Rozprawy doktorskie na temat „Nucleoside Diphosphate Kinase (NDK)”

La nucleoside diphosphate kinase (ndp kinase) est une enzyme du metabolisme qui catalyse la synthese des ribo- et desoxyribo-nucleosides triphosphates a partir de leurs precurseurs diphosphates selon un mecanisme de type ping-pong. C'est un hexamere de sous-unites identiques de 17 kda dont la structure tridimensionnelle est connue. L'implication de ndp kinases dans le developpement et la metastase a ete mise en evidence respectivement chez l'homme et la drosophile. Par ailleurs, il a ete recemment montre que la ndp kinase humaine est capable de se fixer sur le promoteur de l'oncogene c-myc et d'en stimuler la transcription. J'ai etudie plusieurs mutants de la ndp kinase de dictyostelium. L'etude du mutant h55a a montre le role crucial l'his 55 dans les proprietes de fluorescence de l'enzyme phosphorylee, et sa contribution faible dans la catalyse. Le mutant n119a a permis d'obtenir des co-cristaux de l'enzyme avec l'azt diphosphate. La structure obtenue permet d'expliquer la faible efficacite de phosphorylation de l'azt diphosphate in vivo. J'ai par ailleurs etudie la proteine mutante s124g de dictyostelium, correspondant a un mutant isole dans des lignees humaines de neuroblastomes. La mutation n'affecte pas la catalyse, mais modifie les proprietes de repliement du polypeptide. Les fonctions biologiques de la ndp kinase chez dictyostelium ont ete etudiees par surexpression des formes sauvage et mutantes du gene codant pour la ndp kinase cytosolique. J'ai egalement realise l'inactivation de ce gene par recombinaison homologue, et obtenu des transfectants viables. Dans tous les cas, aucune difference de phenotype avec la souche parentale n'a ete observee. Cette absence d'effet chez dictyostelium est discutee dans le contexte des observations realisees chez d'autres organismes.

Les nucleoside diphosphate kinases sont les enzymes responsables in vivo de la synthese des nucleosides triphosphates a partir de l'atp. Elles sont impliquees dans des fonctions cellulaires normales et pathologiques, eventuellement independamment de leur activite enzymatique mais les mecanismes mis en jeu sont actuellement mal definis. Chez l'homme, six isoformes sont decrites a ce jour : nm23-h1, nm23-h2, dr-nm23, nm23-h4, nm23-h5 et nm23-h6. Mon travail a consiste en l'etude de nm23-h4, dont l'existence a ete mise en evidence au laboratoire. J'ai clone l'adnc nm23-h4. Le gene est localise sur le chromosome 16p13. 3. L'expression de l'arnm nm23-h4 est variable selon les tissus et particulierement abondante dans la prostate et le foie. La proteine nm23-h4 (187 acides amines ; mr : 20650) se distingue des autres isoformes humaines, par la presence d'une extension n-terminale caracteristique d'un adressage mitochondrial. La proteine entiere et une forme tronquee sans l'extension ont ete exprimees chez e. Coli et purifiees. La forme tronquee possede l'activite ndp kinase tandis que la forme entiere est inactive, suggerant un effet inhibiteur de l'extremite n-terminale. L'analyse de la structure tridimensionnelle par diffraction aux rayons x, montre que nm23-h4 est hexamerique comme les autres ndp kinases eucaryotes. Nm23-h4 possede un residu serine en position 129 correspondant a la mutation conditionnelle letale dominante de la ndp kinase de la drosophile. Le mutant s129p, beaucoup plus stable aux agents denaturants, confirme l'importance de ce residu dans la stabilite de la proteine. Nous montrons par transfert de western et analyse confocale de cellules humaines sur-exprimant la proteine fusionnee a la gfp, que nm23-h4 est une ndp kinase mitochondriale, la premiere caracterisee chez les mammiferes. Son import dans l'organite s'accompagne d'un clivage proteolytique, vraisemblablement de l'extension n-terminale. Nm23-h4 semble etre associee aux sites de contact mitochondriaux entre les membranes interne et externe. Le role de nm23-h4 dans la mitochondrie est discutee dans le contexte des etudes anterieures sur l'activite ndp kinase mitochondriale et de son association aux sites de contact.

