'glutamic finger,' conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.« less

Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor.

PubMed

Masilamani, Madhan; Narayanan, Sriram; Prieto, Martha; Borrego, Francisco; Coligan, John E

2008-06-01

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic-like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1(+), Rab5(+) early endosomes. It does appear in Rab4(+) early/sorting endosome, but, in the time period examined, fails to reach Rab11(+) recycling or Rab7(+) late endosomes or lysosome-associated membrane protein-1(+) lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity.

PubMed Central

Olson, J C

1993-01-01

Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A have the same molecular mechanism of toxicity; both toxins ADP-ribosylate a modified histidine residue in elongation factor 2. To help identify amino acids involved in this reaction, sequences in DT that share homology with P. aeruginosa exotoxin A were synthesized and examined for a role in the ADP-ribosyltransferase reaction. By using this approach, residues 32 to 54 of DT were found to define an epitope associated with antibody-mediated inhibition of DT enzyme activity. This lends further support to the notion that residues in this region of DT are involved in the enzymatic reaction. PMID:8423159

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

PubMed

Lambeir, A M; Durinx, C; Proost, P; Van Damme, J; Scharpé, S; De Meester, I

2001-11-02

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation.

PubMed

Smith, Rebecca; Sellou, Hafida; Chapuis, Catherine; Huet, Sébastien; Timinszky, Gyula

2018-05-04

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

HIV-1 infection depletes human CD34+CD38- hematopoietic progenitor cells via pDC-dependent mechanisms.

PubMed

Li, Guangming; Zhao, Juanjuan; Cheng, Liang; Jiang, Qi; Kan, Sheng; Qin, Enqiang; Tu, Bo; Zhang, Xin; Zhang, Liguo; Su, Lishan; Zhang, Zheng

2017-07-01

Chronic human immunodeficiency virus-1 (HIV-1) infection in patients leads to multi-lineage hematopoietic abnormalities or pancytopenia. The deficiency in hematopoietic progenitor cells (HPCs) induced by HIV-1 infection has been proposed, but the relevant mechanisms are poorly understood. We report here that both human CD34+CD38- early and CD34+CD38+ intermediate HPCs were maintained in the bone marrow (BM) of humanized mice. Chronic HIV-1 infection preferentially depleted CD34+CD38- early HPCs in the BM and reduced their proliferation potential in vivo in both HIV-1-infected patients and humanized mice, while CD34+CD38+ intermediate HSCs were relatively unaffected. Strikingly, depletion of plasmacytoid dendritic cells (pDCs) prevented human CD34+CD38- early HPCs from HIV-1 infection-induced depletion and functional impairment and restored the gene expression profile of purified CD34+ HPCs in humanized mice. These findings suggest that pDCs contribute to the early hematopoietic suppression induced by chronic HIV-1 infection and provide a novel therapeutic target for the hematopoiesis suppression in HIV-1 patients.

Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

PubMed

Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

2013-05-13

Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

CD34+CD38-CD123+ Cells Are Present in Virtually All Acute Myeloid Leukaemia Blasts: A Promising Single Unique Phenotype for Minimal Residual Disease Detection.

PubMed

Al-Mawali, Adhra; Pinto, Avinash Daniel; Al-Zadjali, Shoaib

In CD34-positive acute myeloid leukaemia (AML), the leukaemia-initiating event likely takes place in the CD34+CD38- cell compartment. CD123 has been shown to be a unique marker of leukaemic stem cells within the CD34+CD38- compartment. The aim of this study was to identify the percentage of CD34+CD38-CD123+ cells in AML blasts, AML CD34+CD38- stem cells, and normal and regenerating bone marrow CD34+CD38- stem cells from non-myeloid malignancies. Thirty-eight adult de novo AML patients with intention to treat were enrolled after the application of inclusion criteria from February 2012 to February 2017. The percentage of the CD34+CD38-CD123+ phenotype in the blast population at diagnosis was determined using a CD45-gating strategy and CD34+ backgating by flow cytometry. We studied the CD34+CD38-CD123+ fraction in AML blasts at diagnosis, and its utility as a unique phenotype for minimal residual disease (MRD) of AML patients. CD123+ cells were present in 97% of AML blasts in patients at diagnosis (median 90%; range 21-99%). CD123+ cells were also present in 97% of the CD34+CD38- compartment (median 0.8164%, range 0.0262-39.7%). Interestingly, CD123 was not present in normal and regenerating CD34+CD38- bone marrow stem cells (range 0.002- 0.067 and 0.004-0.086, respectively). The CD34+CD38-CD123+ phenotype is present in virtually all AML blasts and it may be used as a unique single phenotype for MRD detection in AML patients. © 2017 The Author(s) Published by S. Karger AG, Basel.

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia.

PubMed

Plesa, Adriana; Dumontet, Charles; Mattei, Eve; Tagoug, Ines; Hayette, Sandrine; Sujobert, Pierre; Tigaud, Isabelle; Pages, Marie Pierre; Chelghoum, Youcef; Baracco, Fiorenza; Labussierre, Helene; Ducastelle, Sophie; Paubelle, Etienne; Nicolini, Franck Emmanuel; Elhamri, Mohamed; Campos, Lydia; Plesa, Claudiu; Morisset, Stéphane; Salles, Gilles; Bertrand, Yves; Michallet, Mauricette; Thomas, Xavier

2017-12-26

To evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival. Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia (AML) were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8C panels and FACS CANTO and Diva software (BD Bioscience). We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38- population. Using a cut-off value of 1% of CD34+CD38- from total "bulk leukemic cells" we found that a high (> 1%) level of CD34+CD38- blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38- leukemic cells > 1% was an independent predictor of DFS [HR = 2.8 (1.02-7.73), P = 0.04] and OS [HR = 2.65 (1.09-6.43), P = 0.03]. Taken together, these results show that a CD34/CD38 "backbone" for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.

Peripheral Blood CD38 Bright CD8+ Effector Memory T Cells Predict Acute Graft-versus-Host Disease.

PubMed

Khandelwal, Pooja; Lane, Adam; Chaturvedi, Vijaya; Owsley, Erika; Davies, Stella M; Marmer, Daniel; Filipovich, Alexandra H; Jordan, Michael B; Marsh, Rebecca A

2015-07-01

Acute graft-versus-host disease (aGVHD) is mediated by allogeneic T cell responses. We hypothesized that increases of peripheral blood-activated CD8+ effector memory T (TEM) cells would be observed after hematopoietic stem cell transplantation (HSCT) before onset of aGVHD symptoms. Blood was collected twice weekly after HSCT for 7 weeks in 49 consecutive pediatric and adult HSCT recipients. Samples were incubated with fluorochrome-conjugated antibodies against CD45, CD3, CD8, CD38, CD45RA, and CCR7 and analyzed using flow cytometry. TEM cells were defined as CD3+ CD8+ CCR7- CD45RA(-) lymphocytes. CD38 expression was used as a marker of T cell activation. Patients were followed for 100 days for development of aGVHD. Twenty-three patients developed grade 1 to 4 aGVHD at a median of 37 days (range, 15 to 79 days) after HCST. Absolute CD38 bright CD8+ TEM of > 35 cells/μL predicted aGVHD at a median of 8 days (range, 1 to 34) before aGVHD onset with a sensitivity of 82.6% and specificity of 91.6%. The cumulative incidence of aGVHD was 90% in patients with absolute CD38 bright CD8+ TEM >35 cells/μL and 15% in patients without (P < .0001). Quantification of CD38 bright CD8+ TEM cells may predict aGVHD in children and young adult HSCT recipients. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

PubMed

Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M; Villamil, Silvia H Fernández; Lehtiö, Lari

2012-01-01

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease.

PubMed

Hamblin, Terry J; Orchard, Jenny A; Ibbotson, Rachel E; Davis, Zadie; Thomas, Peter W; Stevenson, Freda K; Oscier, David G

2002-02-01

Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgV(H)) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgV(H) mutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage, IgV(H) mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgV(H) genes and expressed CD38 was 8 years; in those with mutated IgV(H) genes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgV(H) mutations in CLL, it is an independent risk factor that can be used with IgV(H) mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.

CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells.

PubMed

Deaglio, Silvia; Capobianco, Andrea; Bergui, Luciana; Dürig, Jan; Morabito, Fortunato; Dührsen, Ulrich; Malavasi, Fabio

2003-09-15

The prognosis for patients with B-cell chronic lymphocytic leukemia (B-CLL) is generally less favorable for those expressing CD38. Our working hypothesis is that CD38 is not merely a marker in B-CLL, but that it plays a receptor role with pathogenetic potential ruling the proliferation of the malignant clone. CD38 levels were generally low in the patients examined and monoclonal antibody (mAb) ligation was inefficient in signaling. Other cellular models indicated that molecular density and surface organization are critical for CD38 functionality. Interleukin 2 (IL-2) induced a marked up-modulation and surface rearrangement of CD38 in all the patients studied. On reaching a specific expression threshold, CD38 becomes an efficient receptor in purified B-CLL cells. Indeed, mAb ligation is followed by Ca2+ fluxes and by a markedly increased proliferation. The unsuitability of CD38 to perform as a receptor is obviated through close interaction with the B-cell-receptor (BCR) complex and CD19. On mAb binding, CD38 translocates to the membrane lipid microdomains, as shown by a colocalization with the GM1 ganglioside and with CD81, a raft-resident protein. Finally, CD38 signaling in IL-2-treated B-CLL cells prolonged survival and induced the appearance of plasmablasts, providing a pathogenetic hypothesis for the occurrence of Richter syndrome.

Milatuzumab-SN-38 conjugates for the treatment of CD74+ cancers.

PubMed

Govindan, Serengulam V; Cardillo, Thomas M; Sharkey, Robert M; Tat, Fatma; Gold, David V; Goldenberg, David M

2013-06-01

CD74 is an attractive target for antibody-drug conjugates (ADC), because it internalizes and recycles after antibody binding. CD74 mostly is associated with hematologic tumors but is expressed also in solid cancers. Therefore, ADCs of the humanized anti-CD74 antibody, milatuzumab, were examined for the therapy of CD74-expressing solid tumors. Milatuzumab-doxorubicin and two milatuzumab-SN-38 conjugates with cleavable linkers, differing in their stability in serum and how they release SN-38 in the lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology. In vitro cytotoxicity and in vivo therapeutic studies were conducted in the human cancer cell lines A-375 (melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic), NCI-N87 (gastric), and Raji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared with SN-38 ADCs prepared with anti-Trop-2 and anti-CEACAM6 antibodies in xenografts expressing their target antigens. Milatuzumab-doxorubicin was most effective in the lymphoma model, whereas in A-375 and Capan-1 solid tumors, only milatuzumab-SN-38 showed a therapeutic benefit. Despite much lower surface expression of CD74 than Trop-2 or CEACAM6, milatuzumab-SN-38 had similar efficacy in Capan-1 as anti-Trop-2-SN-38, but in NCI-N87, anti-CEACAM6 and anti-Trop-2 conjugates were superior. Studies in two hepatoma lines at a single dose level showed significant benefit over saline controls but not against an irrelevant immunoglobulin G conjugate. CD74 is a suitable target for ADCs in some solid tumor xenografts, with efficacy largely influenced by uniformity of CD74 expression and with SN-38 conjugates providing the best therapeutic responses; SN-38 conjugates were preferable in solid cancers, whereas doxorubicin ADC was better in lymphoma tested. ©2013 AACR

Increased numbers of CD38 molecules on bright CD8+ T lymphocytes in infectious mononucleosis caused by Epstein–Barr virus infection

PubMed Central

ŽIDOVEC LEPEJ, S; VINCE, A; ÐAKOVIĆ RODE, O; REMENAR, A; JEREN, T

2003-01-01

The aim of this study was to quantify the expression of CD38 on CD8+ T lymphocytes of patients with infectious mononucleosis (IM) caused by Epstein–Barr virus (EBV) and cytomegalovirus (CMV). CD38 quantification technique chosen for this study was based on the enumeration of CD38 antibody binding sites in comparison to the quantification standards rather than determining relative fluorescence, which is difficult to standardize. The study enrolled 19 patients with typical clinical and laboratory parameters compatible with EBV-induced IM as well as 10 patients with atypical clinical presentation of this disease. Furthermore, CD38 expression was analysed in a group of 13 patients with IM caused by CMV infection. CD38 quantification was performed within 6 days of the presentation of symptoms. All three groups of IM patients showed a statistically significant increase in the number of anti-CD38 antibody binding sites (which correspond to the number of CD38 molecules) on bright CD8+ T lymphocytes compared to healthy controls. The numbers of CD38 molecules expressed on CD8+ T lymphocytes did not differ significantly between IM patients with typical and atypical clinical presentation of the disease. Patients with CMV-induced IM had significantly lower numbers of CD38 molecules expressed on CD8+ T lymphocytes. Therefore, we conclude that CD38 quantification could be helpful in differential diagnostics of IM cases with atypical clinical presentation. PMID:12930365

Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38

PubMed Central

Escande, Carlos; Nin, Veronica; Price, Nathan L.; Capellini, Verena; Gomes, Ana P.; Barbosa, Maria Thereza; O’Neil, Luke; White, Thomas A.; Sinclair, David A.; Chini, Eduardo N.

2013-01-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD+ metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD+ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD+ase in mammals. Moreover, CD38 knockout mice have higher NAD+ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD+ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD+ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD+ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD+-dependent pathways. PMID:23172919

An age-related numerical and functional deficit in CD19(+) CD24(hi) CD38(hi) B cells is associated with an increase in systemic autoimmunity.

PubMed

Duggal, Niharika A; Upton, Jane; Phillips, Anna C; Sapey, Elizabeth; Lord, Janet M

2013-10-01

Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19(+) CD24(hi) CD38(hi) phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19(+) CD24(hi) CD38(hi) cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll-like receptors was also impaired in CD19(+) CD24(hi) CD38(hi) B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19(+) CD24(hi) CD38(hi) B-cell function was compromised by age-related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19(+) CD24(hi) CD38(hi) B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age-associated change in expression of costimulatory molecules CD80 and CD86 on CD19(+) CD24(hi) CD38(hi) cells, suggesting IL10-dependent immune suppression is impaired, but contact-dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19(+) CD24(hi) CD38(hi) B-cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age-related decline in CD19(+) CD24(hi) CD38(hi) B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging. © 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Regulatory B cells (CD19(+)CD38(hi)CD24(hi)) in alloimmunized and non-alloimmunized children with β-thalassemia major.

PubMed

Zahran, Asmaa M; Elsayh, Khalid I; Saad, Khaled; Embaby, Mostafa; Ali, Ahmed M

2016-03-01

β-Thalassemia major (BTM) is considered the most common hemoglobinopathy in Egypt and is one of the major health problems in our locality. We investigated the frequency of B-regulatory cells (CD19(+)CD38(hi)CD24(hi)); (Bregs) among polytransfused alloimmunized and non-alloimmunized children with BTM. The study included 110 polytransfused pediatric patients with β-thalassemia major. Clinical and transfusion records of all studied patients were reviewed. Indirect antiglobulin test was performed to detect the presence of alloantibodies. We used flow cytometry for detection of CD19(+)CD38(hi)CD24(hi) regulatory B cells. Alloimmunization was detected in 35.5% of thalassemic patients (39/110). The analysis of our data showed a significantly higher frequency of Bregs (CD19(+)CD38(hi)CD24(hi)) in the peripheral blood of both alloimmunized and non-alloimmunized patients as compared to healthy controls. Our data showed that the frequencies of CD19(+)CD24(hi)CD38(hi) Bregs cells were significantly increased in children with BTM. Our data suggested that Bregs cells could play a role in the clinical course of BTM. The relationship of Bregs to immune disorders in BTM children remains to be determined. Further longitudinal study with a larger sample size is warranted to explore the mechanisms of Breg cells in the disease process in BTM patients. Copyright © 2016 Elsevier Inc. All rights reserved.

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients.

PubMed

Karakasheva, Tatiana A; Dominguez, George A; Hashimoto, Ayumi; Lin, Eric W; Chiu, Christopher; Sasser, Kate; Lee, Jae W; Beatty, Gregory L; Gabrilovich, Dmitry I; Rustgi, Anil K

2018-03-22

Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

NASA Astrophysics Data System (ADS)

Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

2013-12-01

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

PubMed Central

Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

2016-01-01

Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

Role of the CD34+ 38- cells in posttransplant hematopoietic recovery.

PubMed

Hénon, P; Sovalat, H; Bourderont, D; Ojeda-Uribe, M; Arkam, Y; Wunder, E; Raidot, J P; Husseini, F; Audhuy, B

1998-01-01

Using three different statistical tests in parallel, we showed in a preliminary study that neither mononuclear cells, CD34+ 33+ or 33- cells, nor CD34+ 38+ cells significantly correlated with engraftment kinetics following autologous blood cell transplantation (ABCT). We additionally demonstrated here, in a series of patients suffering from malignant diseases, that the graft content in CD34+ 38- cells is individually a more sensitive indicator of the earliest, as well as the latest post-ABCT trilineage hematopoietic recovery than the colony-forming units-granulocyte-macrophage and even the total CD34+ cell content. This suggests that the CD34+ 38- cell population is itself subdivided into two more subsets, one being already lineage-committed and responsible for short-term engraftment, the other containing only very primitive hematopoietic cells responsible for sustained engraftment. Strong arguments favor the probability that these subsets correspond to HLA-DR+ and DR cells, respectively. We also defined an optimal threshold value of 0.05 x 10(6) CD34+ 38- cells/kg of the patient's body weight (b.w.) above which a rapid and sustained trilineage engraftment safely occurs. In fact, infusion of lower numbers of cells seems to have a more significant impact on long-term compared to short-term neutrophil recovery and on platelet kinetics engraftment. We additionally looked for the eventual influence on engraftment time of the type of disease, and of post-ABCT administration of hematopoietic growth factors (HGF). When the type of disease appeared to have no influence on the engraftment time, posttransplant HGF administration significantly reduced the time to trilineage engraftment in patients transplanted with < 0.05 x 10(6) CD34+ 38- cells, thus justifying it in case of reinfusion of low numbers of CD34+ 38- cells. On the other hand, the administration of HGF after infusion of more than 0.05 x 10(6) CD34+ 38- cells/kg b.w. did not hasten more, or only very little, the

Prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients.

