The NAD glycohydrolase (NADase) from Bungarus fasciatus venom was purified over 1000-fold to electrophoretic homogeneity through a 3-step procedure which included affinity chromatography on Cibacron Blue agarose. The enzyme exhibited a broad pH profile with the optimum range between 7-8. Studies on the substrate specificity of B. fasciatus venom NADase demonstrated that alterations in the purine ring were less pronounced then alterations in the pyridinium moiety of NAD. Product inhibition studies indicated nicotinamide to be a noncompetitive inhibitor with a K_i = 1.4 mM and ADP-ribose to be a competitive inhibitor with a K_i =0.4 mM. The purified enzyme was inactivated by both 2,4-pentane-dione and Woodward's Reagent K suggesting the involvement of a lysine and carboxyl group in the catalytic process. In contrast to other known NADases, the snake venom enzyme did not self-inactivate. The purified B. fasciatus venom NADase catalyzed a transglycosidation reaction (ADP-ribose transfer) with a number of acceptor molecules. The functioning of a variety of substituted pyridine bases as acceptor molecules was demonstrated through the formation of the corresponding NAD analogs. The enzyme also catalyzed the transfer of ADP-ribose to aliphatic alcohols (methanol to hexanol, inclusive) and a positive chainlength effect was observed in the functioning of these acceptors. Kinetic studies of transglycosidation reactions were consistent with the partitioning of an enzyme-ADP-ribose intermediate between water and nucleophilic acceptors as has been proposed in earlier studies of mammalian NADases. The partitioning of this intermediate between water and pyridine bases can be correlated with the basicity of the ring nitrogen of the pyridine derivative. The K_i of pyridine bases in the hydrolytic reaction did not equate to the K_m of these bases in the pyridine base exchange reaction suggesting two forms of the NADase with varying affinity for the pyridine bases. This implys the pyridine base exchange reaction to be more complicated than originally proposed. / Ph. D.