La nucleoside diphosphate kinase (ndpk) est un enzyme ubiquitaire, qui catalyse la formation des nucleoside triphosphates a partir des diphosphates et maintient l'equilibre de leurs concentrations aux depens de l'atp. La reaction de type ping-pong est tres efficace et passe par un intermediaire phospho-histidine. La ndpk est un tetramere chez certaines bacteries, un hexamere chez les eucaryotes. La sous-unite comporte un domaine a/b organise autour d'un feuillet-b a 4 brins antiparalleles. Outre son role dans la biosynthese des nucleotides, l'enzyme humain aurait une activite histidine kinase et lierait l'adn. De plus, ell serait impliquee dans la proliferation des metastases et dans l'activation de la transcription de certains genes. Dans cette these, nous presentons six structures cristallines de la ndpk de dictyostelium complexee avec des substrats, des analogues ou des inhibiteurs. Elles permettent de mieux comprendre le mecanisme de la catalyse et l'interaction avec des analogues de nucleotides anti-viraux, en visant a la conception de medicaments et a la mutagenese fondee sur la structure. La structure du complexe ndpk. Adp. Alf3 decrit la geometrie de l'etat de transition de la reaction de transfert de phosphate. Alf3 mime le phosphate-g de l'atp lors de son transfert sur l'histidine du site actif par un mecanisme d'attaque en ligne passant par un intermediaire bipyramid trigonal. Des complexes ndp. Aif3 similaires ont depuis ete decrits dans d'autres kinases. La structure du complexe ndpk. Adp. Bef3 illustre le mode fixation de l'atp. Ces deux structures definissent les interactions enzyme-substrat et mettent en evidence deux elements essentiels de la catalyse : l'ion mg++ et le oh en 3 du sucre sur le nucleotide. Nous avons analyse les cinetiques de phosphorylation et dephosphorylation des nucleotides par la ndpk aal'aide de la fluorescence intrinseque de la proteine. Les analogues antiviraux derives de l'azt et de la ddt sont de mauvais substrats de la ndpk en comparaison du substrat naturel tdp. La faible phosphorylation de l'azt s'explique par deux facteurs structuraux : l'absence de la liaison hydrogene entre le 3oh et l'oxygene pontant les phosphates b et g ; le deplacement de la chaine laterale du residu lys 16. La structure cristalline du complexe avec l'azt diphosphate donne une base structurale a la conception de nucleosides antiviraux qui soient de meilleurs substrats de la ndpk. Celles des complexes ndpk avec les deux antiviraux 3-fluoro-udp et 3-amino-adp permet de comprendre pourquoi ce sont aussi de mauvais substrats ; dans les deux cas, la modification en 3 du sucre change l'interaction avec la proteine. Le 3'-phospho-adenosine-5-phosphosulfate (paps) est un inhibiteur puissant de l'activite enzymatique de la ndpk. La structure cristalline du complexe revele qu'il se fixe sur le meme site que l'adp, mais d'une facon differente, qui donne des indications sur le mode de fixation de l'adn.

Les ndp kinases sont des enzymes ubiquitaires et sont la principale source des nucleosides triphosphates autre que l'atp. Elles catalysent le transfert du -phosphate d'un nucleoside triphosphate sur un nucleoside diphosphate avec une faible specificite de substrat et une forte activite. Le mecanisme de la reaction est dit de type ping-pong, avec la formation d'un intermediaire phosphohistidine (h122) au cours de la catalyse. En plus de leur role dans le metabolisme des nucleosides triphosphates, les ndp kinases sont impliquees dans le developpement chez la drosophile et dans les metastases de tumeurs chez l'homme. La ndp kinase de dictyostelium est un hexamere compose de six sous-unites identiques de 17 kda chacune. Grace au modele du mutant h122c, j'ai resolu les structures de l'enzyme sauvage et d'un complexe avec l'adp a respectivement 1,8 a et 2,2 a de resolution. Une sous-unite est constituee d'un domaine globulaire / avec un feuillet antiparallele a quatre brins. Sa topologie est differente de celle des autres phosphotransferases, cependant on la retrouve dans des proteines fixant des mononucleotides, de l'arn ou de l'adn. Chaque sous-unite possede un site actif. Le mode de fixation du nucleotide est different de celui observe chez les proteines fixant des nucleotides. En effet, il n'y a pas de liaisons polaires du nucleotide avec les atomes de la chaine principale et pas de sequence riche en glycines. Le manque de specificite est du a l'absence d'interaction polaire entre la base et l'enzyme. Le ribose et les phosphates font des liaisons hydrogenes avec les chaines laterales. Le monomere, le dimere et le site actif de la ndp kinase de dictyostelium sont les memes chez toutes les ndp kinases. Nous avons propose un mecanisme catalytique du transfert du phosphate sur le n de h122. Nous ne savons toujours pas si les autres fonctions des ndp kinases humaines et de drosophile dependent de leur structure quaternaire