PubMed

Gogia, A; Sharma, A; Raina, V; Kumar, L; Gupta, R; Kumar, R

2013-01-01

Chronic lymphocytic leukemia (CLL) is the most common chronic lympho-proliferative disorder. This study was undertaken to know the prevalence of ZAP-70 and CD 38 in the treatment naive patients of CLL seen at a tertiary care centre of north India. ZAP-70 and CD 38 were tested by flow cytometry on peripheral blood samples. ZAP-70 positive and CD 38 positivity was defined as positive expression on 20% and 30% of CLL cells, respectively. Clinico-hematological profile and its correlation with ZAP-70 and CD 38 were assessed in consecutive 80 CLL patients. There were 64 males and median age of the group was 58 years. Sixteen patients (20%) were asymptomatic and diagnosed incidentally. Median total lymphocyte count (TLC) at presentation was 62 × 10 9 /L. Rai stage distribution was: Stage 0-6, stage I-20, stage II-36, stage III-5, and stage IV-13. ZAP-70 and CD 38 positivity were detected in 20 patients (25%) and 29 patients (36%), respectively. Eleven patients were positive and 34 were negative for both ZAP-70 and CD 38 yielding a concordance rate of 56%. There was no statistically significant difference between ZAP-70 and CD 38 positivity and negativity with regard to age, sex, Lymphocyte count, lymphadenopathy, organomegaly, and Rai staging. ZAP-70 and CD 38 positivity were detected 25% and 36%, respectively, with concordance rate of 56%, which is higher than Western literature. There was no correlation of ZAP-70 and CD 38 positivity with age, sex, lymphadenopathy, organomegaly, and Rai staging.

Inhibiting the CD38/cADPR pathway protected rats against sepsis associated brain injury.

PubMed

Peng, Qian-Yi; Wang, Yi-Min; Chen, Cai-Xia; Zou, Yu; Zhang, Li-Na; Deng, Song-Yun; Ai, Yu-Hang

2018-01-01

The CD38/cADPR pathway has been found to play roles in various inflammatory conditions. However, whether CD38 plays a protective or detrimental effect in the central nervous system (CNS) is controversial. The aim of this study was to determine the effect of CD38/cADPR pathway in sepsis associated brain injury. Male Sprague-Dawley rats were undergone cecal ligation and puncture (CLP) or sham laparotomies. NAD + , cADPR and CD38 were measured in the hippocampus of septic rats at 0, 6, 12, 24, and 48h after CLP surgery. Rats were divided into the sham, CLP group, CLP+ CD38 expression lentivirus (CLP+ CD38 LV), CLP+ CD38 interference lentivirus (CLP+ CD38 Ri), CLP+ negative control lentivirus (CLP+NC) and the CLP+8-Br-cADPR groups. The Western blots of Bcl-2, Bax and iNOS, TUNEL assays, malondialdehyde (MDA) and superoxide dismutase (SOD) assays, transmission electron microscope analysis were performed in the hippocampus of rats. NAD + , cADPR and CD38 levels increased significantly in the hippocampus of septic rats as early as 12-24h after CLP surgery. CD38 knockdown or blocking cADPR with 8-Br-cADPR significantly reduced apoptosis, MDA and SOD activity, iNOS expression and ultrastructural morphology damages in the hippocampus of septic rats. In this study, we found that the CD38/cADPR pathway was activated in sepsis associated brain injury. Blocking this pathway protected the hippocampus from apoptosis, oxidative stress and ultrastructural morphology damages in septic rats. Copyright © 2017. Published by Elsevier B.V.

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

PubMed

Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

2013-06-01

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells

PubMed Central

Zhou, Y; Jiang, H; Gu, J; Tang, Y; Shen, N; Jin, Y

2013-01-01

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy, drug screening and toxicity testing of neural degenerative diseases. However, the molecular regulation of their proliferation and apoptosis, which needs to be revealed before clinical application, is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here, we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells, hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently, global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically, a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression, revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover, forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly, we found that paraquat, a neurotoxin, not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs, indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably, inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively, miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

PubMed

Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J

1992-04-15

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

Upregulation of CD19⁺CD24(hi)CD38(hi) regulatory B cells is associated with a reduced risk of acute lung injury in elderly pneumonia patients.

PubMed

Song, Haihan; Xi, Jianjun; Li, Guang-Gang; Xu, Shumin; Wang, Chunmei; Cheng, Tingting; Li, Hongqiang; Zhang, Ying; Liu, Xiandong; Bai, Jianwen

2016-04-01

Acute lung injury (ALI) is a common complication in elderly pneumonia patients who have a rapid progression, and is accompanied by a high mortality rate. Because the treatment options of ALI are limited to supportive care, identifying pneumonia patients who are at higher risk of ALI development is the emphasis of many studies. Here, we approach this problem from an immunological perspective by examining CD19(+)CD24(hi)CD38(hi) B cells, an important participant in acute and chronic inflammation. We find that elderly pneumonia patients have elevated CD19(+)CD24(hi)CD38(hi) B cell frequency compared to healthy individuals. This B cell population may express a higher level of IL-10, which has been was shown to suppress CD4(+) T cell-mediated proinflammatory cytokine interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) production, through an IL-10-dependent mechanism. We also observe that the frequency of CD19(+)CD24(hi)CD38(hi) B cell is positively correlated with the frequency of CD4(+)CD25(+)Foxp3(+)Tregs in peripheral blood. Moreover, consistent with CD19(+)CD24(hi)CD38(hi) B cell's anti-inflammatory role, we find that pneumonia patients who later developed ALI have reduced level of CD19(+)CD24(hi)CD38(hi) B cells. Together, our results demonstrated that CD19(+)CD24(hi)CD38(hi) B cells in pneumonia patients possess regulatory function in vivo, and are associated with a reduced ALI risk.

Fuzhengpaidu granule regulates immune activation molecules CD38 and human leukocyte antigen-D related on CD4+ and CD8+ T cells in patients with acquired immunodeficiency syndrome/human immunodeficiency virus.

PubMed

Jiang, Feng; Zhang, Rongxin; Gu, Zhenfang; Zhang, Huailing; Guo, Huijun; Deng, Xin; Liang, Jian

2013-08-01

To evaluate the effect of Fuzhengpaidu granule (FZPDG) on immune activation molecules CD38 and human leukocyte antigen-D related (HLA-DR) on CD4+ and CD8+ cells in HIV/AIDS patients, and to explore the underlying mechanism of this therapy. Plasma changes in CD3+, CD4+, CD8+, CD3 + CD4 + CD38 +, CD3 + CD4 + HLA-DR+, CD3 + CD8+CD38+, and CD3+CD8+HLA-DR+ levels in HIV/ AIDS patients treated with FZPDG for six months were examined by flow cytometry and compared with levels in healthy controls. The clinical trial included 34 outpatients with HIV/AIDS. Before treatment, plasma levels of CD38+ and HLA-DR+ on CD4/CD8 cells were higher than those in 28 health controls (P < 0.05). There were no significant changes in serum levels of CD3+, CD4+, and CD8+ T cells between pretreatment baseline versus after treatment, which were 82.85% +/- 5.41%, 14.57% +/- 10.31% and 54.55% +/- 11.43% before treatment and 79.15% +/- 8.21%, 19.96% +/- 9.58% and 56.36% +/- 11.67% after treatment, respectively (P > 0.05). Plasma levels of CD3+ CD4+CD38+ and CD3+CD4+HLA-DR+ were 2.3% +/-2.2% and 7.8% +/- 5.5% before treatment and 1.2% +/-0.8% and 2.6% +/- 1.0% after treatment, respectively. Plasma levels of CD3+CD8+CD38+ and CD3+CD8+ HLA-DR+ were 41.4% +/- 13.4% and 17.8% +/- 11.3% before treatment, which changed to 27.1% +/- 10.2% and 3.8% +/- 2.4% after treatment, respectively (P < 0.05). HIV/AIDS patients exhibited an immune activation profile following FZPDG treatment. A potential mechanism of action for FZPDG appears to lie in its ability to up-regulate CD38 and HLA-DR levels on CD4+ T cells, and down-regulate them on CD8+ cells, thereby modulating immune activation of CD4+and CD8+T cells.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

PubMed Central

Mazaki, Yuichi; Hashimoto, Shigeru; Okawa, Katsuya; Tsubouchi, Asako; Nakamura, Kuniaki; Yagi, Ryohei; Yano, Hajime; Kondo, Akiko; Iwamatsu, Akihiro; Mizoguchi, Akira; Sabe, Hisataka

2001-01-01

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during

Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

PubMed

Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

2016-04-01

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

PubMed

Aybar, L T; McGregor, J G; Hogan, S L; Hu, Y; Mendoza, C E; Brant, E J; Poulton, C J; Henderson, C D; Falk, R J; Bunch, D O

2015-05-01

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production. © 2014 British Society for Immunology.

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

PubMed

Neer, E J

1976-01-01

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-04-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed Central

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-01-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor. PMID:9119480

Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

PubMed Central

Cardoso, A A; Li, M L; Batard, P; Hatzfeld, A; Brown, E L; Levesque, J P; Sookdeo, H; Panterne, B; Sansilvestri, P; Clark, S C

1993-01-01

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation. PMID:7690969

CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer

PubMed Central

Wang, Weiwei; Yuan, Xiangliang; Chen, Hui; Xie, Guohua; Ma, Yanhui; Zheng, Yingxia; Zhou, Yunlan; Shen, Lisong

2015-01-01

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19+CD24hiCD38hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3+T cells or CD4+ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4+Th cells. CD19+CD24hiCD38hiBregs were also found to correlate positively with CD4+FoxP3+ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4+CD25− effector T cells to CD4+FoxP3+Tregs via TGF-β1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer. PMID:26378021

Poly(ADP-ribose) polymerase-1 (Parp-1)-deficient mice demonstrate abnormal antibody responses

PubMed Central

Ambrose, Helen E; Willimott, Shaun; Beswick, Richard W; Dantzer, Françoise; de Murcia, Josiane Ménissier; Yelamos, José; Wagner, Simon D

2009-01-01

Poly(ADP-ribosylation) of acceptor proteins is an epigenetic modification involved in DNA strand break repair, recombination and transcription. Here we provide evidence for the involvement of poly(ADP-ribose) polymerase-1 (Parp-1) in antibody responses. Parp-1−/− mice had increased numbers of T cells and normal numbers of total B cells. Marginal zone B cells were mildly reduced in number, and numbers of follicular B cells were preserved. There were abnormal levels of basal immunoglobulins, with reduced levels of immunoglobulin G2a (IgG2a) and increased levels of IgA and IgG2b. Analysis of specific antibody responses showed that T cell-independent responses were normal but T cell-dependent responses were markedly reduced. Germinal centres were normal in size and number. In vitro purified B cells from Parp-1−/− mice proliferated normally and showed normal IgM secretion, decreased switching to IgG2a but increased IgA secretion. Collectively our results demonstrate that Parp-1 has essential roles in normal T cell-dependent antibody responses and the regulation of isotype expression. We speculate that Parp-1 forms a component of the protein complex involved in resolving the DNA double-strand breaks that occur during class switch recombination. PMID:18778284

Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aksoy, Pinar; Escande, Carlos; Seccion Biologia Celular, Facultad de Ciencias, Universidad de la Republica, Igua 4225, Montevideo

2006-10-13

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1more » enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.« less

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

PubMed

Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

2016-05-01

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling.

PubMed

Futrega, Kathryn; Yu, Jianshi; Jones, Jace W; Kane, Maureen A; Lott, William B; Atkinson, Kerry; Doran, Michael R

2016-04-21

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.

All-trans Retinoic Acid Upregulates Reduced CD38 Transcription in Lymphoblastoid Cell Lines from Autism Spectrum Disorder

PubMed Central

Riebold, Mathias; Mankuta, David; Lerer, Elad; Israel, Salomon; Zhong, Songfa; Nemanov, Luba; Monakhov, Mikhail V; Levi, Shlomit; Yirmiya, Nurit; Yaari, Maya; Malavasi, Fabio; Ebstein, Richard P

2011-01-01

Deficits in social behavior in mice lacking the CD38 gene have been attributed to impaired secretion of oxytocin. In humans, similar deficits in social behavior are associated with autistic spectrum disorder (ASD), for which genetic variants of CD38 have been pinpointed as provisional risk factors. We sought to explore, in an in vitro model, the feasibility of the theory that restoring the level of CD38 in ASD patients could be of potential clinical benefit. CD38 transcription is highly sensitive to several cytokines and vitamins. One of these, all-trans retinoic acid (ATRA), a known inducer of CD38, was added during cell culture and tested on a large sample of N = 120 lymphoblastoid cell (LBC) lines from ASD patients and their parents. Analysis of CD38 mRNA levels shows that ATRA has an upmodulatory potential on LBC derived from ASD patients as well as from their parents. The next crucial issue addressed in our study was the relationship between levels of CD38 expression and psychological parameters. The results obtained indicate a positive correlation between CD38 expression levels and patient scores on the Vineland Adaptive Behavior Scale. In addition, analysis of the role of genetic polymorphisms in the dynamics of the molecule revealed that the genotype of a single-nucleotide polymorphism (rs6449182; C>G variation) in the CpG island of intron 1, harboring the retinoic-acid response element, exerts differential roles in CD38 expression in ASD and in parental LBC. In conclusion, our results provide an empirical basis for the development of a pharmacological ASD treatment strategy based on retinoids. PMID:21528155

CD38-NAD+Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response.

PubMed

Chatterjee, Shilpak; Daenthanasanmak, Anusara; Chakraborty, Paramita; Wyatt, Megan W; Dhar, Payal; Selvam, Shanmugam Panneer; Fu, Jianing; Zhang, Jinyu; Nguyen, Hung; Kang, Inhong; Toth, Kyle; Al-Homrani, Mazen; Husain, Mahvash; Beeson, Gyda; Ball, Lauren; Helke, Kristi; Husain, Shahid; Garrett-Mayer, Elizabeth; Hardiman, Gary; Mehrotra, Meenal; Nishimura, Michael I; Beeson, Craig C; Bupp, Melanie Gubbels; Wu, Jennifer; Ogretmen, Besim; Paulos, Chrystal M; Rathmell, Jeffery; Yu, Xue-Zhong; Mehrotra, Shikhar

2018-01-09

Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD + -dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD + , enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD + axis could increase the efficacy of anti-tumor adoptive T cell therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Principal component for metabolic syndrome risk maps to chromosome 4p in Mexican Americans: the San Antonio Family Heart Study.

PubMed

Cai, Guowen; Cole, Shelley A; Freeland-Graves, Jeanne H; MacCluer, Jean W; Blangero, John; Comuzzie, Anthony G

2004-10-01

Metabolic syndrome refers to the clustering of disease conditions such as insulin resistance, hyperinsulinemia, dyslipidemia, hypertension, and obesity. To explore the genetic predispositions of this complex syndrome, we conducted a principal components analysis using data on 14 phenotypes related to the risk of developing metabolic syndrome. The subjects were 566 nondiabetic Mexican Americans, distributed in 41 extended families from the San Antonio Family Heart Study. The factor scores obtained from these 14 phenotypes were used in multipoint linkage analysis using SOLAR. Factors were identified that accounted for 73% of the total variance of the original variables: body size-adiposity, insulin-glucose, blood pressure, and lipid levels. Each factor exhibited evidence for either significant or suggestive linkage involving four factor-specific chromosomal regions relating to chromosomes 1, 3, 4, and 6. Significant evidence for linkage of the lipid factor was found on chromosome 4 near marker D4S403 (LOD = 3.52), where the cholecystokinin A receptor (CCKAR) and ADP-ribosyl cyclase 1 (CD38) genes are located. Suggestive evidence for linkage of the body size-adiposity factor to chromosome 1 near marker D1S1597 (LOD = 2.53) in the region containing the nuclear receptor subfamily 0, group B, member 2 gene (NROB2) also was observed. The insulin-glucose and blood pressure factors were linked suggestively to regions on chromosome 3 near marker D3S1595 (LOD = 2.20) and on chromosome 6 near marker D6S 1031 (LOD = 2.08), respectively. In summary, our findings suggest that the factor structures for the risk of metabolic syndrome are influenced by multiple distinct genes across the genome.

Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes

PubMed Central

Xiong, Jing; Xia, Min; Xu, Ming; Zhang, Yang; Abais, Justine M; Li, Guangbi; Riebling, Christopher R; Ritter, Joseph K; Boini, Krishna M; Li, Pin-Lan

2013-01-01

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes. PMID:24238063

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

PubMed Central

Sahay, Bikash; Patsey, Rebeca L.; Eggers, Christian H.; Salazar, Juan C.; Radolf, Justin D.; Sellati, Timothy J.

2009-01-01

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes. PMID:20011115

CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism.

PubMed

Camacho-Pereira, Juliana; Tarragó, Mariana G; Chini, Claudia C S; Nin, Veronica; Escande, Carlos; Warner, Gina M; Puranik, Amrutesh S; Schoon, Renee A; Reid, Joel M; Galina, Antonio; Chini, Eduardo N

2016-06-14

Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

PubMed Central

Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

2009-01-01

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.

PubMed

Mabuchi, I; Hamaguchi, Y; Fujimoto, H; Morii, N; Mishima, M; Narumiya, S

1993-11-01

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia.

