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Thiamine Triphosphate and Their Hydrolyzing Enzymes

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Université de Liège Faculté de Médecine GIGA-Neurosciences Laboratoire du Vieillissement Pathologique et Epilepsies Professeur Lucien Bettendorff Metabolic significance of inorganic triphosphate, thiamine triphosphate and their hydrolyzing enzymes Mémoire présenté par Grégory Kohn, Titulaire d’un Master en Sciences Biomédicales, en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques ____________________________________________________ Année académique 2015-2016 Université de Liège Faculté de Médecine GIGA-Neurosciences Laboratoire du Vieillissement Pathologique et Epilepsies Professeur Lucien Bettendorff Metabolic significance of inorganic triphosphate, thiamine triphosphate and their hydrolyzing enzymes Mémoire présenté par Grégory Kohn, Titulaire d’un Master en Sciences Biomédicales, en vue de l’obtention du grade de Docteur en Sciences Biomédicales et Pharmaceutiques ____________________________________________________ Année académique 2015-2016 “La Science n’est plus à même de fournir aucune certitude, mais des propositions temporaires qui se métamorphoseront aussi vite que nos certitudes d’hier.” Ilya Prigogine (1917-2003, physicien Belge, Prix Nobel de Chimie en 1977) Cover picture modified from : Okuno D., Iino R., Noji H. 2011. Rotation and structure of FoF1-ATP synthase. J. Biochem. 149 (6) 655-64. doi: 10.1093/jb/mvr049. Abstract Our laboratory has been interested for many years in thiamine triphosphate (ThTP), an unusual triphosphate derivative of thiamine (vitamin B1) found in nearly all organisms. In mammalian tissues, ThTP is hydrolyzed by a very specific cytosolic thiamine triphosphatase (ThTPase) belonging to an ancient superfamily of proteins called CYTH. Several members of this superfamily have been characterized and they all have in common that they act on triphosphorylated substrates. Some bacterial members (the N. europeae specifically) hydrolyze inorganic triphosphate (PPPi) which raises the question of the physiological significance of this compound. We first studied the tripolyphosphatase activity in mammals and in bacteria and we showed that it is widely distributed in all organisms. We attempted to identify the enzymes responsible for this activity. We showed that the E. coli CYTH enzyme, ygiF, although highly specific for PPPi plays only a minor role in the cytosolic PPPase activity, while most of the activity is due to inorganic pyrophosphatase. In animal tissues, most PPPase activity is due to the short-chain exopolyphosphatase prune, which hydrolyzes PPPi with high catalytic efficiency. We hypothesize that PPPi may be formed as a by-product of metabolism and the major role of PPPase activities may be to keep PPPi concentrations very low to avoid toxic effects linked to interference with Ca2+ metabolism. In order to check this hypothesis, it would be important to have a specific and sensitive method for measuring intracellular PPPi concentrations. Such a technique is presently not available. In the second part of our work, we tried to manipulate intracellular ThTP concentrations in order to get insight into the physiological role of this compound. Expression of mammalian ThTPase in E. coli prevented ThTP accumulation, but did not affect the growth of the bacteria. We then reduced ThTPase expression in zebrafish embryos using morpholino oligomers. This led to severe malformations of the embryos. Finally, we attempted to produce a ThTPase knockout mouse. However, we found that the spermatogenesis of ThTPase-null sperm cells was impaired and the chimerae were unable to transmit. In conclusion, our results suggest that ThTPase inactivation results in heavy developmental consequences, possibly as a result of ThTP toxicity. List of abbreviations List of abbreviations : ADP Adenosine diphosphate AMP Adenosine monophosphate AThDP Adenosine thiamine diphosphate AThTP Adenosine thiamine triphosphate ATP Adenosine triphosphate CoA Coenzyme A CYTH CyaB-Thiamine triphosphatase E1o 2-Oxoglutarate dehydrogense E1p Pyruvate