Les ndpk sont des enzymes qui catalysent le transfert du phosphate gamma d'un nucleoside triphosphate sur un nucleoside diphosphate avec une faible specificite pour le substrat. Elles sont une source de nucleoside triphosphates et sont impliquees dans l'activation des analogues de nucleoside utilises contre le sida. L'activite antivirale de ces analogues est due a l'absence d'hydroxyle en 3 du sucre, cette particularite en fait de mauvais substrats de la ndpk. Cet hydroxyle essentiel a la catalyse forme une liaison hydrogene avec l'oxygene 07 pontant les phosphates beta et gamma. Nous avons determine la structure de trois complexes de la ndpk de dictyostelium avec des analogues de nucleotide. La premiere decrit l'interaction du mutant h122g avec le d4t triphosphate. Une liaison hydrogene c3h---o7 permet d'expliquer l'augmentation d'efficacite catalytique de la ndpk sur cet analogue par rapport a l'azt. Les deux autres structures decrivent l'interaction de la ndpk sauvage avec les nucleotides alpha-borano-phosphate qui sont de meilleurs substrats que leurs nucleotides parents. Les borano-phosphate conferent une resistance de la liaison phosphodiester a la pyrophosphorolyse, or celle-ci expliquerait la resistance du vih aux analogues actuellement utilises. La structure du complexe avec l'alpha-borano-tdp de configuration rp a permis d'expliquer la stereospecificite de la ndpk pour ce compose. La structure avec l'alpha-borano-d4t diphosphate de configuration sp explique pourquoi la ndpk n'a qu'une stereospecificite partielle sur les composes alpha-borano-phosphate derives du d4t. Pour etendre l'etude structurale aux ndpk humaines recemment decouvertes nous avons determine la structure de la ndpk mitochondriale h4. Cette structure a mis en evidence le changement conformationnel induit par la presence d'une serine en position 96 a la place d'une proline dans les autres ndpk et a permis de suggerer un role pour la serine 113 dans la reconnaissance de la guanine.