PubMed

Lin, Ke; Sherrington, Paul D; Dennis, Michael; Matrai, Zoltan; Cawley, John C; Pettitt, Andrew R

2002-08-15

Established adverse prognostic factors in chronic lymphocytic leukemia (CLL) include CD38 expression, relative lack of IgV(H) mutation, and defects of the TP53 gene. However, disruption of the p53 pathway can occur through mechanisms other than TP53 mutation, and we have recently developed a simple screening test that detects p53 dysfunction due to mutation of the genes encoding either p53 or ATM, a kinase that regulates p53. The present study was conducted to examine the predictive value of this test and to establish the relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation. CLL cells from 71 patients were examined for IgV(H) mutation, CD38 expression, and p53 dysfunction (detected as an impaired p53/p21 response to ionizing radiation). Survival data obtained from 69 patients were analyzed according to each of these parameters. Relative lack of IgV(H) mutation (less than 5%; n = 45), CD38 positivity (antigen expressed on more than 20% of malignant cells; n = 19), and p53 dysfunction (n = 19) were independently confirmed as adverse prognostic factors. Intriguingly, all p53-dysfunctional patients and all but one of the CD38(+) patients had less [corrected] than 5% IgV(H) mutation. Moreover, patients with p53 dysfunction and/or CD38 positivity (n = 31) accounted for the short survival of the less mutated group. These findings indicate that the poor outcome associated with having less than 5% IgV(H) mutation may be due to the overrepresentation of high-risk patients with p53 dysfunction and/or CD38 positivity within this group, and that CD38(-) patients with functionally intact p53 may have a prolonged survival regardless of the extent of IgV(H) mutation.

Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neuhof, Dirk; Zwicker, Felix; Kuepper, Jan-Heiner

2007-11-01

Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10more » Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.« less

Glucose and cyclic adenosine monophosphate stimulate activities of adenylate cyclase and guanylate cyclase of Tetrahymena pyriformis infusoria.

PubMed

Shpakov, A O; Derkach, K V; Uspenskaya, Z I

2012-02-01

The sensitivities of cyclase enzymes adenylate cyclase and guanylate cyclase to glucose and extracellular cAMP were studied in Tetrahymena pyriformis infusoria. Glucose effectively stimulated activities of both cyclase enzymes, while cAMP more effectively stimulated adenylate cyclase. It was shown that [6-(14)C]glucose specifically bound to Tetrahymena pyriformis infusoria at dissociation constant (K(D)) and number of binding sites (B(max)) 43 nM and 7.53 fmol glucose per 100,000 cells and [8-(3)H]cAMP bound at 19 nM and 4.46 fmol cAMP per 100,000 cells, respectively. Hence, glucose and cAMP specifically bound to Tetrahymena pyriformis cells and stimulated activities of cyclases in these infusoria.

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

PubMed Central

Ostolaza, Helena; Martín, César; González-Bullón, David; Uribe, Kepa B.; Etxaniz, Asier

2017-01-01

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered. PMID:28934133

Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

PubMed

Han, P; Lucero, M T

2005-01-01

Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

A preclinical model of CD38-pretargeted radioimmunotherapy for plasma cell malignancies.

PubMed

Green, Damian J; Orgun, Nural N; Jones, Jon C; Hylarides, Mark D; Pagel, John M; Hamlin, Donald K; Wilbur, D S; Lin, Yukang; Fisher, Darrell R; Kenoyer, Aimee L; Frayo, Shani L; Gopal, Ajay K; Orozco, Johnnie J; Gooley, Theodore A; Wood, Brent L; Bensinger, William I; Press, Oliver W

2014-02-15

The vast majority of patients with plasma cell neoplasms die of progressive disease despite high response rates to novel agents. Malignant plasma cells are very radiosensitive, but the potential role of radioimmunotherapy (RIT) in the management of plasmacytomas and multiple myeloma has undergone only limited evaluation. Furthermore, CD38 has not been explored as a RIT target despite its uniform high expression on malignant plasma cells. In this report, both conventional RIT (directly radiolabeled antibody) and streptavidin-biotin pretargeted RIT (PRIT) directed against the CD38 antigen were assessed as approaches to deliver radiation doses sufficient for multiple myeloma cell eradication. PRIT demonstrated biodistributions that were markedly superior to conventional RIT. Tumor-to-blood ratios as high as 638:1 were seen 24 hours after PRIT, whereas ratios never exceeded 1:1 with conventional RIT. (90)Yttrium absorbed dose estimates demonstrated excellent target-to-normal organ ratios (6:1 for the kidney, lung, liver; 10:1 for the whole body). Objective remissions were observed within 7 days in 100% of the mice treated with doses ranging from 800 to 1,200 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in treated mice compared with tumors that were 2,982% ± 2,834% of initial tumor volume in control animals) by day 23. Furthermore, 100% of animals bearing NCI-H929 multiple myeloma tumor xenografts treated with 800 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin achieved long-term myeloma-free survival (>70 days) compared with none (0%) of the control animals. ©2013 AACR.

Sensitive parenting is associated with plasma oxytocin and polymorphisms in the OXTR and CD38 genes.

PubMed

Feldman, Ruth; Zagoory-Sharon, Orna; Weisman, Omri; Schneiderman, Inna; Gordon, Ilanit; Maoz, Rina; Shalev, Idan; Ebstein, Richard P

2012-08-01

Research in mammals has demonstrated the involvement of oxytocin (OT) in social bond formation; yet, its role in human bonding remains unclear. Plasma OT has been used as a proxy for central activity and studies indicate its association with human affiliative behaviors. Molecular genetic studies also reveal a role for OT neuropathways in shaping the social brain. However, the links between peripheral OT, genetic markers, and their combined contribution to human parenting are unknown. Participants included 352 individuals: 272 mothers and fathers and their 4- to 6-month-old-infants and 80 nonparents. Plasma OT was assayed from adults who were genotyped for oxytocin receptor (OXTR) and CD38 risk alleles associated with social dysfunctions. CD38 is an ectoenzyme that mediates the release of brain OT. Parent-infant interactions were microcoded for parental touch and gaze synchrony and participants reported on parental care in childhood. OXTR (rs2254298 and rs1042778) and CD38 (rs3796863) risk alleles were each associated with lower plasma OT. Reduced plasma OT and both OXTR and CD38 risk alleles were related to less parental touch. The interaction of high plasma OT and low-risk CD38 alleles predicted longer durations of parent-infant gaze synchrony. Parents reporting greater parental care showed higher plasma OT, low-risk CD38 alleles, and more touch toward their infants. Results indicate that peripheral and genetic markers of the extended OT pathway are interrelated and underpin core behaviors associated with human parenting and social engagement. These findings may have important implications for understanding neuropsychiatric disorders marked by early social dysfunctions. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

PubMed

Chillemi, Antonella; Zaccarello, Gianluca; Quarona, Valeria; Ferracin, Manuela; Ghimenti, Chiara; Massaia, Massimo; Horenstein, Alberto L; Malavasi, Fabio

2013-05-20

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

Inhibiting poly(ADP-ribose) polymerase: a potential therapy against oligodendrocyte death

PubMed Central

Veto, Sara; Acs, Peter; Bauer, Jan; Lassmann, Hans; Berente, Zoltan; Setalo, Gyorgy; Borgulya, Gabor; Sumegi, Balazs; Komoly, Samuel; Gallyas, Ferenc; Illes, Zsolt

2010-01-01

Oligodendrocyte loss and demyelination are major pathological hallmarks of multiple sclerosis. In pattern III lesions, inflammation is minor in the early stages, and oligodendrocyte apoptosis prevails, which appears to be mediated at least in part through mitochondrial injury. Here, we demonstrate poly(ADP-ribose) polymerase activation and apoptosis inducing factor nuclear translocation within apoptotic oligodendrocytes in such multiple sclerosis lesions. The same morphological and molecular pathology was observed in an experimental model of primary demyelination, induced by the mitochondrial toxin cuprizone. Inhibition of poly(ADP-ribose) polymerase in this model attenuated oligodendrocyte depletion and decreased demyelination. Poly(ADP-ribose) polymerase inhibition suppressed c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation, increased the activation of the cytoprotective phosphatidylinositol-3 kinase-Akt pathway and prevented caspase-independent apoptosis inducing factor-mediated apoptosis. Our data indicate that poly(ADP-ribose) polymerase activation plays a crucial role in the pathogenesis of pattern III multiple sclerosis lesions. Since poly(ADP-ribose) polymerase inhibition was also effective in the inflammatory model of multiple sclerosis, it may target all subtypes of multiple sclerosis, either by preventing oligodendrocyte death or attenuating inflammation. PMID:20157013

Staphylococcal leukotoxins trigger free intracellular Ca2+ rise in neurones, signalling through acidic stores and activation of store-operated channels

PubMed Central

Jover, Emmanuel; Tawk, Mira Y; Laventie, Benoît-Joseph; Poulain, Bernard; Prévost, Gilles

2013-01-01

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca2+ concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca2+ indicator dye. Using pharmacological antagonists of receptors and Ca2+ channels, the variations in intracellular Ca2+ concentration were found independent of the activation of voltage-operatedCa2+ channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) or H+-ATPase and antagonists of the store-operated Ca2+ entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca2+. Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca2+ from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca2+ from lysosomes, modifies the steady-state level of reticular Ca2+ stores and finally activates the Store-Operated Calcium Entry complex. PMID:23152983

Staphylococcal leukotoxins trigger free intracellular Ca(2+) rise in neurones, signalling through acidic stores and activation of store-operated channels.

PubMed

Jover, Emmanuel; Tawk, Mira Y; Laventie, Benoît-Joseph; Poulain, Bernard; Prévost, Gilles

2013-05-01

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca(2+) concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca(2+) indicator dye. Using pharmacological antagonists of receptors and Ca(2+) channels, the variations in intracellular Ca(2+) concentration were found independent of the activation of voltage-operated Ca(2+) channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca(2+)-ATPase (SERCA) or H(+)-ATPase and antagonists of the store-operated Ca(2+) entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca(2+). Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca(2+) from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca(2+) from lysosomes, modifies the steady-state level of reticular Ca(2+) stores and finally activates the Store-Operated Calcium Entry complex. © 2012 Blackwell Publishing Ltd.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

PubMed

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

PubMed

Preugschat, Frank; Carter, Luke H; Boros, Eric E; Porter, David J T; Stewart, Eugene L; Shewchuk, Lisa M

2014-12-15

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+). Copyright © 2014 Elsevier Inc. All rights reserved.

Feasibility of controlling CD38-CAR T cell activity with a Tet-on inducible CAR design

PubMed Central

Poels, Renée; Mulders, Manon J.; van de Donk, Niels W. C. J.; Themeli, Maria; Lokhorst, Henk M.; Mutis, Tuna

2018-01-01

Recent clinical advances with chimeric antigen receptor (CAR) T cells have led to the accelerated clinical approval of CD19-CARs to treat acute lymphoblastic leukemia. The CAR T cell therapy is nevertheless associated with toxicities, especially if the CARs are not entirely tumor-specific. Therefore, strategies for controlling the CAR T cell activity are required to improve their safety profile. Here, by using the multiple myeloma (MM)-associated CD38 molecule as target molecule, we tested the feasibility and utility of a doxycycline (DOX) inducible Tet-on CD38-CAR design to control the off-target toxicities of CAR T cells. Using CARs with high affinity to CD38, we demonstrate that this strategy allows the proper induction of CD38-CARs and CAR-mediated T cell cytotoxicity in a DOX-dose dependent manner. Especially when the DOX dose was limited to 10ng/ml, its removal resulted in a relatively rapid decay of CAR- related off-tumor effects within 24 hours, indicating the active controllability of undesired CAR activity. This Tet-on CAR design also allowed us to induce the maximal anti-MM cytotoxic activity of affinity-optimized CD38-CAR T cells, which already display a low toxicity profile, hereby adding a second level of safety to these cells. Collectively, these results indicate the possibility to utilize this DOX inducible CAR-design to actively regulate the CAR-mediated activities of therapeutic T cells. We therefore conclude that the Tet-on system may be more advantageous above suicide-genes to control the potential toxicities of CAR T cells without the need to destroy them permanently. PMID:29847570

Comparative Analysis of Pharmacophore Features and Quantitative Structure-Activity Relationships for CD38 Covalent and Non-covalent Inhibitors.

PubMed

Zhang, Shuang; Xue, Xiwen; Zhang, Liangren; Zhang, Lihe; Liu, Zhenming

2015-12-01

In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non-covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure-based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure-activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non-covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non-bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross-validated correlation coefficient values (q(2)) of 0.564 and 0.571, and non-cross-validated values (r(2)) of 0.967 and 0.971, respectively. The CD38 non-covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non-covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross-validated correlation coefficient values (q(2)) of 0.469 and 0.454, and non-cross-validated values (r(2)) of 0.814 and 0.819, respectively. © 2015 John Wiley & Sons A/S.

PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

PubMed Central

Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

2010-01-01

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PMID:20228053

Bifunctional Homodimeric Triokinase/FMN Cyclase

PubMed Central

Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

2014-01-01

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

Small interfering RNA mediated Poly (ADP-ribose) Polymerase-1 inhibition upregulates the heat shock response in a murine fibroblast cell line

PubMed Central

2011-01-01

Poly (ADP-ribose) polymerase-1 (PARP-1) is a highly conserved multifunctional enzyme, and its catalytic activity is stimulated by DNA breaks. The activation of PARP-1 and subsequent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) contributes to significant cytotoxicity in inflammation of various etiologies. On the contrary, induction of heat shock response and production of heat shock protein 70 (HSP-70) is a cytoprotective defense mechanism in inflammation. Recent data suggests that PARP-1 modulates the expression of a number of cellular proteins at the transcriptional level. In this study, small interfering RNA (siRNA) mediated PARP-1 knockdown in murine wild-type fibroblasts augmented heat shock response as compared to untreated cells (as evaluated by quantitative analysis of HSP-70 mRNA and HSP-70 protein expression). These events were associated with increased DNA binding of the heat shock factor-1 (HSF-1), the major transcription factor of the heat shock response. Co-immunoprecipitation experiments in nuclear extracts of the wild type cells demonstrated that PARP-1directly interacted with HSF-1. These data demonstrate that, in wild type fibroblasts, PARP-1 plays a pivotal role in modulating the heat shock response both through direct interaction with HSF-1 and poly (ADP-ribosylation). PMID:21345219

Structural and Chemical Biology of Terpenoid Cyclases

PubMed Central

2017-01-01

The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function. PMID:28841019

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity

PubMed Central

Hess, Kenneth C.; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A.; Buck, Jochen; Levin, Lonny R.; Barrientos, Antoni

2014-01-01

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.—Hess, K. C., Liu, J., Manfredi, G., Mühlschlegel, F. A., Buck, J., Levin, L. R., Barrientos, A. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. PMID:25002117

REGULATION OF THE T-CELL RESPONSE BY CD39

PubMed Central

Takenaka, Maisa C.; Robson, Simon; Quintana, Francisco J.

2016-01-01

The ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, or CD39) catalyzes the phosphohydrolysis of extracellular adenosine triphosphate (eATP) and diphosphate (eADP) released under conditions of inflammatory stress and cell injury. CD39 generates adenosine monophosphate (AMP), which is in turn used by the ecto-5’-nucleotidase CD73 to synthesize adenosine. These ectonucleotidases have major impacts on the dynamic equilibrium of pro-inflammatory eATP and ADP nucleotides vs. immunosuppressive adenosine nucleosides. Indeed, CD39 plays a dominant role in the purinergic regulation of inflammation and the immune response because its expression is influenced by genetic and environmental factors. Here, we review the specific role of CD39 in the kinetic regulation of cellular immune responses in the evolution of disease. We focus on the effects of CD39 on T cells and explore potential clinical applications in autoimmunity, chronic infections and cancer. PMID:27236363

Calmodulin-regulated adenylyl cyclases and neuromodulation.

PubMed

Xia, Z; Storm, D R

1997-06-01

Coincidence detection and crosstalk between signal transduction systems play very important regulatory roles in the nervous system, particularly in the regulation of transcription. Coupling of the Ca2+ and cAMP regulatory systems by calmodulin-regulated adenylyl cyclases is hypothesized to be important for some forms of synaptic plasticity, neuroendocrine function, and olfactory detection. Recent studies of a mutant mouse deficient in type I calmodulin-sensitive adenylyl cyclase have provided the first evidence that adenylyl cyclases are important for synaptic plasticity, as well as for learning and memory in vertebrates.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in zebrafish models of nephrotic syndrome

PubMed Central

van den Heuvel, Lambertus P.; Khodaparast, Laleh; Khodaparast, Ladan; van Geet, Chris; Freson, Kathleen

2017-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish. PMID:28759637

Methotrexate induces poly(ADP-ribose) polymerase-dependent, caspase 3-independent apoptosis in subsets of proliferating CD4+ T cells.

PubMed

Nielsen, C H; Albertsen, L; Bendtzen, K; Baslund, B

2007-05-01

The mechanism of action of methotrexate (MTX) in autoimmune diseases (AID) is unclear. A pro-apoptotic effect has been demonstrated in mitogen-stimulated peripheral blood mononuclear cells (PBMC), but studies employing conventional antigens have disputed a pro-apoptotic effect. CD4+ T helper (Th) cells play a significant role in most AID. We therefore examined directly, by flow cytometry, the uptake of MTX by the T helper (Th) cells stimulated for 6 days with Candida albicans (CA) or tetanus toxoid (TT), and its consequences with respect to induction of apoptosis. While none of the resting Th cells took up MTX, nearly all the dividing Th cells did, and this abrogated further cell division. Among dividing Th cells, MTX induced an approximately sixfold increase over baseline levels in the proportion of apoptotic cells. This proportion could be reverted to baseline by the addition of folic acid. Exposure of CA-stimulated PBMC to MTX significantly increased their level of cleaved poly(ADP-ribose) polymerase (PARP), and a similar tendency was observed in TT-stimulated cells. Unlike CA and TT, the mitogen phytohaemagglutinin (PHA) induced proliferation of both CD4- and CD4+ T cells, and induced apoptosis in both undivided and divided Th cells. PHA-induced apoptosis involved activation of caspase-3 and the anti-apoptotic protein Bcl-2 in addition to PARP cleavage, suggesting that PHA induces apoptosis via different pathways than CA and TT. We suggest that the latter are more representative of stimulation with self-antigens in AID, and that a pro-apoptotic effect of MTX on self-antigen-stimulated Th cells contributes to the effect of MTX in the treatment of AID.

The Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

PubMed Central

Covarrubias, R; Chepurko, E; Reynolds, A; Huttinger, ZM; Huttinger, R; Stanfill, K; Wheeler, DG; Novitskaya, T; Robson, SC; Dwyer, KM; Cowan, PJ; Gumina, RJ

2016-01-01

Objective Circulating blood cells and endothelial cells express Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5’-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results To determine how CD73 activity influences in vivo thrombosis, the time to FeCl3-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared to wild-type (WT) mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in hC39-Tg/CD73-null mice. To determine if hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted WT mice, but not on WT reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared to hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis. PMID:27417582

Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

PubMed

Escande, Carlos; Nin, Veronica; Price, Nathan L; Capellini, Verena; Gomes, Ana P; Barbosa, Maria Thereza; O'Neil, Luke; White, Thomas A; Sinclair, David A; Chini, Eduardo N

2013-04-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways.

Integrative neurochemistry and neurobiology of social recognition and behavior analyzed with respect to CD38-dependent brain oxytocin secretion.

PubMed

Salmina, Alla B; Lopatina, Olga; Kuvacheva, Natalia V; Higashida, Haruhiro

2013-01-01

This review summarizes the literature and our own data regarding the role of NAD⁺-glycohydrolase/CD38-controlled molecular mechanisms of hypothalamic and pituitary oxytocin secretion in social behavior regulation. Current approaches to the modulation of both CD38 expression and brain cell activity that represent prospective treatments for disorders associated with altered social behavior are discussed.

Role of peroxynitrite and poly (ADP-ribosyl) synthetase activation in cardiovascular derangement induced by zymosan in the rat.

PubMed

Cuzzocrea, S; Zingarelli, B; Caputi, A P

1998-01-01

Peritoneal administration of zymosan in the rat induced a severe inflammatory process characterised by an increase in the plasma levels of nitrite and nitrate, stable metabolites of nitric oxide (NO) and in the levels of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, at 18 hours zymosan challenge. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the aorta of zymosan-shocked rats. In ex vivo experiments, thoracic aorta rings of zymosan-treated rats showed a reduced contraction to noradrenaline and reduced responsiveness to the relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). Treatment of zymosan-shocked rats with 3-aminobenzamide or Nicotinamide, inhibitors of poly ADP-ribosil synthetase (PARS) activity reduced the production of peroxynitrite and significantly prevented the cardiovascular dysfunction. Our data suggest that peroxynitrite and PARS activation play a role in the zymosan-induced cardiovascular derangements in the rat.

Mitochondrial Free [Ca2+] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

PubMed Central

Haumann, Johan; Dash, Ranjan K.; Stowe, David F.; Boelens, Age D.; Beard, Daniel A.; Camara, Amadou K.S.

2010-01-01

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+]m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+]m is affected by H2PO4− (Pi), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+]m, membrane potential, redox state, matrix volume, pHm, and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and Pi. Energized mitochondria showed a dose-dependent increase in [Ca2+]m after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+]e); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+]m up to twofold. The ADP effect on increasing [Ca2+]m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or Pi. Oligomycin largely reduced the increase in [Ca2+]m by ADP compared to control, and [Ca2+]m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+]m increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+]e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+]m largely due to the interaction of matrix Ca2+ with ATP, ADP, Pi, and cation buffering proteins in the matrix. PMID:20712982

Increased numbers of circulating ICOS⁺ follicular helper T and CD38⁺ plasma cells in patients with newly diagnosed primary biliary cirrhosis.

PubMed

Wang, Li; Sun, Xiguang; Qiu, Jinpeng; Cai, Yanjun; Ma, Liang; Zhao, Pingwei; Jiang, Yanfang

2015-02-01

Aberrant activation of follicular helper T (TFH) and B cells is associated with the development of autoimmune diseases. However, little is known about the potential role of these cells in the development of primary biliary cirrhosis (PBC). This study aimed at characterizing the numbers of different subsets of circulating Tfh and B cells as well as evaluating their potential association with the levels of immunoglobulins and autoantibodies in newly diagnosed PBC patients. The numbers of circulating CD27(+), CD38(+), CD86(+) and CD95(+) B cells as well as inducible T cell costimulator (ICOS)(+) and programmed death-1 (PD-1)(+), IL-21(+) TFH cells were examined in 58 patients with newly diagnosed PBC and 30 matched healthy controls (HCs). The numbers of circulating CD38(+)CD19(+), CD86(+)CD19(+), and CD95(+)CD19(+) B cells; CD3(+)CD4(+)CXCR5(+)ICOS(+) and CD3(+)CD4(+)CXCR5(+)PD-1(+) Tfh cells; and the levels of serum IL-21 in the PBC patients were significantly greater, but the numbers of CD27(+)CD19(+) B cells were significantly less than those in the HCs (p < 0.05). The numbers of CD3(+)CD4(+)CXCR5(+)ICOS(+) Tfh cells were positively correlated with the numbers of CD38(+)CD19(+) and CD86(+)CD38(+)CD19(+) B cells and the levels of serum anti-mitochondrial antibodies against M2 antigen (AMA-M2), AMA and immunolgubin M (IgM) in the PBC patients. The levels of serum IL-21 were positively correlated with the levels of serum AMA-M2, AMA, IgG and IgM, but negatively with the numbers of CD27(+)CD19(+) B cells in the PBC patients. Increased numbers of circulating ICOS(+) and IL-21(+) Tfh and CD38(+) plasma cells may be exhibited by patients with recent diagnoses of PBC.

Integrative analysis reveals CD38 as a therapeutic target for plasma cell-rich pre-disease and established rheumatoid arthritis and systemic lupus erythematosus.

PubMed

Cole, Suzanne; Walsh, Alice; Yin, Xuefeng; Wechalekar, Mihir D; Smith, Malcolm D; Proudman, Susanna M; Veale, Douglas J; Fearon, Ursula; Pitzalis, Costantino; Humby, Frances; Bombardieri, Michele; Axel, Amy; Adams, Homer; Chiu, Christopher; Sharp, Michael; Alvarez, John; Anderson, Ian; Madakamutil, Loui; Nagpal, Sunil; Guo, Yanxia

2018-05-02

Plasmablasts and plasma cells play a key role in many autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study was undertaken to evaluate the potential of targeting CD38 as a plasma cell/plasmablast depletion mechanism by daratumumab in the treatment of patients with RA and SLE. RNA-sequencing analysis of synovial biopsies from various stages of RA disease progression, flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from patients with RA or SLE and healthy donors, immunohistochemistry assessment (IHC) of synovial biopsies from patients with early RA, and ex vivo immune cell depletion assays using daratumumab (an anti-CD38 monoclonal antibody) were used to assess CD38 as a therapeutic target. We demonstrated that the plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from patients with arthralgia, undifferentiated arthritis (UA), early RA and established RA as compared to healthy controls and control patients with osteoarthritis. In addition, the highest CD38 expression was observed on plasma cells and plasmablasts compared to natural killer (NK) cells, classical dendritic cells (DCs), plasmacytoid DCs (pDCs) and T cells, in blood from healthy controls and patients with SLE and RA. Furthermore, IHC showed CD38 staining in the same region as CD3 and CD138 staining in synovial tissue biopsies from patients with early RA. Most importantly, our data show for the first time that daratumumab effectively depletes plasma cells/plasmablasts in PBMC from patients with SLE and RA in a dose-dependent manner ex vivo. These results indicate that CD38 may be a potential target for RA disease interception and daratumumab should be evaluated clinically for the treatment of both RA and SLE.

45 CFR 95.621 - ADP reviews.

Code of Federal Regulations, 2011 CFR

2011-10-01

... use; (C) Software and data security; (D) Telecommunications security; (E) Personnel security; (F... Federal review. (f) ADP System Security Requirements and Review Process—(1) ADP System Security Requirement. State agencies are responsible for the security of all ADP projects under development, and...

Regulation and therapeutic targeting of peptide-activated receptor guanylyl cyclases

PubMed Central

Potter, Lincoln R.

2016-01-01

Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP. PMID:21185863

Genetic variation in CD38 and breastfeeding experience interact to impact infants’ attention to social eye cues

PubMed Central

Krol, Kathleen M.; Monakhov, Mikhail; Lai, Poh San; Ebstein, Richard P.; Grossmann, Tobias

2015-01-01

Attending to emotional information conveyed by the eyes is an important social skill in humans. The current study examined this skill in early development by measuring attention to eyes while viewing emotional faces in 7-mo-old infants. In particular, we investigated individual differences in infant attention to eyes in the context of genetic variation (CD38 rs3796863 polymorphism) and experiential variation (exclusive breastfeeding duration) related to the oxytocin system. Our results revealed that, whereas infants at this age show a robust fear bias (increased attention to fearful eyes), their attention to angry and happy eyes varies as a function of exclusive breastfeeding experience and genetic variation in CD38. Specifically, extended exclusive breastfeeding duration selectively enhanced looking preference to happy eyes and decreased looking to angry eyes. Importantly, however, this interaction was impacted by CD38 variation, such that only the looking preferences of infants homozygous for the C allele of rs3796863 were affected by breastfeeding experience. This genotype has been associated with reduced release of oxytocin and higher rates of autism. In contrast, infants with the CA/AA genotype showed similar looking preferences regardless of breastfeeding exposure. Thus, differences in the sensitivity to emotional eyes may be linked to an interaction between the endogenous (CD38) and exogenous (breastfeeding) availability of oxytocin. These findings underline the importance of maternal care and the oxytocin system in contributing to the early development of responding to social eye cues. PMID:26371313

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

PubMed

Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

2016-06-10

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement satisfies the ADP...

Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

PubMed Central

Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos

2015-01-01

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K m and unchanged k cat of F37A-ADPRibase-Mn, while the K m values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type. PMID:25692488

Clostridium difficile binary toxin CDT

PubMed Central

Gerding, Dale N; Johnson, Stuart; Rupnik, Maja; Aktories, Klaus

2014-01-01

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed. PMID:24253566

The human NAD metabolome: Functions, metabolism and compartmentalization

PubMed Central

Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

2015-01-01

Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

Time course and strain dependence of ADP release during contraction of permeabilized skeletal muscle fibers.

PubMed

West, Timothy G; Hild, Gabor; Siththanandan, Verl B; Webb, Martin R; Corrie, John E T; Ferenczi, Michael A

2009-04-22

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of

The KRAS Promoter Responds to Myc-associated Zinc Finger and Poly(ADP-ribose) Polymerase 1 Proteins, Which Recognize a Critical Quadruplex-forming GA-element*

PubMed Central

Cogoi, Susanna; Paramasivam, Manikandan; Membrino, Alexandro; Yokoyama, Kazunari K.; Xodo, Luigi E.

2010-01-01

The murine KRAS promoter contains a G-rich nuclease hypersensitive element (GA-element) upstream of the transcription start site that is essential for transcription. Pulldown and chromatin immunoprecipitation assays demonstrate that this GA-element is bound by the Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) proteins. These proteins are crucial for transcription, because when they are knocked down by short hairpin RNA, transcription is down-regulated. This is also the case when the poly(ADP-ribosyl)ation activity of PARP-1 is inhibited by 3,4-dihydro-5-[4-(1-piperidinyl) butoxyl]-1(2H) isoquinolinone. We found that MAZ specifically binds to the duplex and quadruplex conformations of the GA-element, whereas PARP-1 shows specificity only for the G-quadruplex. On the basis of fluorescence resonance energy transfer melting and polymerase stop assays we saw that MAZ stabilizes the KRAS quadruplex. When the capacity of folding in the GA-element is abrogated by specific G → T or G → A point mutations, KRAS transcription is down-regulated. Conversely, guanidine-modified phthalocyanines, which specifically interact with and stabilize the KRAS G-quadruplex, push the promoter activity up to more than double. Collectively, our data support a transcription mechanism for murine KRAS that involves MAZ, PARP-1 and duplex-quadruplex conformational changes in the promoter GA-element. PMID:20457603

Effects of Lubrol detergents on adenylate cyclases.

PubMed

Bär, H P; Kulshrestha, S

1975-04-01

The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects onbasal and stimulated adenylate cyclases. Above concentrations of 0.001-0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibed about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer centain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

PubMed Central

Maresca, Julia A.; Graham, Joel E.; Wu, Martin; Eisen, Jonathan A.; Bryant, Donald A.

2007-01-01

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria. PMID:17606904

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

PubMed

Maresca, Julia A; Graham, Joel E; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A

2007-07-10

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Agents targeting lymphoid or myeloid cells surface antigens [II]: CD22, CD30, CD33, CD38, CD40, SLAMF-7 and CCR4).

PubMed

Drgona, L; Gudiol, C; Lanini, S; Salzberger, B; Ippolito, G; Mikulska, M

2018-03-20

The present review is part of the ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies. To review, from an Infectious Diseases perspective, the safety profile of agents targeting CD22, CD30, CD33, CD38, CD40, SLAMF-7 and CCR4 and to suggest preventive recommendations. Computer-based MEDLINE searches with MeSH terms pertaining to each agent or therapeutic family. The risk and spectrum of infections in patients receiving CD22-targeted agents (i.e. inotuzumab ozogamicin) are similar to those observed with anti-CD20 antibodies. Anti-Pneumocystis prophylaxis and monitoring for cytomegalovirus (CMV) infection is recommended for patients receiving CD30-targeted agents (brentuximab vedotin). Due to the scarcity of data, the risk posed by CD33-targeted agents (gemtuzumab ozogamicin) cannot be assessed. Patients receiving CD38-targeted agents (i.e. daratumumab) face an increased risk of varicella-zoster virus (VZV) infection. Therapy with CD40-targeted agents (lucatumumab or dacetuzumab) is associated with opportunistic infections similar to those observed in hyper-IgM syndrome, and prevention strategies (including anti-Pneumocystis prophylaxis and pre-emptive therapy for CMV infection) are warranted. SLAMF-7 (CD319)-targeted agents (elotuzumab) induce lymphopenia and increase the risk of infection (particularly due to VZV). The impact of CCR4-targeted agents (mogamulizumab) on infection susceptibility is difficult to distinguish from the effect of underlying diseases and concomitant therapies. However, anti-Pneumocystis and anti-herpesvirus prophylaxis and screening for chronic hepatitis B virus (HBV) infection are recommended. Specific management strategies should be put in place to reduce the risk and/or the severity of infectious complications associated to the reviewed agents. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 5 2013-04-01 2013-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement...

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement...

ADP--A Must in the Secondary School

ERIC Educational Resources Information Center

Majernik, John A.

1974-01-01

The rationale for including automated data processing (ADP) in secondary schools is given. ADP instruction: prepares students for data processing employment and for advanced ADP study, aids all students preparing for business careers, aids students in choosing a career, provides consumer information, and adds realism to other classroom…

Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

PubMed

Marriott, Andrew S; Copeland, Nikki A; Cunningham, Ryan; Wilkinson, Mark C; McLennan, Alexander G; Jones, Nigel J

2015-09-01

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

The guanylyl cyclase family at Y2K.

PubMed

Wedel, B; Garbers, D

2001-01-01

During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model

PubMed Central

Romeo Cardeillac, Carlos J.; Cal Castillo, Karina B.; Vilchez Larrea, Salomé C.; Sotelo Sosa, José R.; Folle Ungo, Gustavo A.; Fernández Villamil, Silvia H.

2017-01-01

Background Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. Methods Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. Results We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. Discussion The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR

Adenylyl cyclase and G-proteins in Phytomonas.

PubMed

Farber, M D; Montagna, A E; Paveto, C; Dollet, M; Sanchex-Moreno, M; Osuna, A; Torres, H N; Flawia, M M

1995-01-01

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.

Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV Cross-Linking and Luciferase Reporter Assay.

PubMed

Ji, Yingbiao

2017-01-01

The RNA-binding proteins (RBPs) play a pivotal role in controlling gene expression through posttranscriptional processes. As the trans-acting factors, RBPs interact with the cis-regulatory elements located within mRNAs to regulate mRNA translational efficiency. Adding a new-layer regulation, recent studies suggest that poly(ADP-ribosyl)ation of the RNA-binding proteins often inhibit the RNA-binding ability of RBPs, thus regulating RBP-dependent mRNA metabolism including translational control. Here, we describe a biotin-based UV cross-linking method to determine if excessive accumulation of pADPr in the cell disrupts the interaction between RBPs and their target mRNAs. In addition, we illustrate the protocol of using the luciferase reporter assay to determine the effect of poly(ADP-ribosyl)ation on mRNA translation.

Relationship of calcium and membrane guanylate cyclase in adrenocorticotropin-induced steroidogenesis.