Dans le cadre des recherches menées au sein du laboratoire de Physiologie Animale le gène codant pour la nicotinamide phosphorybosyltransférase (NAmPT) à été identifié et cloné. Au cours de ce travail, nous avons étudié le rôle de cette enzyme du métabolisme du Nicotinamide Adénine Dinucléotide ainsi que celui des enzymes dépendantes du NAD (PARP et sirtuines) dans le contrôle de la réponse inflammatoire. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Nicotinamide adenine dinucleotide (NAD+) represents a long-known key molecule in cellular metabolism. It was initially identified for its ability to convey electrons and protons between redox partners in multiple bioenergetic and biosynthetic reactions. In addition, NAD+ also serves as a substrate for NAD+-consuming enzymes such as sirtuins and poly ADP-ribose polymerases (PARPs). These latter enzymes catalyze dynamic post-translational modifications that control virtually every signaling pathway orchestrating cell fate. The aim of this work was to analyze the role of NAD+ in the context of programmed cell death mechanisms.<p>Our findings indicate that NAD+ is protective against DNA damage-induced cell death and FAS-induced apoptosis, while, unexpectedly, it promotes TNF-induced necroptosis, a regulated form of necrosis. Indeed raising NAD+ cellular levels sensitized culture cells to necroptosis, while NAD+ depletion protected cells from this form of cell death. Furthermore, specific silencing of NAD+-dependent sirtuins was also found to be protective against TNF-induced necroptosis. Consistently, a pharmacological pan-sirtuin inhibitor called cambinol protected cells from necroptosis. Then, as necroptosis represents a back-up mechanism that may have evolved in response to viral pathogens expressing anti-apoptotic proteins, we demonstrated in an in vitro model mimicking viral infection that pharmacological sirtuin inhibition protected cells from poly I:C-induced necroptotic cell death. In vivo, we demonstrated that cambinol partially protected kidney from necrosis after ischemia/reperfusion. We have also shown that enhancing liver NAD+ concentration via isonicotinamide increases the susceptibility of mice to systemic inflammatory response syndrome (SIRS). Moreover, our preliminary data show that isonicotinamide substantially improves the ability of cyclophosphamide to trigger the rejection of the murine mastocytoma P815 tumor cell line.<p>Collectively, our observations point to a role for NAD+ in the control of necroptosis in a sirtuin-dependent manner. These observations may bear relevance to the better understanding of the pathophysiological consequences of excessive production of the pro-inflammatory cytokine TNF and the control of viral infections and tumor progression/immunotherapy. & / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le nicotinamide est un des deux constituants de la vitamine B3. C’est aussi un agent pharmacologique qui a été testé dans le traitement du HIV chez l’homme, et comme traitement contre diverses pathologies. Il présente également des capacités cytoprotectrices dans plusieurs modèles de stress cellulaire et est considéré comme un agent pharmacologique prometteur dans le traitement des maladies de dégénérescence cérébrale d’origine vasculaire. Il module la réponse innée en inhibant notamment la synthèse de molécules pro-inflammatoires. Toutefois, son influence sur la réponse adaptative n’a pas encore été analysée. <p>Le nicotinamide intervient dans la biosynthèse du NAD comme précurseur dans la voie de « sauvetage ». Le rôle du NAD comme coenzyme dans de nombreuses réactions enzymatiques du métabolisme de la cellule est bien connu. De plus, le NAD peut être dégradé par différentes enzymes impliquées dans différentes modifications post-traductionnelles de protéines (PARPs, Sirtuines et MARTs). Le nicotinamide est un produit de la dégradation du NAD mais également un inhibiteur de ces enzymes et constitue donc un outil permettant d’étudier le rôle de ces enzymes dans la transduction de signaux intracellulaires. <p>Nous avons utilisé le nicotinamide comme un inhibiteur non toxique des différentes enzymes impliquées dans les réactions d’ADP-ribosylation et étudié son effet sur l’activation des lymphocytes B. Le nicotinamide inhibe la prolifération et la différentiation de ces cellules. Il ne module pas les étapes précoces du BCR mais inhibe l’activation des MAPKs et de la kinase Akt. L’inhibition des MAPKs Erk est corrélée avec une réduction de l’expression de la cycline D2 et du marqueur d’activation CD69. L’utilisation d’un inhibiteur des PARPs ne nous a pas permis de reproduire les effets du NAm sur la voie MAPK Erk et CD69. Par contre, le MIBG, un inhibiteur des MARTs inhibe bien la surexpression du CD69 ainsi que la phosphorylation des kinases Erk. Bien que le nicotinamide soit capable d’inhiber l’expression du CD69 in vivo, nos expériences ne nous ont pas permis de moduler la réponse immune adaptative in vivo. <p>Ceci suggère dès lors que l’utilisation de fortes doses de nicotinamide comme traitement pharmacologique de certaines affections chez l’homme ne devrait pas poser de problème au niveau de la réponse adaptative. De plus, notre mise en évidence des MARTs dans le contrôle de l’activation des lymphocytes B ouvre des perspectives encourageantes pour de nouveaux traitements modulant la réponse adaptative. Cette réponse étant particulièrement impliquée dans certaines maladies auto-immunes, il est potentiellement intéressant de trouver des inhibiteurs de ces enzymes plus puissants que le nicotinamide afin de moduler la réponse immune adaptative in vivo<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Atherosclerosis occurs preferentially at branches and curves in arteries exposed to disturbed flow while sparing straight portions of arteries exposed to undisturbed flow. In vivo and in vitro studies have implicated NADPH oxidases in atherosclerosis and hypertension. Shear stress can induce reactive oxygen species production in endothelial cells from a variety of sources, including NADPH oxidases. Here, we examined the hypothesis that unidirectional laminar shear (LS) and oscillatory shear (OS) would differentially regulate gene expression profiles in NADPH oxidase-dependent and -independent manners, and that these genes would provide novel molecular targets in understanding endothelial cell biology and vascular disease. The p47phox subunit of the NADPH oxidase can be an important regulator of certain Nox isoforms, including Nox1 and Nox2 which may be responsible for shear-induced superoxide production. In order to isolate p47phox-dependent shear responses, we took advantage of the p47phox-/- transgenic mouse model which lacks a functional p47phox subunit. We developed a method to isolate murine aortic endothelial cells using an enzymatic digestion technique. These cells expressed characteristic endothelial markers, including VE-cadherin, PECAM1, and eNOS, and aligned in the direction of flow. We successfully isolated primary murine aortic endothelial cells from both wild-type C57BL/6 mice (MAE-WT) and p47phox-/- mice (MAE-p47). Furthermore, we established an immortalized cell line from each of these cell types, iMAE-WT and iMAE-p47. We carried out microarray studies using Affymetrix Mouse Genome 430 2.0 Arrays (39,000+ transcripts) on MAE-WT and MAE-p47 that were exposed to atheroprotective LS or atherogenic OS for 24 hours. In comparison to LS, OS significantly changed the expression of 187 and 298 genes in MAE-WT and MAE-p47, respectively. Of those, 23 genes showed similar gene expression patterns in both cell types while 462 genes showed different gene expression patterns in the two cell types, demonstrating a considerable role for p47phox-based NADPH oxidases in shear-dependent gene expression. Changes in expression of several genes, including Kruppel-like factor 2 (Klf2), endothelial nitric oxide synthase (eNOS), angiopoietin 2 (Ang2), junctional adhesion molecule 2 (Jam2), bone morphogenic receptor type II (Bmpr2), and bone morphogenic protein 4 (Bmp4) were confirmed by quantitative PCR and/or immunoblotting using both primary cells and immortalized cells. Of these genes, our data suggest that Jam2, Bmpr2, and Bmp4 may be shear-sensitive in a p47phox-dependent manner. Taken together, our studies have identified a set of shear- and p47phox-sensitive genes, including unexpected and novel targets, which may play critical roles in vascular cell biology and pathobiology.

The mechanism by which nicotinamide adenine dinucleotide (NAD) stimulates the activity of adenylate cyclase (AC) in canine plasma membrane has been studied. Using [3 2P]-NAD, the activation by NAD was correlated with the radiolabeling of the stimulatory guanosine triphosphate (GTP) binding protein Gsa. Further characterization demonstrated that the modification occurred only in the presence of G-protein activators and that arginine residue(s) were modified by ADP-ribose by the action of a mono-ADP-ribosyltransferase. Inhibitors of the transferase blocked both the modification of Gsa and the activation of AC. Collectively, these studies suggest that ADP-ribosylation of Gsa by an endogenous mono-ADP-ribosyltransferase may regulate cardiac AC.

Abstract not available

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 29, 2010). Vita. Thesis advisor: Grace Y. Sun. "May 2008" Includes bibliographical references.