Les analogues de nucleotides administres dans les therapies antiretrovirales (ddn, azt, d4t, 3tc) sont transformes dans la cellule en triphosphate par des kinases cellulaires avant d'atteindre leur cible, la transcriptase inverse du virus vih. Cette activation implique les enzymes de la voie de recuperation des nucleotides. Le dernier phosphate est ajoute par la nucleoside diphosphate (ndp) kinase, veritable enzyme de menage, avec une specificite large pour tous les nucleotides. La reactivite de la ndp kinase avec les analogues antiretroviraux s'est averee beaucoup plus faible que supposee. A l'aide d'une methode mesurant la fluorescence intrinseque de la proteine, nous avons etudie la reactivite de l'enzyme avec differents analogues de nucleotides donneur (ntp) ou accepteur (ndp) de phosphate. Bien que l'equilibre de la reaction avec les ntp et ndp soit le meme que pour les nucleotides naturels, la vitesse de transfert de phosphate est tres diminuee (1000 a 10000 fois) pour les analogues antiviraux, substrats lents de la ndp kinase. L'affinite relative des ntp a ete mesuree directement pour un mutant inactif de l'enzyme. Tous les antiviraux se lient moins bien au site actif que leurs homologues naturels a l'exception du d4t triphosphate qui se lie a l'enzyme avec la meme affinite que la thymidine triphosphate. L'absence de 3'oh sur le ribose empeche l'etablissement d'une liaison hydrogene intramoleculaire avec l'o 7 du phosphate , cruciale pour une catalyse efficace. Seul le d4t est capable d'effectuer partiellement une interaction de ce type. C'est le meilleur antiviral phosphoryle par la ndp kinase. L'examen de la structure d'un nucleotide lie au site actif montre que l'oxygene pro-rp du phosphate du nucleotide n'interagit ni avec le mg 2 +, ni avec la proteine. En collaboration avec l'unite de chimie organique de l'institut pasteur, un groupement borano (-bh 3) a ete introduit a la place de cet oxygene. Les derives rp--bh 3 de l'azt et du d4t di- et triphosphate sont dix fois mieux phosphoryles par la ndp kinase, en raison de leur meilleure affinite pour l'enzyme. Le diastereoisomere rp est aussi un meilleur terminateur de chaine de l'activite transcriptase inverse : les nucleotides -borano sont plus efficacement incorpores dans l'adn viral. Ils sont egalement moins sensibles a l'excision (pyrophosphorolyse) par l'enzyme sauvage comme par des variants resistants de la polymerase virale. A cause du role essentiel de la fonction 3'oh du nucleotide, nous avons essaye de compenser son absence dans les analogues antiviraux en introduisant une fonction - oh dans le site actif de la ndp kinase. Plusieurs mutants ont ete crees par mutagenese dirigee : un seul mutant pour lequel l'asn 119 a ete substituee par une ser phosphoryle mieux les antiviraux avec une efficacite catalytique entre deux et dix fois superieure, due a une fixation plus aisee au site actif. L'introduction de la ser en 119 permet de restaurer le reseau de liaisons hydrogene indispensable au transfert de phosphate. En parallele, nous avons recherche de nouveaux inhibiteurs de la ndp kinase : l'adenosine 3 phosphate 5 phosphosulfate (paps) inhibe l'activite ndp kinase avec une affinite de 10 m. L'analyse des cocristaux de l'enzyme complexee avec le paps montre que l'analogue de nucleotide se lie au site actif selon un mode different du nucleotide naturel. Le paps represente ainsi l'inhibiteur de plus forte affinite connu pour cette enzyme.

Mycobacterium tuberculosis (Mtb) evasion of the host immune response leads to its resilient persistence within the macrophage ultimately triggering active tuberculosis, a deadly infectious disease afflicting millions of people globally. To combat this disease, there is an urgent need to reveal the underlying mechanisms of pathogenesis by understanding how mycobacteria interact with the host macrophage. The work of this thesis contributes to this understanding through the identification of Mtb nucleoside diphosphate kinase (Ndk) as a virulence factor that contributes to pathogenesis by interfering with key host small G-proteins essential for proper function of the innate immune response. Combining biochemical interaction assays with GTPase activation assays, this work demonstrated that Mtb Ndk interacts with host Rab5, Rab7, and Rac1 to inactivate their downstream signaling. The consequence of interfering with Rab5 and Rab7 signaling is a dramatic decrease in phagolysosome fusion of mycobacteria phagosomes, while inactivation of Rac1 led to NADPH oxidase assembly defect causing impaired reactive oxygen species production. Additional studies utilizing mycobacteria strains with knocked down Ndk further demonstrated its contribution to virulence. Indeed, strains with knocked down Ndk were substantially more prone to intracellular killing within the macrophage, which correlated with increased levels of phagolysosome fusion, raised levels of macrophage oxidative burst, and an increase in programmed cell death. Consistent with these findings, in vivo virulence of Mtb with knocked down Ndk was reduced significantly compared to wild type Mtb when monitoring mice survival. Altogether, the work of this thesis demonstrated that Mtb Ndk is a bona fide mycobacterial virulence factor that attenuates host innate immunity.