PubMed

Nambi, P; Aiyar, N V; Roberts, A N; Sharma, R K

1982-07-01

Chlorpromazine, when incubated with isolated adrenal cells, inhibited the ACTH-stimulated formation of cGMP and corticosterone production. It also inhibited the ACTH-stimulated membrane guanylate cyclase, but did not affect the binding of ACTH to the membrane receptors. cGMP-induced steroidogenesis was not affected by the drug. These data indicate that chlorpromazine interferes with adrenal steroid metabolism at a site between the hormone receptor and guanylate cyclase and also show that guanylate cyclase is composed of separate receptor and catalytic components. Furthermore, based on the premise that chlorpromazine exerts its inhibitory action by blocking the binding of a calcium receptor protein, such as calmodulin, to the receptor-coupled guanylate cyclase, it is proposed that the interaction of calcium, presumably through a calcium-binding protein, is essential for ACTH-dependent guanylate cyclase.

Pituitary adenylate cyclase-activating polypeptide: a novel peptide with protean implications.

PubMed

Pisegna, Joseph R; Oh, David S

2007-02-01

The purpose of this review is to highlight the importance of pituitary adenylate cyclase-activating polypeptide in physiological processes and to describe how this peptide is becoming increasingly recognized as having a major role in the body. Since its discovery in 1989, investigators have sought to determine the site of biological activity and the function of pituitary adenylate cyclase-activating polypeptide in maintaining homeostasis. Since its discovery, pituitary adenylate cyclase-activating polypeptide appears to play an important role in the regulation of processes within the central nervous system and gastrointestinal tract, as well in reproductive biology. Pituitary adenylate cyclase-activating polypeptide has been shown to regulate tumor cell growth and to regulate immune function through its effects on T lympocytes. These discoveries suggest the importance of pituitary adenylate cyclase-activating polypeptide in neuronal development, neuronal function, gastrointestinal tract function and reproduction. Future studies will examine more closely the role of pituitary adenylate cyclase-activating polypeptide in regulation of malignantly transformed cells, as well as in regulation of immune function.

Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent mu to gamma1 class switch recombination in CD38-activated B cells.

PubMed

Tsukamoto, Yumiko; Uehara, Shoji; Mizoguchi, Chieko; Sato, Atsushi; Horikawa, Keisuke; Takatsu, Kiyoshi

2005-10-21

Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to mu to gamma1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce mu to gamma1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces mu to gamma1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce mu to gamma1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce mu to gamma1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR.

DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.

PubMed

Cohen-Armon, Malka; Visochek, Leonid; Rozensal, Dana; Kalal, Adi; Geistrikh, Ilona; Klein, Rodika; Bendetz-Nezer, Sarit; Yao, Zhong; Seger, Rony

2007-01-26

PolyADP-ribose polymerases (PARPs) catalyze a posttranslational modification of nuclear proteins by polyADP-ribosylation. The catalytic activity of the abundant nuclear protein PARP-1 is stimulated by DNA strand breaks, and PARP-1 activation is required for initiation of DNA repair. Here we show that PARP-1 also acts within extracellular signal-regulated kinase (ERK) signaling cascade that mediates growth and differentiation. The findings reveal an alternative mode of PARP-1 activation, which does not involve binding to DNA or DNA damage. In a cell-free system, recombinant PARP-1 was intensively activated and thereby polyADP-ribosylated by a direct interaction with phosphorylated ERK2, and the activated PARP-1 dramatically increased ERK2-catalyzed phosphorylation of the transcription factor Elk1. In cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes, PARP-1 activation enhanced ERK-induced Elk1-phosphorylation, core histone acetylation, and transcription of the Elk1-target gene c-fos. These findings constitute evidence for PARP-1 activity within the ERK signal-transduction pathway.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

PubMed Central

Skopova, Karolina; Tomalova, Barbora; Kanchev, Ivan; Rossmann, Pavel; Svedova, Martina; Adkins, Irena; Bibova, Ilona; Tomala, Jakub; Masin, Jiri; Guiso, Nicole; Osicka, Radim; Sedlacek, Radislav; Kovar, Marek

2017-01-01

ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. PMID:28396322

ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

PubMed

Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

2016-08-02

ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

Structural basis for lack of ADP-ribosyltransferase activity in poly(ADP-ribose) polymerase-13/zinc finger antiviral protein.

PubMed

Karlberg, Tobias; Klepsch, Mirjam; Thorsell, Ann-Gerd; Andersson, C David; Linusson, Anna; Schüler, Herwig

2015-03-20

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual...(k)(3)(F), the ADP test is performed under the plan (determined without regard to disaggregation...

26 CFR 1.401(k)-2 - ADP test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 5 2011-04-01 2011-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual...(k)(3)(F), the ADP test is performed under the plan (determined without regard to disaggregation...

The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

PubMed Central

Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

2015-01-01

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

Increased expression of activation antigens on CD8+ T lymphocytes in Myalgic Encephalomyelitis/chronic fatigue syndrome: inverse associations with lowered CD19+ expression and CD4+/CD8+ ratio, but no associations with (auto)immune, leaky gut, oxidative and nitrosative stress biomarkers.

PubMed

Maes, Michael; Bosmans, Eugene; Kubera, Marta

2015-01-01

There is now evidence that specific subgroups of patients with Myalgic Encephalomyelitis / chronic fatigue syndrome (ME/CFS) suffer from a neuro-psychiatric-immune disorder. This study was carried out to delineate the expression of the activation markers CD38 and human leukocyte antigen (HLA) DR on CD4+ and CD8+ peripheral blood lymphocytes in ME/CFS. Proportions and absolute numbers of peripheral lymphocytes expressing CD3+, CD19+, CD4+, CD8+, CD38+ and HLA-DR+ were measured in ME/CFS (n=139), chronic fatigue (CF, n=65) and normal controls (n=40). The proportions of CD3+, CD8+, CD8+CD38+ and CD8+HLA-DR+ were significantly higher in ME/CFS patients than controls, while CD38+, CD8+CD38+, CD8+HLA-DR+ and CD38+HLA-DR+ were significantly higher in ME/CFS than CF. The percentage of CD19+ cells and the CD4+/CD8+ ratio were significantly lower in ME/CFS and CF than in controls. There were highly significant inverse correlations between the increased expression of CD38+, especially that of CD8+CD38+, and the lowered CD4+/CD8+ ratio and CD19+ expression. There were no significant associations between the flow cytometric results and severity or duration of illness and peripheral blood biomarkers of oxidative and nitrosative stress (O&NS, i.e. IgM responses to O&N modified epitopes), leaky gut (IgM or IgA responses to LPS of gut commensal bacteria), cytokines (interleukin-1, tumor necrosis factor-α), neopterin, lysozyme and autoimmune responses to serotonin. The results support that a) increased CD38 and HLA-DR expression on CD8+ T cells are biomarkers of ME/CFS; b) increased CD38 antigen expression may contribute to suppression of the CD4+/CD8+ ratio and CD19+ expression; c) there are different immune subgroups of ME/CFS patients, e.g. increased CD8+ activation marker expression versus inflammation or O&NS processes; and d) viral infections or reactivation may play a role in a some ME/CFS patients.

45 CFR 95.621 - ADP reviews.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false ADP reviews. 95.621 Section 95.621 Public Welfare....621 ADP reviews. The Department will conduct periodic onsite surveys and reviews of State and local... the Department and State or local agencies prior to conducting such surveys or reviews, which may...

Bordetella Adenylate Cyclase-Hemolysin Toxins

PubMed Central

Guiso, Nicole

2017-01-01

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

General base-general acid catalysis by terpenoid cyclases.

PubMed

Pemberton, Travis A; Christianson, David W

2016-07-01

Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

PubMed

Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

2015-08-07

The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Adenylate cyclase from rabbit heart: substrate binding site].

PubMed

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

Adenovirus Death Protein (ADP) Is Required for Lytic Infection of Human Lymphocytes

PubMed Central

Murali, V. K.; Ornelles, D. A.; Gooding, L. R.; Wilms, H. T.; Huang, W.; Tollefson, A. E.; Wold, W. S. M.

2014-01-01

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection. PMID:24198418

Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

PubMed Central

Bitensky, M W; Wheeler, M A; Mehta, H; Miki, N

1975-01-01

Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from, and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD. PMID:1058474

Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34+CD38- stem and progenitor cells in chronic myeloid leukemia.

PubMed

Dolinska, Monika; Piccini, Alexandre; Wong, Wan Man; Gelali, Eleni; Johansson, Anne-Sofie; Klang, Johannis; Xiao, Pingnan; Yektaei-Karin, Elham; Strömberg, Ulla Olsson; Mustjoki, Satu; Stenke, Leif; Ekblom, Marja; Qian, Hong

2017-08-19

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL + CD34 + CD38 - cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34 + CD38 - cells from 7 CML patients. The majority of the single leukemic BCR-ABL + CD34 + CD38 - cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

PubMed

Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

2017-02-01

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. Copyright © 2017 American Society for Microbiology.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

PubMed Central

Truong-Bolduc, Q. C.; Khan, N. S.

2016-01-01

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. PMID:27956597

Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1988-01-01

The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

CD40 engagement on dendritic cells induces cyclooxygenase-2 and EP2 receptor via p38 and ERK MAPKs.

PubMed

Harizi, Hedi; Limem, Ilef; Gualde, Norbert

2011-02-01

We have previously reported that cyclooxygenase (COX)-2-derived prostaglandin (PG)E2 critically regulates dendritic cell (DC) inflammatory phenotype and function through EP2/EP4 receptor subtypes. As genes activated by CD40 engagement are directly relevant to inflammation, we examined the effects of CD40 activation on inflammatory PGs in murine bone marrow-derived DC (mBM-DC). We showed for the first time that activation of mBM-DC with agonist anti-CD40 monoclonal antibody (anti-CD40 mAb) dose dependently induces the synthesis of significant amounts of PGE2 via inducible expression of COX-2 enzyme, as NS-398, a COX-2-selective inhibitor reduces this upregulation. In contrast to lipopolysaccharide, which upregulates mBM-DC surface levels of EP2 and EP4 receptors, CD40 crosslinking on mBM-DC increases EP2, but not EP4, receptor expression. Flow cytometry analysis and radioligand-binding assay showed that EP2 was the major EP receptor subtype, which binds to PGE2 at the surface of anti-CD40-activated mBM-DC. Upregulation of COX-2 and EP2 levels by CD40 engagement was accompanied by dose-dependent phosphorylation of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) and was abrogated by inhibitors of both pathways. Collectively, we demonstrated that CD40 engagement on mBM-DC upregulates COX-2 and EP2 receptor expression through activation of p38 and ERK1/2 MAPK signaling. Triggering the PGE2/EP2 pathway by anti-CD40 mAb resulted on the induction of Th2 immune response. Thus, CD40-induced production of PGE2 by mBM-DC could represent a negative feedback mechanism involving EP2 receptor and limiting the propagation of Th1 responses. Blocking CD40 pathway may represent a novel therapeutic pathway of inhibiting COX-2-derived prostanoids in chronically inflamed tissues (that is, arthritis).

Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

PubMed

Stiles, Bradley G; Pradhan, Kisha; Fleming, Jodie M; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R

2014-09-05

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.

Modification of adenylate cyclase by photoaffinity analogs of forskolin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, L.T.; Nie, Z.M.; Mende, T.J.

1989-01-01

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography followingmore » SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.« less

Enhanced levels of soluble CD40 ligand exacerbate platelet aggregation and thrombus formation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway.

PubMed

Yacoub, Daniel; Hachem, Ahmed; Théorêt, Jean-François; Gillis, Marc-Antoine; Mourad, Walid; Merhi, Yahye

2010-12-01

CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40(-/-) mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor-associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40(-/-) mice. sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases.

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encodedmore » by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.« less

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

PubMed Central

Yoshimura, M; Cooper, D M

1992-01-01

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+. Images PMID:1379717

Downregulated expression of the cyclase-associated protein 1 (CAP1) reduces migration in esophageal squamous cell carcinoma.

PubMed

Li, Mei; Yang, Xiaojing; Shi, Hui; Ren, Hanru; Chen, Xueyu; Zhang, Shu; Zhu, Junya; Zhang, Jianguo

2013-09-01

Overexpression of cyclase-associated proteins has been associated with poor prognosis in several human cancers. Cyclase-associated protein 1 is a member of the cyclase-associated proteins which contributes to tumor progression. The aim of the present study was to examine the expression of cyclase-associated protein 1 and to elucidate its clinicopathologic significance in a larger series of esophageal squamous cell carcinoma. Immunohistochemical and western blot analyses were performed in esophageal squamous cell carcinoma tissues. Survival analyses were performed by using the Kaplan-Meier method. The role of cyclase-associated protein 1 in migration was studied in esophageal squamous cell carcinoma cell lines of TE1 through knocking down cyclase-associated protein 1 with siRNA and overexpression of cyclase-associated protein 1. The regulation of cyclase-associated protein 1 on migration was determined by transwell and wound-healing assays. Immunohistochemical analysis showed that cyclase-associated protein 1 expression was negatively associated with E-cadherin and significantly associated with lymph node metastases. Survival analysis revealed that cyclase-associated protein 1 overexpression was significantly associated with overall survival (P = 0.011). Knock down of cyclase-associated protein 1 in TE1 cells resulted in decreased vimentin and F-actin levels and the capability for migration. In addition, overexpression of cyclase-associated protein 1 promoted the migration of TE1 cells. These findings suggest that cyclase-associated protein 1 is involved in the metastasis of esophageal squamous cell carcinoma, and that elevated levels of cyclase-associated protein 1 expression may indicate a poor prognosis for patients with esophageal squamous cell carcinoma.

Neuroblastoma differentiation involves the expression of two isoforms of the alpha-subunit of Go.

PubMed

Brabet, P; Pantaloni, C; Rodriguez, M; Martinez, J; Bockaert, J; Homburger, V

1990-04-01

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.

Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer

NASA Astrophysics Data System (ADS)

Yang, Zhe; Luo, Huiyan; Cao, Zhong; Chen, Ya; Gao, Jinbiao; Li, Yingqin; Jiang, Qing; Xu, Ruihua; Liu, Jie

2016-06-01

Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases andmore » bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.« less

General Base-General Acid Catalysis by Terpenoid Cyclases§

PubMed Central

Pemberton, Travis A.; Christianson, David W.

2016-01-01

Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains. PMID:27072285

Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

PubMed

Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

2013-06-01

Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of age and posture on human lymphocyte adenylate cyclase activity.

PubMed

Mader, S L; Robbins, A S; Rubenstein, L Z; Tuck, M L; Scarpace, P J

1988-03-01

1. A number of age-related changes have been reported in the catecholamine-adrenoceptor-adenylate cyclase system. Most of the data available on these alterations come from resting subjects; the response to acute stress may provide additional insights into the age effect on these responses. 2. We measured supine and 10 min upright plasma noradrenaline and lymphocyte adenylate cyclase activity in ten healthy elderly subjects (age 66-80 years) and seven healthy young subjects (age 27-34 years). 3. Isoprenaline stimulation of lymphocyte adenylate cyclase activity was not significantly different between supine and upright positions or between elderly and young subjects. There was a marked increase in forskolin-stimulated adenylate cyclase activity in the upright posture in both elderly and young subjects. The increment over supine levels was 70% in the elderly (P less than 0.025) and 73% in the young (P less than 0.05). This enhanced forskolin activity was not seen in two young subjects who became syncopal. 4. These data suggest that enhanced forskolin-stimulated adenylate cyclase activity occurs after 10 min of upright posture in both elderly and young subjects, and may be relevant to immediate blood pressure regulation. We were unable to demonstrate any age-related differences in these acute adrenergic responses.

ADP Analysis project for the Human Resources Management Division

NASA Technical Reports Server (NTRS)

Tureman, Robert L., Jr.

1993-01-01

The ADP (Automated Data Processing) Analysis Project was conducted for the Human Resources Management Division (HRMD) of NASA's Langley Research Center. The three major areas of work in the project were computer support, automated inventory analysis, and an ADP study for the Division. The goal of the computer support work was to determine automation needs of Division personnel and help them solve computing problems. The goal of automated inventory analysis was to find a way to analyze installed software and usage on a Macintosh. Finally, the ADP functional systems study for the Division was designed to assess future HRMD needs concerning ADP organization and activities.

Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

PubMed Central

Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

2012-01-01

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

Therapeutic Approaches to the Treatment of Botulism

DTIC Science & Technology

1989-10-01

of the clostridial neurotoxins (last report). It must now be reported that there is another unusual quality to the data on interactions. As just...636-640, 1986. Matsuoka, I., Syuto, B., Kurihara, K. and Kubo, S.: ADP- ribosylation of specific membrane proteins in pheochromocytoma and primary

Therapeutic Approaches to the Treatment of Botulism

DTIC Science & Technology

1987-10-01

dependence of various preparations of type C toxin. An absence of toxin-induced pa alysis, or an unusually long time for onset of paralysis, at low...Kurihara, K. and Kubo, S.: ADP- ribosylation of specific membrane proteins in pheochromocytoma and primary-cultured brain cells by botulinum neurotoxins

Toll-like receptor 7 cooperates with IL-4 in activated B cells through antigen receptor or CD38 and induces class switch recombination and IgG1 production.