dehydrogenase E2o Dihydrolipoamide succinyltransferase E2p Dihydrolipoamide acetyltransferase E3 Dihydrolipoamide dehydrogenase EDTA Ethylenediaminetetraacetic acid ES cell Embryonnic stem cell GC Glycine cleage complex Hpf Hour post-fertilization IPTG Isopropyl β-D-1-thiogalactopyranoside kcat Catalytic constant Km Michaelis constant KOMP Knockout Mouse Project LC-ESI/MS/MS Liquid chromatography Electrospray ionization mass spectrometry NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information NMP Nucleotide monophosphate NMR Nuclear magnetic resonance NTP Nucleotide triphosphate OGDH 2-Oxoglutarate dehydrogenase complex PCR Polymerase chain reaction PDH Pyruvate dehydrogenase complex Pi Inorganic phosphate PolyP Inorganic polyphosphate PPase Pyrophosphatase PPi Inorganic diphosphate PPK Polyphosphate kinase PPPase Tripolyphosphatase PPPi Inorganic triphosphate PPX Exopolyphosphatase List of abbreviations qPCR quantitative PCR SD Standard deviation SDS-PAGE Sodium dodecyl sulfate Polyacrylamide gel electrophoresis SL Standard length SLC Solute carrier (gene) SOC Swim-out - central part SOP Swim-out - peripheral part SUL Swim-up - lower part SUS Swim-up - superior part TCA Trichloroacetic acid ThDP Thiamine diphosphate ThMP Thiamine monophosphate ThTP Thiamine triphosphate ThTPase Thiamine triphosphatase THTR Thiamine transporter (protein) TTM Triphosphate tunnel metalloenzyme WT Wild-type Acknowledgements Après 10 ans passés à écumer les couloirs, les amphis et les laboratoires de l’université, il y a beaucoup de personne que je me dois de remercier pour le soutien qu’ils m’ont apporté de différentes manières. J’oublierai sûrement de citer certains d’entre eux mais ils savent qu’ils sont malgré tout dans mes pensées. Tout d’abord, bien entendu, je tiens à remercier ma famille et surtout mes parents à qui revient tout le mérite de mon parcours, même si je ne leur ai probablement pas souvent fait ressentir. Et un merci tout spécial à mon grand-père qui n’est malheureusement plus là pour assister à l’aboutissement de cette entreprise. Il y a quelques personnes à qui mon parcours scientifique doit énormément. Tout d’abord, le Pr Lucien Bettendorff qui m’a conseillé et guidé au cours de toutes mes années de doctorat ; le Dr Pierre Wins, la bibliothèque vivante perpétuellement remise à jour que tout jeune chercheur devrait avoir la chance de rencontrer ; le Dr Bernard Lakaye et ses précieux conseils qui m’ont permis d’éviter nombre de pièges au cours de ces cinq années et Ilca pour sa gentillesse. Ensuite, bien entendu, le Dr David Delvaux pour m’avoir appris les bases du travail scientifique et pour toujours avoir été de bon conseil quand je débutais ma carrière. Egalement les Dr Marjorie Gangolf et Tiziana Gigliobinaco avec qui j’ai partagé bien plus qu’un bureau lors de mes premiers pas de chercheur et Arlette Minet et son esprit d’aventure. Un merci spécial va ensuite à Julie Vignisse avec qui j’ai partagé pas mal des joies et des peines survenues aux cours de mon master d’abord et de ma thèse ensuite. Et bien sûr, un grand merci à Coralie Pasquet pour les pauses-café et Margaux Sambon pour ses “Greeeeg” présageant toujours une question. Une dédicace spéciale Acknowledgements à mon admiratrice secrète pour avoir refait la décoration du bureau et pour son “ théobromine-tactisme” exacerbé. Je tiens aussi à remercier les Dr Laurence de Nijs et Nathalie Wolkoff pour avoir contribué à la bonne atmosphère de notre équipe ainsi que Sophie Harray qui a partagé avec moi une première expérience d’élevage de N.A.C. et Laurie Medard pour sa folie et sa bonne humeur. Je tiens à remercier tous les étudiants qui ont transité pour quelques mois par notre labo et tout spécialement Franciska, pour m’avoir fait découvrir et repousser les limites de la patience, ainsi que Cindy pour m’avoir rendu foi dans le travail fourni par la nouvelle génération d’étudiant. Je ne peux pas oublier non plus ceux avec qui j’ai collaboré, Marc Muller et tous les membres de l’équipe du Laboratoire de Biologie Moléculaire et Génie Génétique, surtout Marie Winandy pour son écolage ; les membres du Laboratoire de Génétique et de