Les nucleosides diphosphates (ndp) kinases sont des enzymes ubiquitaires qui catalysent la synthese des nucleosides triphosphates. L'adnc codant pour la ndp kinase de dictyostelium discoideum (d. D. ) a ete isole par criblage fonctionnel. Puis l'enzyme, exprimee chez e. Coli, a ete purifiee a homogeneite, ses proprietes biochimiques ont ete comparees a celles de l'enzyme partiellement purifiee a partir de cellules de d. D. Au stade vegetatif. Leurs proprietes analogues indiquent que la ndp kinase de d. D. Ne subit pas de modifications post traductionnelles susceptibles de modifier son activite. Contrairement a l'enzyme humaine, constituee de deux types de sous-unites, la proteine de d. D. Est un homohexamere de 103 kda. La structure hexamerique de l'enzyme est confirmee par les premiers resultats de l'etude de la structure cristallographique. Son mecanisme reactionnel est de type ping-pong avec formation d'un intermediaire phosphoryle sur l'histidine 122. Bien que le taux d'arnm, tres eleve au stade vegetatif, diminue des le debut du developpement pour atteindre un niveau tres bas, le taux de proteine ainsi que l'activite ndp kinase decroissent peu au cours du cycle. La sequence de la ndp kinase de d. D. Est similaire a 75% avec les produits de deux genes: nm23, propose comme suppresseur du potentiel metastatique de cellules tumorales chez les mammiferes et awd, implique dans l'embryogenese de la drosophile. Cette similitude a permis d'attribuer une fonction a ces proteines et suggere que les ndp kinases, pourraient intervenir dans le controle de processus tels que le developpement ou la proliferation tumorale

La nucleoside diphosphate kinase (ndp kinase) est une proteine ubiquitaire composee, chez les eucaryotes, de six sous-unites de 17 a 20 kda. Cette enzyme catalyse le transfert du phosphate d'un nucleoside triphosphate vers un nucleoside diphosphate via un intermediaire phospho-enzyme. En plus de son role dans le metabolisme des nucleotides, la ndp kinase est impliquee dans d'autres fonctions cellulaires comme le developpement chez la drosophile ou la proliferation cellulaire chez l'humain. Par des techniques d'immuno-fluorescence, la presence de la ndp kinase b a pu etre mise en evidence dans le cytoplasme mais aussi dans le noyau de cellules humaines. Cette enzyme, qui est capable de se fixer sur le promoteur de l'oncogene c-myc et d'activer sa transcription, possede des proprietes de liaison a divers oligonucleotides, avec une meilleure affinite pour les sequences d'adn simple brin riches en pyrimidines. J'ai etudie le role de la structure quaternaire dans les differentes activites de la ndp kinase de dictyostelium. Apres avoir modifie par mutagenese dirigee plusieurs residus impliques dans les contacts entre sous-unites, j'ai obtenu une proteine mutante dimerique, p100s-n150stop, depourvue d'activite enzymatique. En presence de nucleotide substrat, cette proteine se reassocie en hexameres ayant des proprietes cinetiques identiques a celles de l'enzyme sauvage alors que la proteine mutante dimerique est incapable de s'autophosphoryler. La reassociation en hexamere fait apparaitre un intermediaire transitoire phosphorylable mais peu actif. L'ensemble de ces resultats demontre que l'activite catalytique est portee exclusivement par la forme hexamerique. Alors que l'enzyme sauvage de dictyostelium n'interagit pas avec l'adn, la forme dimerique est capable de former un complexe adn/proteine. De meme, pour la ndpk-b humaine, cette fonction est dependante de l'etat oligomerique. Ces resultats supportent l'hypothese selon laquelle les differents etats oligomeriques de la ndp kinase sont responsables de fonctions cellulaires differentes.