PubMed

Tsukamoto, Yumiko; Nagai, Yoshinori; Kariyone, Ai; Shibata, Takuma; Kaisho, Tsuneyasu; Akira, Shizuo; Miyake, Kensuke; Takatsu, Kiyoshi

2009-04-01

IL-4 and 8-mercaptoguanosine (8-SGuo) stimulation of CD38-activated B cells induces mu to gamma1 class switch recombination (CSR) at the DNA level leading to a high level of IgG1 production. Although some of signaling events initiated by IL-4 in activated B cells have been characterized, the involvement of TLR/MyD88 and Btk pathway in IL-4-dependent mu to gamma1 CSR has not been thoroughly evaluated. In this study, we characterized receptors for 8-SGuo and differential roles of 8-SGuo and IL-4 in the induction and mu to gamma1 CSR and IgG1 production. The role of TLR7 and MyD88 in 8-SGuo-induced AID expression and mu to gamma1 CSR was documented, as 8-SGuo did not act on CD38-stimulated splenic B cells from Tlr7(-/-) and Myd88(-/-) mice. CD38-activated B cells from Btk-deficient mice failed to respond to TLR7 ligands for the AID expression and CSR, indicating that Btk is also indispensable for the system. Stimulation of CD38-activated B cells with 8-SGuo induced significant AID expression and DNA double strand breaks, but IL-4 stimulation by itself did not trigger mu to gamma1 CSR. Intriguingly, the mu to gamma1 CSR in the B cells stimulated with CD38 and 8-SGuo totally depends on IL-4 stimulation. Similar results were obtained in the activated B cells through BCR and loxoribine, a well-known TLR7 ligand, in place of 8-SGuo. In vivo administration of TLR7 ligand and anti-CD38 antibody induced the generation of CD138(+) IgG1(+) cells. These results indicate that TLR7 is a receptor for 8-SGuo and plays an essential role in the AID and Blimp-1 expression; however it is not enough to complete mu to gamma1 CSR in CD38-activated B cells. IL-4 may be required for the induction of DNA repair system together with AID for the completion of CSR.

Pituitary adenylate cyclase-activating polypeptide is a potent broad-spectrum antimicrobial peptide: Structure-activity relationships.

PubMed

Starr, Charles G; Maderdrut, Jerome L; He, Jing; Coy, David H; Wimley, William C

2018-06-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring cationic peptide with potent immunosuppressant and cytoprotective activities. We now show that full length PACAP38 and to a lesser extent, the truncated form PACAP27, and the closely related vasoactive intestinal peptide (VIP) and secretin had antimicrobial activity against the Gram-negative bacteria Escherichia coli in the radial diffusion assay. PACAP38 was more potent than either the bovine neutrophil antimicrobial peptide indolicidin or the synthetic antimicrobial peptide ARVA against E. coli. PACAP38 also had activity against the Gram-positive bacteria Staphylococcus aureus in the same assay with comparable potency to indolicidin and ARVA. In the more stringent broth dilution assay, PACAP38 had moderate sterilizing activity against E. coli, and potent sterilizing activity against the Gram-negative bacteria Pseudomonas aeruginosa. PACAP27, VIP and secretin were much less active than PACAP38 in this assay. PACAP38 also had some activity against the Gram-positive bacteria Bacillus cereus in the broth dilution assay. Many exopeptidase-resistant analogs of PACAP38, including both receptor agonists and antagonists, had antimicrobial activities equal to, or better than PACAP38, in both assays. PACAP38 made the membranes of E. coli permeable to SYTOX Green, suggesting a classical membrane lytic mechanism. These data suggest that analogs of PACPAP38 with a wide range of useful biological activities can be made by judicious substitutions in the sequence. Copyright © 2018 Elsevier Inc. All rights reserved.

Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masure, H.R.; Donovan, M.G.; Storm, D.R.

1991-01-01

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increasesmore » in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.« less

The temperature-dependence of adenylate cyclase from baker's yeast.

PubMed Central

Londesborough, J; Varimo, K

1979-01-01

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism. PMID:391221

Requirement of GM2 ganglioside activator for phospholipase D activation

PubMed Central

Nakamura, Shun-ichi; Akisue, Toshihiro; Jinnai, Hitoshi; Hitomi, Tomohiro; Sarkar, Sukumar; Miwa, Noriko; Okada, Taro; Yoshida, Kimihisa; Kuroda, Shun’ichi; Kikkawa, Ushio; Nishizuka, Yasutomi

1998-01-01

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown. PMID:9770472

Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

PubMed Central

Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

2014-01-01

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

PubMed Central

Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Membrane guanylate cyclase is a beautiful signal transduction machine: overview.

PubMed

Sharma, Rameshwar K

2010-01-01

This article is a sequel to the four earlier comprehensive reviews which covered the field of membrane guanylate cyclase from its origin to the year 2002 (Sharma in Mol Cell Biochem 230:3-30, 2002) and then to the year 2004 (Duda et al. in Peptides 26:969-984, 2005); and of the Ca(2+)-modulated membrane guanylate cyclase to the year 1997 (Pugh et al. in Biosci Rep 17:429-473, 1997) and then to 2004 (Sharma et al. in Curr Top Biochem Res 6:111-144, 2004). This article contains three parts. The first part is "Historical"; it is brief, general, and freely borrowed from the earlier reviews, covering the field from its origin to the year 2004 (Sharma in Mol Cell Biochem, 230:3-30, 2002; Duda et al. in Peptides 26:969-984, 2005). The second part focuses on the "Ca(2+)-modulated ROS-GC membrane guanylate cyclase subfamily". It is divided into two sections. Section "Historical" and covers the area from its inception to the year 2004. It is also freely borrowed from an earlier review (Sharma et al. in Curr Top Biochem Res 6:111-144, 2004). Section "Ca(2+)-modulated ROS-GC membrane guanylate cyclase subfamily" covers the area from the year 2004 to May 2009. The objective is to focus on the chronological development, recognize major contributions of the original investigators, correct misplaced facts, and project on the future trend of the field of mammalian membrane guanylate cyclase. The third portion covers the present status and concludes with future directions in the field.

Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

PubMed

Hungen, K V; Roberts, S; Hill, D F

1975-08-22

Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

PubMed Central

Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

2016-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat.

PubMed

Deguchi, T; Amano, E; Nakane, M

1976-11-01

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.

Trial watch – inhibiting PARP enzymes for anticancer therapy

PubMed Central

Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

2016-01-01

ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed Central

Adams, A F; Haslam, R J

1978-01-01

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed

Adams, A F; Haslam, R J

1978-07-15

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by

The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

PubMed Central

Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

1972-01-01

Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

Dysfunction of outer segment guanylate cyclase caused by retinal disease related mutations

PubMed Central

Zägel, Patrick; Koch, Karl-Wilhelm

2014-01-01

Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca2+-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca2+-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca2+-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases. PMID:24616660

Over-expression of CD8+ T-cell activation is associated with decreased CD4+ cells in patients seeking treatment of Alcohol Use Disorder.

PubMed

Zuluaga, Paola; Sanvisens, Arantza; Martínez-Cáceres, Eva; Teniente, Aina; Tor, Jordi; Muga, Robert

2017-11-01

Harmful alcohol consumption may have an impact on the adaptive immune system through an imbalance in T cell subpopulations and changes in cell activation. We aimed to analyze profiles of CD4 and CD8T cell activation in patients with alcohol use disorder (AUD). We used a cross-sectional study with patients seeking treatment of the disorder. Blood samples for immunophenotyping were obtained at admission. Profiles of T cell activation were defined: (I) CD38 + /HLA-DR + , (II) CD38 + /HLA-DR - , (III) CD38 - /HLA-DR + , (IV) CD38 - /HLA-DR - and compared with healthy controls. We calculated a CD8 + T cell activation indicator (AI) that was defined as the quotient of non-activated cells (CD38 - /HLA-DR - ) and activated cells (CD38 + /HLA-DR + ). 60 patients were eligible (83%M); median age was 49 years [IQR: 44-54] and alcohol consumption was 145g/day [IQR: 90-205]. Mean±SD of CD38 + /HLA-DR - was 50.3±50.6 cells/μL in patients and 33.5±24.5 cells/μL in controls (p=0.03), for the CD38 - /HLA-DR + it was 61±62.2 cells/μL in patients and 21.2±17.3 cells/μL in controls (p<0.001) and for the CD38 + /HLA-DR + it was 20.2±15.6 cells/μL in patients and 10.8±10.3 cells/μL in controls (p<0.001). In patients, an inverse correlation was observed between absolute number and percentage of CD4 + T cells, and the percentage of CD38 + /HLA-DR + CD8 + T cells (r=0.37, p=0.003; r=0.2, p=0.086, respectively). Patients with AUD have an increased expression of immune activation with respect to healthy individuals. This excess of activated CD8 + T cells correlates with the absolute CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Daratumumab resistance is frequent in advanced-stage multiple myeloma patients irrespective of CD38 expression and is related to dismal prognosis.

PubMed

Pick, Marjorie; Vainstein, Vladimir; Goldschmidt, Neta; Lavie, David; Libster, Diana; Gural, Alexander; Grisariu, Sigal; Avni, Batia; Ben Yehuda, Dina; Gatt, Moshe E

2018-05-01

Daratumumab is a promising new antimyeloma agent. We report a single center "real-world" series of multiple myeloma (MM) and amyloidosis (AL) patients treated with daratumumab. Forty-one patients were included: 7 second-line MM, 30 heavily pretreated (median number of therapies of 5) advanced MM, and 4 with AL. Second-line patients and advanced AL showed high rate of durable overall responses. However, advanced MM patients had a dismal prognosis with an overall response rate (ORR) of 36%, and a short median progression-free and overall survival of 2.3 and 6.6 months, respectively. Responses were particularly poor in patients with extramedullary plasmacytomas. Neither the addition of another agent to daratumumab nor changing to the next line of therapy produced significant durable responses in this patient population. Flow cytometry analysis demonstrated that CD38 expression level was not predictive of response. We show that CD38 expression dynamics by a commercially available anti-CD38 antibody after daratumumab administration was hindered by competitive binding of daratumumab. Responses to daratumumab and combinations in patients with advanced MM, particularly with extramedullary disease, are low and short-lived, stressing the administration of this agent should be early in the course of the disease. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparison of the effects of histamine and tolazoline on adenylate cyclase activity from guinea pig heart.

PubMed

Weinryb, I; Michel, I M

1975-01-01

Both histamine and tolazoline (2-benzyl-2-imidazoline) stimulated particulate fractions of adenylate cyclase from guinea pig myocardium. Tolazoline was one-tenth as potent, and about two-thirds as active at maximally effective levels, as was histamine. Enhancement of cyclase activity by tolazoline was additive with that by isoproterenol, and the histamine and tolazoline concentration-response curves were parallel, suggesting that tolazoline acted at the same site as histamine. At maximally effective concentrations, tolazoline did not affect ATPase or cyclic AMP phosphodiesterase activities associated with the cyclase preparations. The H1-receptor antagonist mepyramine, and the H2 antagonist, burimamide, blocked stimulation of cyclase by tolazoline at one-tenth the molarity of agonist. Both antagonists were less effective vs. histamine stimulation of heart cyclase in particulate fractions or whole homogenates, with mepyramine being generally more potent. It is suggested that the molecular basis of the stimulatory effect of tolazoline on cardiac tissue may be histaminergic stimulation of adenylate cyclase. Furthermore, the lack of potency of burimamide as a histamine antagonist and its lack of specificity compared to mepyramine, at the subcellular level, indicate that histamine-responsive adenylate cyclase from heart may not be a satisfactory molecular model for the H2 receptor pharmacology of histamine in cardiac tissue.

Interaction of CD38 Variant and Chronic Interpersonal Stress Prospectively Predicts Social Anxiety and Depression Symptoms Over Six Years

PubMed Central

Tabak, Benjamin A.; Vrshek-Schallhorn, Suzanne; Zinbarg, Richard E.; Prenoveau, Jason M.; Mineka, Susan; Redei, Eva E.; Adam, Emma K.; Craske, Michelle G.

2015-01-01

Variation in the CD38 gene, which regulates secretion of the neuropeptide oxytocin, has been associated with several social phenotypes. Specifically, rs3796863 A allele carriers have demonstrated increased social sensitivity. In 400 older adolescents, we used trait-state-occasion modeling to investigate how rs3796863 genotype, baseline ratings of chronic interpersonal stress, and their gene-environment (GxE) interaction predicted trait social anxiety and depression symptoms over six years. We found significant GxE effects for CD38 A-carrier genotypes and chronic interpersonal stress at baseline predicting greater social anxiety and depression symptoms. A significant GxE effect of smaller magnitude was also found for C/C genotype and chronic interpersonal stress predicting greater depression; however, this effect was small compared to the main effect of chronic interpersonal stress. Thus, in the context of chronic interpersonal stress, heightened social sensitivity associated with the rs3796863 A allele may prospectively predict risk for social anxiety and (to a lesser extent) depression. PMID:26958455

Action of Escherichia coli Enterotoxin: Adenylate Cyclase Behavior of Intestinal Epithelial Cells in Culture

PubMed Central

Kantor, Harvey S.; Tao, Pearl; Wisdom, Charlene

1974-01-01

Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model. PMID:4364505

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Calcium modulates the ATP and ADP hydrolysis in human placental mitochondria.

PubMed

Martínez, Federico; Uribe, Aida; Espinosa-García, M Teresa; Flores-Herrera, Oscar; García-Pérez, Cecilia; Milán, Rebeca

2002-08-01

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.

Increased CD39 Nucleotidase Activity on Microparticles from Patients with Idiopathic Pulmonary Arterial Hypertension

PubMed Central

Visovatti, Scott H.; Hyman, Matthew C.; Bouis, Diane; Neubig, Richard; McLaughlin, Vallerie V.; Pinsky, David J.

2012-01-01

Background Idiopathic pulmonary arterial hypertension (IPAH) is a devastating disease characterized by increased pulmonary vascular resistance, smooth muscle and endothelial cell proliferation, perivascular inflammatory infiltrates, and in situ thrombosis. Circulating intravascular ATP, ADP, AMP and adenosine activate purinergic cell signaling pathways and appear to induce many of the same pathologic processes that underlie IPAH. Extracellular dephosphorylation of ATP to ADP and AMP occurs primarily via CD39 (ENTPD1), an ectonucleotidase found on the surface of leukocytes, platelets, and endothelial cells [1]. Microparticles are micron-sized phospholipid vesicles formed from the membranes of platelets and endothelial cells. Objectives: Studies here examine whether CD39 is an important microparticle surface nucleotidase, and whether patients with IPAH have altered microparticle-bound CD39 activity that may contribute to the pathophysiology of the disease. Methodology/ Principal Findings Kinetic parameters, inhibitor blocking experiments, and immunogold labeling with electron microscopy support the role of CD39 as a major nucleotidase on the surface of microparticles. Comparison of microparticle surface CD39 expression and nucleotidase activity in 10 patients with advanced IPAH and 10 healthy controls using flow cytometry and thin layer chromatograph demonstrate the following: 1) circulating platelet (CD39+CD31+CD42b+) and endothelial (CD39+CD31+CD42b−) microparticle subpopulations in patients with IPAH show increased CD39 expression; 2) microparticle ATPase and ADPase activity in patients with IPAH is increased. Conclusions/ Significance We demonstrate for the first time increased CD39 expression and function on circulating microparticles in patients with IPAH. Further research is needed to elucidate whether these findings identify an important trigger for the development of the disease, or reflect a physiologic response to IPAH. PMID:22792409

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

PubMed

Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Lean, A.; Cantin, M.

1986-03-05

The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levelsmore » of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.« less

Effect of nitroso complexes of some transition metals on the activity of soluble guanylate cyclase.

PubMed

Severina, I S; Bussygina, O G; Grigorjev, N B

1992-03-01

Effects of nitroso complexes of some transition metals (Fe, Co, Cr), differing in the character of NO oxidation on the activity of human and rat platelet guanylate cyclase were studied. 3 types of nitroso complexes were used: (1) NO group carries a positive charge--a nitrosonium cation (Na2[FeNO + (CN)5]-nitroprusside); (2) NO is neutral--(K3[CrNO(CN)5 and [CoNO(NH3)5]SO4) and (3) NO is coordinated as anion NO- (K3[CoNO-(CN)5]. It is shown that the highest stimulatory effect is produced by sodium nitroprusside, whose activating action is due to the interaction of its NO group with the guanylate cyclase heme. Nitroso complexes (Co and Cr) the NO group of which is neutral stimulated guanylate cyclase activity insignificantly and this activation was not guanylate cyclase heme directed. Nitroso complex (Co) with NO coordinated as anion NO(-)--is a guanylate cyclase inhibitor. In contrast to nitroprusside, the nitroso complexes used (Co and Cr) have no hypotensive effect. It was concluded that the essential requirement for the realization of the hypotensive effect of transition metals' nitroso complexes is the ability of these compounds to activate soluble guanylate cyclase solely by the heme-dependent mechanism.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

NASA Astrophysics Data System (ADS)

Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

2017-06-01

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Bosak, T.; Pearson, A.; Losick, R.

2005-12-01

Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

Modification of opiate agonist binding by pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-03-05

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin.more » This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.« less

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

PubMed

Rovira, Alexander R; Fin, Andrea; Tor, Yitzhak

2017-11-08

The synthesis, photophysics, and biochemical utility of a fluorescent NAD + analogue based on an isothiazolo[4,3-d]pyrimidine core (N tz AD + ) are described. Enzymatic reactions, photophysically monitored in real time, show N tz AD + and N tz ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N tz AD + is converted to N tz ADH, reflecting a complementary photophysical behavior to that of the native NAD + /NADH. N tz AD + and N tz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. N tz AD + also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tz ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD + .

Dual functional bioactive-peptide, AIMP1-derived peptide (AdP), for anti-aging.

PubMed

Kim, Jina; Kang, Sujin; Kwon, HanJin; Moon, HoSang; Park, Min Chul

2018-06-19

Human skin aging is caused by several factors, such as UV irradiation, stress, hormone, and pollution. Wrinkle formation and skin pigmentation are representative features of skin aging. Although EGF and arbutin are used as anti-wrinkle and skin whitening agents, respectively, they have adverse effects on skin. When more cosmeceutical ingredients are added to cosmetic product, adverse effects are also accumulated. For these reasons, multifunctional and safe cosmetic ingredients are in demand. The aim of the present study is to investigate the novel anti-aging agents, AIMP1-derived peptide (AdP, INCI name: sh-oligopeptide-5/sh-oligopeptide SP) for cosmetic products. To assess the anti-wrinkle effect of AdP, collagen type I synthesis and fibroblast proliferation were determined on human fibroblasts. The anti-wrinkle effect of AdP was examined by ELISA and cell titer glo assay. To assess the whitening, melanin content and tyrosinase activity were determined on melanocytes. The whitening effect of AdP was examined by melanin measurement and enzyme activity assay. The safety of AdP was determined by cytotoxicity and immunogenicity, CCK-8 and TNF-α ELISA assay, respectively. AdP treatment induced the collagen type I synthesis and fibroblast proliferation. Also, AdP treatment inhibited melanin synthesis by regulating tyrosinase activity. The anti-aging effect of AdP is more potent than EGF and albutin. AdP did not show adverse effects. These results show that AdP can be dual functional and safe cosmeceutical agent to prevent skin aging. © 2018 Wiley Periodicals, Inc.