Physiologie Bactérienne de l’ULB, surtout Johan Timmermans pour son aide, et enfin Fabien Ectors pour la production et le croisement de nos chimères. Enfin, merci à ceux qui étaient là avant, pendant et seront toujours là après tout ça, Pierre et Laulau, Bruno, Jen et Benoit, Loïc, Léon et Cath. And last but not least, un grand merci aux membres du GIGA-Neurosciences : tout d’abord aux techniciens Bernard, Alexandra, Alice, Laurent, P-B, les Patricia et Guérin qui ont toujours été là pour répondre à mes questions ; ensuite à nos dévouées secrétaires Lari et Jess sur qui on peut toujours compter ; Acknowledgements Et enfin à tous les autres, doctorant, PI ou autres qui ont fait et font du centre ce qu’il est. I have now to switch to English to thank many people that were or were not already cited in the first part. To all of you who will be included in the next part, thank you for the different experiences and opinions shared and for the free English class. As I have always said: sorry to fail as a local for not teaching you any French and instead using you to improve my English. First, I want to thank all the “expats” from the GIGA- Neurosciences, and specially the two “douche” Arash and Deb for all the good time and Sita and Asya for all the good music, live or on vinyl. Also all the others members of the so called “Liege Expats group” with a special thanks to Sophie who made me an expat in my own city. Thank you all for the trips, parties, pic-nics, museum visit and so on and so forth that we’ve made together. I also want to thank my climbing partners Todd, Katya, Rohan, Amy, Stephane, Brett and Cyrine; nothing is as good as getting high (15 meters high) with nice people after a good day of work. “But standing in water up to their knees three very unimportant little people were doing the best they could with an idea and that’s about all they had and I don’t know maybe that night these three people were making history but anyway what I’m telling to tell you folks is: here we are!” J.Steinbeck.
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  • Transdifferentiation of Muscle to Electric Organ: Regulation of Muscle-Specific Proteins Is Independent of Patterned Nerve Activity

    Transdifferentiation of Muscle to Electric Organ: Regulation of Muscle-Specific Proteins Is Independent of Patterned Nerve Activity

    DEVELOPMENTAL BIOLOGY 186, 115-126 (1997) ARTICLE NO. DB978580 Transdifferentiation of Muscle to Electric Organ: Regulation of Muscle-Specific Proteins Is Independent of Patterned Nerve Activity John M. Patterson I and Harold H. Zakon 2 Department of Zoology and Center for Developmental Biology, University of Texas at Austin, Austin, Texas 78712 Transdifferentiation is the conversion of one differentiated cell type into another. The electric organ of fishes transdifferen- tiates from muscle but little is known about how this occurs. To begin to address this question, we studied the expression of muscle- and electrocyte-specific proteins with immunohistochemistry during regeneration of the electric organ. In the early stages of regeneration, a blastema forms. Blastemal ceils cluster, express desmin, fuse into myotubes, and then express tr-actinin r tropomyosin, and myosin. Myotubes in the periphery of the blastema continue to differentiate as muscle; those in the center grow in size, probably by fusing with each other, and lose their sarcomeres as they become electrocytes. Tropomyosin is rapidly down.regulated while desmin, tr-actinin, and myosin continue to be diffusely ex- pressed in newly formed electrocytes despite the absence of organized sarcomeres. During this time an isoform of keratin that is a marker for mature electrocytes is expressed. One week later, the immunoreactivities of myosin disappears and t~-actinin weakens, while that of desmin and keratin remain strong. Since nerve fibers grow into the blastema preceding the appearance of any differentiated cells, we tested whether the highly rhythmic nerve activity associated with electromo- tor input plays a role in transdifferentiation and found that electrocytes develop normally in the absence of electromotor neuron activity.