Evidence for a role of the oxytocin system, indexed by genetic variation in CD38, in the social bonding effects of expressed gratitude

PubMed Central

2014-01-01

Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological ‘glue’ to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships. PMID:24396004

Adenylyl cyclase G is activated by an intramolecular osmosensor.

PubMed

Saran, Shweta; Schaap, Pauline

2004-03-01

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers.

PubMed

Karatzaferi, Christina; Myburgh, Kathryn H; Chinn, Marc K; Franks-Skiba, Kathleen; Cooke, Roger

2003-04-01

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, approximately 0.04 N/mm(2), in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still approximately 0.04 N/mm(2). The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K(i), of approximately 240 and 280 microM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.

ADP's ABCs of Training

ERIC Educational Resources Information Center

Weinstein, Margery

2010-01-01

When a company's core competence is processing data, it is sometimes easy to lose sight of the obvious--the information right under its nose. In the case of Automatic Data Processing, Inc. (ADP), a business outsourcing company specializing in human resources, payroll, tax, and benefits administrations solutions, that is not a problem. Through…

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.

PubMed

Blain-Hartung, Matthew; Rockwell, Nathan C; Moreno, Marcus V; Martin, Shelley S; Gan, Fei; Bryant, Donald A; Lagarias, J Clark

2018-06-01

Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleus sp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

Role of CD81 and CD58 in minimal residual disease detection in pediatric B lymphoblastic leukemia.

PubMed

Tsitsikov, E; Harris, M H; Silverman, L B; Sallan, S E; Weinberg, O K

2018-06-01

Minimal residual disease (MRD) in B lymphoblastic leukemia has been demonstrated to be a powerful predictor of clinical outcome in numerous studies in both children and adults. In this study, we evaluated 86 pediatric patients with both diagnostic and remission flow cytometry studies and compared expression of CD81, CD58, CD19, CD34, CD20, and CD38 in the detection of MRD. We evaluated 86 patients with B lymphoblastic leukemia who had both diagnostic studies and remission studies for the presence of MRD using multicolor flow cytometry. We established our detection limit for identifying abnormal lymphoblasts using serial dilutions. We also compared flow cytometry findings with molecular MRD detection in a subset of patients. We found that we can resolve differences between hematogones and lymphoblasts in 85 of 86 cases using a combination of CD45, CD19, CD34, CD10, CD20, CD38, CD58, and CD81. Our detection limit using flow cytometry is 0.002% for detecting a population of abnormal B lymphoblasts. Comparison with MRD assessment by molecular methods showed a high concordance rate with flow cytometry findings. Our study highlights importance of using multiple markers to detect MRD in B lymphoblastic leukemia. Our findings indicate that including both CD58 and CD81 markers in addition to CD19, CD34, CD20, CD38, and CD10 are helpful in MRD detection by flow cytometry. © 2018 John Wiley & Sons Ltd.

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies.

PubMed

Wong, J T; Pinto, C E; Gifford, J D; Kurnick, J T; Kradin, R L

1989-11-15

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36

PubMed Central

Munkonda, Mercedes N.; Akbari, Shareef; Landry, Chloe; Sun, Suzy; Xiao, Fengxia; Turner, Maddison; Holterman, Chet E.; Nasrallah, Rania; Hébert, Richard L.; Kennedy, Christopher R. J.; Burger, Dylan

2018-01-01

ABSTRACT Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100–1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-β) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease. PMID:29435202

Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okajima, F.; Sho, K.; Kondo, Y.

1988-08-01

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrationsmore » below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.« less

Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial.

PubMed

Wayne, Alan S; Kreitman, Robert J; Findley, Harry W; Lew, Glen; Delbrook, Cynthia; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Fitzgerald, David J; Pastan, Ira

2010-03-15

Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. Lymphoblasts from children with B-lineage ALL were assessed for CD22 expression by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non-Hodgkin lymphoma. All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC(50), 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 microg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood.

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

PubMed Central

Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

2013-01-01

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

PubMed Central

Sequeira, Vasco; Najafi, Aref; McConnell, Mark; Fowler, Ewan D; Bollen, Ilse A E; Wüst, Rob C I; dos Remedios, Cris; Helmes, Michiel; White, Ed; Stienen, Ger J M; Tardiff, Jil; Kuster, Diederik W D; van der Velden, Jolanda

2015-01-01

Abstract Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca2+] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca2+ handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca2+] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca2+ both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca2+] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca2+ overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca2+]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca2+, and thereby increase myocardial stiffness. Key points Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin

ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

PubMed Central

Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

2014-01-01

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs. PMID:24391508

ADP1 affects plant architecture by regulating local auxin biosynthesis.

PubMed

Li, Ruixi; Li, Jieru; Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

2014-01-01

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

Evidence for a role of the oxytocin system, indexed by genetic variation in CD38, in the social bonding effects of expressed gratitude.

PubMed

Algoe, Sara B; Way, Baldwin M

2014-12-01

Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological 'glue' to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships. © The Author (2014). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Nitric oxide (NO), the only nitrogen monoxide redox form capable of activating soluble guanylyl cyclase.

PubMed

Dierks, E A; Burstyn, J N

1996-06-28

In the present study, we determined that of the redox forms of nitrogen monoxide, NO-, NO and NO+, only NO significantly activates soluble guanylyl cyclase (GTP pyrophosphate-lyase cyclizing, EC 4.6.1.2). Neither of the NO-donors tested, Angeli's salt (Na2N2O3) or Piloty's acid (C6H5SO2NHOH), caused a change in the guanylyl cyclase activity relative to the basal activity level. Interference by other reaction products was eliminated as a possible explanation for the lack of activation. To the extent that NO+ could be stabilized in aqueous solution, by dissolution of the nitrosonium salt NOPF6 in dry organic solvent prior to addition to the enzyme in buffer, NO+ had no effect on the activity of soluble guanylyl cyclase. The counter-ion, PF6-, had a minimal effect on the enzyme activity and, therefore was, not responsible for the lack of activation by NO+. These observations suggest that NO- is the natural activator of soluble guanylyl cyclase and is reasonably identical with endothelium-derived relaxing factor, the physiological regulator of soluble guanylyl cyclase activity.

Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase.

PubMed Central

Waldman, S A; Kuno, T; Kamisaki, Y; Chang, L Y; Gariepy, J; O'Hanley, P; Schoolnik, G; Murad, F

1986-01-01

A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

PubMed Central

Purohit, Rahul; Weichsel, Andrzej; Montfort, William R

2013-01-01

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ∼150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction. PMID:23934793

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Effects of Forskolin on Kupffer Cell Production of Interleukin-10 and Tumor Necrosis Factor Alpha Differ from Those of Endogenous Adenylyl Cyclase Activators: Possible Role for Adenylyl Cyclase 9

PubMed Central

Dahle, Maria K.; Myhre, Anders E.; Aasen, Ansgar O.; Wang, Jacob E.

2005-01-01

Proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-α expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and β-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 μg/ml) caused attenuated TNF-α levels in culture medium (forskolin/isoproterenol, P ≤ 0.05; prostaglandin E2, P ≤ 0.01). Forskolin also reduced IL-10 mRNA and protein (P ≤ 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P ≤ 0.05). We conclude that regulation of TNF-α and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2 differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9. PMID:16239525

Nicotinamide megadosing increases hepatic poly(ADP-ribose) levels in choline-deficient rats.

PubMed

ApSimon, M M; Rawling, J M; Kirkland, J B

1995-07-01

Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)polymerase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 x 3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

Defense ADP Acquisition Study.

DTIC Science & Technology

1981-11-30

Logistics ALS - Advanced Logistics System AMP - ADPS Master Plan ANSI - American National Standards Institute APR - Agency Procurement Request ASD(C...Computers IRM - Information Resources Management ISO - International Standards Organization L LCC - Life Cycle Costs LCM - Life Cycle Management LE...man- agement in the process * Lack of a mission orientation . Lack of systems management and life cycle perspectives * Lack of effective leadership

Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

PubMed

Cao, Tian-Jun; Huang, Xing-Qi; Qu, Yuan-Yuan; Zhuang, Zhong; Deng, Yin-Yin; Lu, Shan

2017-04-11

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene ( BfLCYB ) encoding a lycopene β-cyclase from Bangia fuscopurpurea , a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

PubMed

Chen, S; Lane, A P; Bock, R; Leinders-Zufall, T; Zufall, F

2000-07-01

Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

Monitoring of the ADP/ATP Ratio by Induced Circularly Polarised Europium Luminescence.

PubMed

Shuvaev, Sergey; Fox, Mark A; Parker, David

2018-06-18

A series of three europium complexes bearing picolyl amine moieties was found to possess differing binding affinities towards Zn 2+ and three nucleotides: AMP, ADP, and ATP. A large increase in the total emission intensity was observed upon binding Zn 2+ , followed by signal amplification upon the addition of nucleotides. The resulting adducts possessed strong induced circularly polarised emission, with ADP and ATP signals of opposite sign. Model DFT geometries of the adducts suggest the Δ diastereoisomer is preferred for ATP and the Λ isomer for ADP/AMP. This change in sign allows the ADP/ATP (or AMP/ATP) ratio to be assessed by monitoring changes in the emission dissymmetry factor, g em . © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue.

PubMed

Macdonald, W A; Stephenson, D G

2006-05-15

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue

PubMed Central

Macdonald, W A; Stephenson, D G

2006-01-01

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 μm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (−log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 μm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 μm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres. PMID:16556653

The Role of Cargo Proteins in GGA Recruitment

PubMed Central

Hirst, Jennifer; Seaman, Matthew N J; Buschow, Sonja I; Robinson, Margaret S

2007-01-01

Coat proteins are recruited onto membranes to form vesicles that transport cargo from one compartment to another, but the extent to which the cargo helps to recruit the coat proteins is still unclear. Here we have examined the role of cargo in the recruitment of Golgi-localized, γ-ear-containing, ADP ribosylation factor (ARF)-binding proteins (GGAs) onto membranes in HeLa cells. Moderate overexpression of CD8 chimeras with cytoplasmic tails containing DXXLL-sorting signals, which bind to GGAs, increased the localization of all three GGAs to perinuclear membranes, as observed by immunofluorescence. GGA2 was also expressed at approximately twofold higher levels in these cells because it was degraded more slowly. However, this difference only partially accounted for the increase in membrane localization because there was a approximately fivefold increase in GGA2 associated with crude membranes and a ∼12-fold increase in GGA2 associated with clathrin-coated vesicles (CCVs) in cells expressing CD8-DXXLL chimeras. The effect of cargo proteins on GGA recruitment was reconstituted in vitro using permeabilized control and CD8-DXXLL-expressing cells incubated with cytosol containing recombinant GGA2 constructs. Together, these results demonstrate that cargo proteins contribute to the recruitment of GGAs onto membranes and to the formation of GGA-positive CCVs. PMID:17451558

Pituitary adenylate cyclase-activating polypeptide stimulates glucose production via the hepatic sympathetic innervation in rats.

PubMed

Yi, Chun-Xia; Sun, Ning; Ackermans, Mariette T; Alkemade, Anneke; Foppen, Ewout; Shi, Jing; Serlie, Mireille J; Buijs, Ruud M; Fliers, Eric; Kalsbeek, Andries

2010-07-01

The unraveling of the elaborate brain networks that control glucose metabolism presents one of the current challenges in diabetes research. Within the central nervous system, the hypothalamus is regarded as the key brain area to regulate energy homeostasis. The aim of the present study was to investigate the hypothalamic mechanism involved in the hyperglycemic effects of the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP). Endogenous glucose production (EGP) was determined during intracerebroventricular infusions of PACAP-38, vasoactive intestinal peptide (VIP), or their receptor agonists. The specificity of their receptors was examined by coinfusions of receptor antagonists. The possible neuronal pathway involved was investigated by 1) local injections in hypothalamic nuclei, 2) retrograde neuronal tracing from the thoracic spinal cord to hypothalamic preautonomic neurons together with Fos immunoreactivity, and 3) specific hepatic sympathetic or parasympathetic denervation to block the autonomic neuronal input to liver. Intracerebroventricular infusion of PACAP-38 increased EGP to a similar extent as a VIP/PACAP-2 (VPAC2) receptor agonist, and intracerebroventricular administration of VIP had significantly less influence on EGP. The PACAP-38 induced increase of EGP was significantly suppressed by preinfusion of a VPAC2 but not a PAC1 receptor antagonist, as well as by hepatic sympathetic but not parasympathetic denervation. In the hypothalamus, Fos immunoreactivity induced by PACAP-38 was colocalized within autonomic neurons in paraventricular nuclei projecting to preganglionic sympathetic neurons in the spinal cord. Local infusion of PACAP-38 directly into the PVN induced a significant increase of EGP. This study demonstrates that PACAP-38 signaling via sympathetic preautonomic neurons located in the paraventricular nucleus is an important component in the hypothalamic control of hepatic glucose production.

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji

2009-05-08

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependentmore » on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.« less

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-08-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed Central

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-01-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes. Images PMID:2545625

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

PubMed Central

Oguchi, Yusuke; Mikhailenko, Sergey V.; Ohki, Takashi; Olivares, Adrian O.; De La Cruz, Enrique M.; Ishiwata, Shin'ichi

2008-01-01

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions. PMID:18509050

ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase

PubMed Central

Mayer, Faith V.; Heath, Richard; Underwood, Elizabeth; Sanders, Matthew J.; Carmena, David; McCartney, Rhonda R.; Leiper, Fiona C.; Xiao, Bing; Jing, Chun; Walker, Philip A.; Haire, Lesley F.; Ogrodowicz, Roksana; Martin, Stephen R.; Schmidt, Martin C.; Gamblin, Steven J.; Carling, David

2011-01-01

Summary The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK. PMID:22019086

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases.

PubMed

Cunningham, F X; Gantt, E

2001-02-27

Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein). Carotenoids with two epsilon-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings. An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

PubMed Central

Cunningham, Francis X.; Gantt, Elisabeth

2001-01-01

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures. PMID:11226339

Control of Herbaspirillum seropedicae NifA Activity by Ammonium Ions and Oxygen

PubMed Central

Souza, E. M.; Pedrosa, F. O.; Drummond, M.; Rigo, L. U.; Yates, M. G.

1999-01-01

The activity of a truncated form of Herbaspirillum seropedicae NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O2 sensitivity or Fe dependence. The model for nitrogen control involving PII could thus apply to the proteobacteria at large. NifA may have a role in controlling ADP-ribosylation of nitrogenase in Azospirillum brasilense. PMID:9882688

Detection of Cholera Toxin by Optical Methods: A Mechanism-Based Approach to the Generic Detection of Protein Toxins.

DTIC Science & Technology

1997-04-01

Reaction of NAD and NBAG ......................... 9 2 Time Course of ADP-Ribosylation of LAME and Agmatine with eNAD and Cholera Toxin Fragment A...11 TABLES 1 Retention of eNAD by Dowex 1 . ................................ 13 2 Reaction of LAME and Agmatine with Fluorochromes...14 3 Sensitivity of Detection of LAME and Agmatine by Fluorescamine ......... 15 4 Retention of LAME and Agmatine by Dowex 50

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

PubMed

Zhang, Jian; Li, Xiaoling; Kwansa, Herman; Kim, Yun Tai; Yi, Liye; Hong, Gina; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M; Koehler, Raymond C; Yang, Zeng-Jin

2017-06-01

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

PubMed Central

Gong, M C; Iizuka, K; Nixon, G; Browne, J P; Hall, A; Eccleston, J F; Sugai, M; Kobayashi, S; Somlyo, A V; Somlyo, A P

1996-01-01

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase. Images Fig. 3 Fig. 4 PMID:8577766

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

PubMed Central

Rotcheewaphan, Suwatchareeporn; Belisle, John T.; Webb, Kristofor J.; Kim, Hee-Jin; Spencer, John S.

2016-01-01

The second messenger, bis-(3′,5′)-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host–pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography–mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae. PMID:27450520

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

PubMed

Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

2016-09-01

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

Characterization of the toxin Plx2A, a RhoA-targeting ADP-ribosyltransferase produced by the honey bee pathogen Paenibacillus larvae.

PubMed

Ebeling, Julia; Fünfhaus, Anne; Knispel, Henriette; Krska, Daniel; Ravulapalli, Ravikiran; Heney, Kayla A; Lugo, Miguel R; Merrill, A Rod; Genersch, Elke

2017-12-01

The toxin Plx2A is an important virulence factor of Paenibacillus larvae, the etiological agent of American Foulbrood, the most destructive bacterial disease of honey bees. Biochemical and functional analyses as well as the crystal structure of Plx2A revealed that it belongs to the C3 mono-ADP-ribosylating toxin subgroup. RhoA was identified as the cellular target of Plx2A activity. The kinetic parameters (K M , k cat ) were established for both the transferase and glycohydrolase reactions. When expressed in yeast, Plx2A was cytotoxic for eukaryotic cells and catalytic variants confirmed that the cytotoxicity of Plx2A depends on its enzymatic activity. The crystal structure of Plx2A was solved to 1.65 Å and confirmed that it is a C3-like toxin, although with a new molecular twist, it has a B-domain. A molecular model of the 'active' enzyme conformation in complex with NAD + was produced by computational methods based on the recent structure of C3bot1 with RhoA. In murine macrophages, Plx2A induced actin cytoskeleton reorganization while in insect cells, vacuolization and the occurrence of bi-nucleated cells was observed. The latter is indicative of an inhibition of cytokinesis. All these cellular effects are consistent with Plx2A inhibiting the activity of RhoA by covalent modification. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Analytical Design Package (ADP2): A computer aided engineering tool for aircraft transparency design

NASA Technical Reports Server (NTRS)

Wuerer, J. E.; Gran, M.; Held, T. W.

1994-01-01

The Analytical Design Package (ADP2) is being developed as a part of the Air Force Frameless Transparency Program (FTP). ADP2 is an integrated design tool consisting of existing analysis codes and Computer Aided Engineering (CAE) software. The objective of the ADP2 is to develop and confirm an integrated design methodology for frameless transparencies, related aircraft interfaces, and their corresponding tooling. The application of this methodology will generate high confidence for achieving a qualified part prior to mold fabrication. ADP2 is a customized integration of analysis codes, CAE software, and material databases. The primary CAE integration tool for the ADP2 is P3/PATRAN, a commercial-off-the-shelf (COTS) software tool. The open architecture of P3/PATRAN allows customized installations with different applications modules for specific site requirements. Integration of material databases allows the engineer to select a material, and those material properties are automatically called into the relevant analysis code. The ADP2 materials database will be composed of four independent schemas: CAE Design, Processing, Testing, and Logistics Support. The design of ADP2 places major emphasis on the seamless integration of CAE and analysis modules with a single intuitive graphical interface. This tool is being designed to serve and be used by an entire project team, i.e., analysts, designers, materials experts, and managers. The final version of the software will be delivered to the Air Force in Jan. 1994. The Analytical Design Package (ADP2) will then be ready for transfer to industry. The package will be capable of a wide range of design and manufacturing applications.

Delayed effects of rhG-CSF mobilization treatment and apheresis on circulating CD34+ and CD34+ Thy-1dim CD38- progenitor cells, and lymphoid subsets in normal stem cell donors for allogeneic transplantation.

PubMed

Körbling, M; Anderlini, P; Durett, A; Maadani, F; Bojko, P; Seong, D; Giralt, S; Khouri, I; Andersson, B; Mehra, R; vanBesien, K; Mirza, N; Przepiorka, D; Champlin, R

1996-12-01

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) is emerging as a new stem cell transplant modality. Rather than undergoing general anesthesia for bone marrow harvest, normal blood stem cell donors are subjected to rhG-CSF mobilization treatment followed by single or multiple apheresis. Whereas the effects of cytokine treatment and apheresis on stem cell peripheralization and collection have been described, little is known about delayed effects of rhG-CSF treatment and apheresis on a normal hematopoietic system, and there are no long-term data that address safety issues. Ten normal, patient-related donors underwent a 3 or 4 day rhG-CSF (filgrastim) treatment (12 micrograms/kg/day) followed by single or tandem apheresis. We monitored peripheral blood (PB) cellularity including CD34+ and lymphoid subsets at baseline, during cytokine treatment, prior to apheresis, and at days 2, 4, 7, 30 and 100 post-apheresis. The PB progenitor cell concentration peak prior to apheresis was followed by a nadir by day 7 and normalized by day 30, with the exception of the most primitive CD34+ Thy-1dim CD38- progenitor subset that reached a nadir by day 30. Lymphoid subsets such as CD3, 4, 8, suppressor cells (CD3+ 4- 8- TCR+ alpha beta), and B cells (CD19+) showed a similar pattern with a nadir concentration by day 7, followed, except for B cells, by a rebound by day 30 and subnormal counts at day 100. The PB concentrations of hemoglobin and platelets dropped mainly due to the apheresis procedure itself, and normalized by day 30. With cytokine treatment, the PB alkaline phosphatase and lactate dehydrogenase concentrations increased 2.2- and 2.8-fold, respectively, over baseline, and returned to normal range by day 30. Based on the preliminary nature of this study, the clinical relevance of these findings is still unclear.

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

1987-06-25

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, M.; Allison, W.S.

1986-05-05

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with (/sup 3/H)ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. (/sup 3/H)ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with (/sup 3/H)ADP in 30more » min with a Kd of 30 microM. (/sup 3/H)ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of (/sup 3/H)ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. (/sup 3/H)ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits.« less

Adenylyl cyclases in the digestive system

PubMed Central

Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

2015-01-01

Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca2+ and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. PMID:24521753

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

PubMed Central

Suzuki, Kano; Mizutani, Kenji; Maruyama, Shintaro; Shimono, Kazumi; Imai, Fabiana L.; Muneyuki, Eiro; Kakinuma, Yoshimi; Ishizuka-Katsura, Yoshiko; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamato, Ichiro; Murata, Takeshi

2016-01-01

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model. PMID:27807367

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

PubMed Central

Skogerson, L; Wakatama, E

1976-01-01

Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. PMID:174100

Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

PubMed

Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

2018-06-01

Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Convenient approaches to synthesis of furanoid sugar-aza-crown ethers from C-ribosyl azido aldehyde via a reductive amination/amidation.

PubMed

Hsieh, Yu-Chi; Chir, Jiun-Ly; Zou, Wei; Wu, Hsiu-Han; Wu, An-Tai

2009-05-26

A short and highly efficient route to the alpha-anomer of a furanoid sugar-aza-crown ether was developed by a one-pot reductive amination of an alpha-anomer C-ribosyl azido aldehyde. In addition, the beta-anomer furanoid sugar-aza-crown ether was synthesized from a linear disaccharide precursor via amidation and then followed by microwave-assisted amide reduction.

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

PubMed

Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

2012-07-31

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelali, Ala

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional proteinmore » 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

PubMed

Etzl, Stefan; Lindner, Robert; Nelson, Matthew D; Winkler, Andreas

2018-06-08

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties. © 2018 Etzl et al.

Are genetic variations in OXTR, AVPR1A, and CD38 genes important to social integration? Results from two large U.S. cohorts.

PubMed

Chang, Shun-Chiao; Glymour, M Maria; Rewak, Marissa; Cornelis, Marilyn C; Walter, Stefan; Koenen, Karestan C; Kawachi, Ichiro; Liang, Liming; Tchetgen Tchetgen, Eric J; Kubzansky, Laura D

2014-01-01

Some evidence suggests that genetic polymorphisms in oxytocin pathway genes influence various social behaviors, but findings thus far have been mixed. Many studies have been based in small samples and there is possibility of publication bias. Using data from 2 large U.S. prospective cohorts with over 11,000 individuals, we investigated 88 SNPs in OXTR, AVPR1A, and CD38, in relation to social integration (measured as social connectedness in both binary and continuous forms and being continuously married). After correction for multiple testing only one SNP in CD38 (rs12644506) was significantly associated with social integration and that SNP predicted when using a dichotomized indicator of social connectedness (adjusted p=0.02), but not a continuous measure of social connectedness or the continuously married outcome. A significant gender-heterogeneous effect was identified in one OXTR SNP on dichotomized social connectedness; specifically, rs4686302 T allele was nominally associated with social connectedness in men, whereas the association direction was opposite in women (adjusted gender heterogeneity p=0.02). Furthermore, the rs53576 A allele was significantly associated with social connectedness only in women, and the effect magnitude was stronger in a dominant genetic model (adjusted p=0.003). In summary, our findings suggested that common genetic variants of OXTR, CD38, and AVPR1A are not associated with social integration as measured in this study using the simplified Berkman-Syme Social Network Index, but these findings and other work hint that effects may be modified by gender or other social experiences. Further work considering genetic pathways in relation to social integration may be more fruitful if these additional factors can be more comprehensively evaluated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Are genetic variations in OXTR, AVPR1A, and CD38 genes important to social integration? Results from two large U.S. cohorts

PubMed Central

Chang, Shun-Chiao; Glymour, M Maria; Rewak, Marissa; Cornelis, Marilyn; Walter, Stefan; Koenen, Karestan C; Kawachi, Ichiro; Liang, Liming; Tchetgen, Eric Tchetgen; Kubzansky, Laura D.

2013-01-01

Some evidence suggests that genetic polymorphisms in oxytocin pathway genes influence various social behaviors, but findings thus far have been mixed. Many studies have been based in small samples and there is possibility of publication bias. Using data from 2 large U.S. prospective cohorts with over 11,000 individuals, we investigated 88 SNPs in OXTR, AVPR1A, and CD38, in relation to social integration (measured as social connectedness in both binary and continuous forms and being continuously married). After correction for multiple testing only one SNP in CD38 (rs12644506) was significantly associated with social integration and that SNP predicted when using a dichotomized indicator of social connectedness (adjusted p=0.02), but not a continuous measure of social connectedness or the continuously married outcome. A significant gender-heterogeneous effect was identified in one OXTR SNP on dichotomized social connectedness; specifically, rs4686302 T allele was nominally associated with social connectedness in men, whereas the association direction was opposite in women (adjusted gender heterogeneity p=0.02). Furthermore, the rs53576 A allele was significantly associated with social connectedness only in women, and the effect magnitude was stronger in a dominant genetic model (adjusted p=0.003). In summary, our findings suggested that common genetic variants of OXTR, CD38, and AVPR1A are not associated with social integration as measured in this study using the simplified Berkman-Syme Social Network Index, but these findings and other work hint that effects may be modified by gender or other social experiences. Further work considering genetic pathways in relation to social integration may be more fruitful if these additional factors can be more comprehensively evaluated. PMID:24209975

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

PubMed Central

Barnett, J. E. G.; Rasheed, A.; Corina, D. L.

1973-01-01

After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105. PMID:4352864

An ADPE Protest Primer: Lessons Learned from GSBCA Protest Decisions

DTIC Science & Technology

1991-06-01

reverse if necessary and identify, by block number) The General services Administration’s Board of Contract Appeals (GSBCA) is a significant venue for...David R. Whipple,i D ep a rtm en t of A d i i t a i eS ci ce iim ABSTRACT The General Services Administration’s Board of Contract Appeals (GSBCA) is a...Administration Board of Contract Appeals (GSBCA) ADPE protest decisions. In effect this study will serve as a primer to familiarize new Federal ADPE

CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways.

PubMed

Li, Jibin; Huang, Qichao; Long, Xiaoyu; Zhang, Jing; Huang, Xiaojun; Aa, Jiye; Yang, Hushan; Chen, Zhinan; Xing, Jinliang

2015-12-01

CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive. We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms. Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells. Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Elevated IL-17A and IL-22 regulate expression of inducible CD38 and Zap-70 in chronic lymphocytic leukemia.

PubMed

Kouzegaran, Samaneh; Siroosbakht, Soheila; Farsad, Bahram Fariborz; Rezakhaniha, Bijan; Dormanesh, Banafshe; Behnod, Vahid; Tanha, Amir Saber

2018-01-01

In this study, we investigated the role and expression of interleukin (IL)-17A and IL-22 in chronic lymphocytic leukemia. We evaluated the expression of markers above on CLL by ELISA, qRT-PCR, flow cytometric analysis and nonparametric Kruskal-Wallis test. Quantitative RT-PCR revealed that the mRNA levels of IL-17A and IL-22 in PBMCs of CLL patients were upregulated compared with those from healthy subjects (mean ± SD: 1.96 ± 0.232 vs.0.72 ± 0.15, P < 0.001 and mean ± SD: 2.45 ± 0.534 vs.0.81 ± 0.26, P < 0.001, respectivily). In addition, findings showed that the IL-17A and IL-22 plasma level was significantly elevated than that from healthy control group (P  < 0.001). The median IL-17A and IL-22 in CLL patients and healthy control group were 48.28 ± 17.2 pg mL -1 ; 20.01 ± 11.16 pg mL -1 and 58.68 ± 23.4 pg mL -1 ;16.47 ± 10.31 P < 0.001, respectively. The levels of IL-17A and IL-22 was not significantly associated with the different stages of disease (Rai stages; Kruskal-Wallis test P > 0.05).No significant relationship was found between expression of CD38 and higher median serum levels of IL-17A in patients, but patients with negative expression of ZAP-70 showed a significant association with higher median serum levels of IL-17A compared with healthy subjects. (57.84 pg mL -1 vs. 31.67 pg mL -1 ; P = 0.016). IL-22 is elevated and associated with CD38 and Zap-70 expression in patients with CLL. No significant correlation was found between expression of CD38 and increased levels of IL-17A, negative expression of ZAP-70 showed a significant association with increased levels of IL-17A. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Study of linear optical parameters of sodium sulphide nano-particles added ADP crystals

NASA Astrophysics Data System (ADS)

Kochuparampil, A. P.; Joshi, J. H.; Dixit, K. P.; Jethva, H. O.; Joshi, M. J.

2017-05-01

Ammonium Dihydrogen Phosphate (ADP) is one of the nonlinear optical crystals. It is having various applications like optical mixing, electro-optical modulator, harmonic generators, etc. Chalcogenide compounds are poorly soluble in water and difficult to add in the water soluble ADP crystals. The solubility of Chalcogenide compounds can be increased by synthesizing the nano-structured samples with suitable capping agent. In the present study sodium sulphide was added in to ADP to modify its linear optical parameters. Sodium sulphide nano particles were synthesized by co-precipitation technique using Ethylene diamine as capping agent followed by microwave irradiation. The powder XRD confirmed the nano-structured nature of sodium sulphide nano particles. The solubility of nanoparticles of sodium sulphide increased significantly in water compared to the bulk. Pure and Na2S added ADP crystals were grown by slow solvent evaporation method at room temperature. The presence of sodium in ADP was confirmed by AAS. The UV-Vis spectra were recorded for all crystals. Various optical parameters like, transmittance, energy band gap, extinction coefficient, refractive index, optical conductivity, etc. were evaluated. The electronic polarizibility of pure and doped crystals calculated from energy band gap. The effect of doping concentration was found on various parameters.

Adenylyl cyclases in the digestive system.

PubMed

Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

2014-06-01

Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Blockade of CD26 signaling inhibits human osteoclast development.

PubMed

Nishida, Hiroko; Suzuki, Hiroshi; Madokoro, Hiroko; Hayashi, Mutsumi; Morimoto, Chikao; Sakamoto, Michiie; Yamada, Taketo

2014-11-01

Bone remodeling is maintained by the delicate balance between osteoblasts (OBs) and osteoclasts (OCs). However, the role of CD26 in regulating bone remodeling has not yet been characterized. We herein show that CD26 is preferentially expressed on normal human OCs and is intensely expressed on activated human OCs in osteolytic bone alterations. Macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (sRANKL) induced human OC differentiation, in association with CD26 expression on monocyte-macrophage lineage cells. CD26 expression was accompanied by increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which is crucial for early human OC differentiation. The humanized anti-CD26 monoclonal antibody, huCD26mAb, impaired the formation and function of tartrate-resistant acid phosphatase (TRAP)/CD26 positive multi-nucleated (nuclei > 3) OCs with maturation in the manner of dose-dependency. It was revealed that huCD26mAb inhibits early OC differentiation via the inactivation of MKK3/6, p38 MAPK and subsequent dephosphorylation of microphthalmia-associated transcription factor (mi/Mitf). These inhibitions occur immediately after RANKL binds to RANK on the human OC precursor cells and were demonstrated using the OC functional assays. huCD26mAb subsequently impaired OC maturation and bone resorption by suppressing the expression of TRAP and OC fusion proteins. In addition, p38 MAPK inhibitor also strongly inhibited OC formation and function. Our results suggest that the blockade of CD26 signaling impairs the development of human functional OCs by inhibiting p38 MAPK-mi/Mitf phosphorylation pathway and that targeting human OCs with huCD26mAb may have therapeutic potential for the treatment of osteolytic lesions following metastasis to alleviate bone destruction and reduce total skeletal-related events (SREs). © 2014 American Society for Bone and Mineral Research.

FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms.

PubMed

Mita, Tomohiro; Furuhashi, Masato; Hiramitsu, Shinya; Ishii, Junnichi; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Tanaka, Marenao; Ohno, Kohei; Akasaka, Hiroshi; Ohnishi, Hirofumi; Yoshida, Hideaki; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

2015-02-01

Fatty acid-binding protein 4 (FABP4) is expressed in adipocytes, and elevated plasma FABP4 level is associated with obesity-mediated metabolic phenotype. Postprandial regulation and secretory signaling of FABP4 has been investigated. Time courses of FABP4 levels were examined during an oral glucose tolerance test (OGTT; n=53) or a high-fat test meal eating (n=35). Effects of activators and inhibitors of adenyl cyclase (AC)-protein kinase A (PKA) signaling and guanylyl cyclase (GC)-protein kinase G (PKG) signaling on FABP4 secretion from mouse 3T3-L1 adipocytes were investigated. FABP4 level significantly declined after the OGTT or a high-fat meal eating, while insulin level was increased. Treatment with low and high glucose concentration or palmitate for 2 h did not affect FABP4 secretion from 3T3-L1 adipocytes. FABP4 secretion was increased by stimulation of lipolysis using isoproterenol, a β3 -adrenoceptor agonist (CL316243), forskolin, dibutyryl-cAMP and atrial natriuretic peptide, and the induced FABP4 secretion was suppressed by insulin or an inhibitor of PKA (H-89), PKG (KT5823) or hormone sensitive lipase (CAY10499). FABP4 is secreted from adipocytes in association with lipolysis regulated by AC-PKA- and GC-PKG-mediated signal pathways. Plasma FABP4 level declines postprandially, and suppression of FABP4 secretion by insulin-induced anti-lipolytic signaling may be involved in this decline in FABP4 level. © 2014 The Obesity Society.

The first structure of a bacterial diterpene cyclase: CotB2.

PubMed