Les isoenzymes de nucléoside diphosphate kinase (NDPK) sont connues depuis maintenant presque 60 ans et n'ont été considérées que pour leur activité catalytique de transfert de groupement phosphoryle. La découverte du gène nme, un gène antimétastatique codant une NDPK, a renouvelé l'intérêt scientifique pour cette famille d'enzymes. Il est désormais connu que la multiplication des gènes durant l'évolution a été accompagnée de diversifications structurales et fonctionnelles. J'ai étudié la fixation des NDPK-A, -B et –D (retrouvées associées aux membranes biologiques, bien que le rôle de cette association soit encore méconnu) à des membranes modèles, et j'ai trouvé des différences dans les mécanismes de fixation. J'ai montré la capacité de la NDPK-D, isoforme mitochondriale, à interagir avec des membranes anioniques ou zwitterioniques, à augmenter leur fluidité et à former des domaines protéolipidiques en présence de CL, lipide anionique spécifique de la membrane mitochondriale interne. J'ai observé cette capacité à former des domaines protéolipidiques avec d'autres protéines interagissant avec la CL, comme la créatine kinase mais pas le cytochrome C. La NDPK-A ne se fixe pas aux phospholipides du feuillet interne de la membrane plastique, ce qui suggère un autre partenaire in vivo. La NDPK-B n'interagit qu'avec des membranes anioniques via un processus en deux étapes, provoque une diminution de fluidité et est capable de former des domaines protéolipidiques. La ségrégation des lipides anioniques induite par la fixation de protéines pourrait contribuer à la formation de plateformes au sein de la membrane susceptibles de servir de point d'ancrage à de nombreuses molécules, modulant ainsi les fonctions cellulaires
Nucleoside diphosphate kinase isoenzymes (NDPK) have been known for nearly 60 years and, until recently, have been considered as housekeeping enzymes. The discovery of a nme gene, an antimetastatic gene that codes for a NDPK, revived the interest for this family. It is now known that the multiplication of nme genes throughout evolution has been accompanied with structural and functional diversification. I studied the binding of NDPK-A, -B and –D (which ae retrieved associated to cellular membranes where they are thought to play several roles) to model membranes and found differences in their behavior towards different compositions of phospholipids. I showed the ability of the NDPKD mitochondrial isoform to interact with both anionic and zwitterionic membranes, to modify their fluidity and to form proteolipidic domains in presence of CL, a mitochondrial inner membrane specific anionic lipid. I observed this ability to form proteo-cardiolipin domains with other CL interacting protein like creatine kinase but not with cytochrome c. NDPK-A was not able to bind to inner leaflet plasma membrane mimicking systems suggesting another partner in vivo. Concerning NDPK-B, it interacted only with anionic membranes via a two step-process, induced a decrease of the membrane fluidity and was able to form proteolipidic domains. Such anionic lipid segregation triggered by protein binding may contribute to platforms formation within membranes. Those platforms are then susceptible to provide a functional docking platform for numerous molecules and thus to modulate cellular functions

Biological processes are governed by highly specific macromolecular interactions. Understanding the precise mechanism of ligand recognition in proteins is essential for deriving features responsible for such recognition capabilities. Although protein sequences give first-hand information about their function, their three-dimensional structures, which are the evolutionary units, convey the function better. Three-dimensional structures of many proteins determined through X-ray crystallography and/or NMR are available in the Protein Data Bank, a public repository. This resource has stimulated the development of computational techniques to read and analyse the wealth of structural data. Structural bioinformatics is an area that provides a means to transform information in the protein structures into functional insights and enable addressing a variety of questions about what defines and dictates ligand recognition. Large-scale analyses of several protein-ligand complexes have indicated that both one-to-many and many-to-one relationships of protein-folds and ligand-types are widely seen in the PDB. This means that a given ligand can be recognized by diverse proteins and a given protein can recognize different types of ligands at the same location, ligands referring to endogenous ligands, natural metabolites as well as small molecule inhibitors, and drugs. Given this, it is important to understand the determinants of recognition of a given ligand. This becomes important for applications in drug discovery that includes, lead design and lead optimization, assessment of draggability of a target, identification of off-target effects, polypharmacological targets and drug repurposing. The present work utilizes the information present at the functional sites, rationalizing many examples of ligand binding and deriving useful patterns that can be used for genome-wide function annotation and drug discovery applications. A large-scale analysis of the binding of two important classes of ligands, a sugar and nucleotides was carried out by analysing the sub-structures at their binding sites by matching, aligning and clustering. The two ligands studied are sialic acid, and nucleoside mon/di/tri phosphates (nucleotides or NTPs), for their binding to many proteins reported in the PDB. Sialic acid was found to be recognized by 170 different proteins representing 17 unique sequence families. Our approach deciphered a unified understanding of the basis of recognition of this ligand and showed six structural motifs, which contained different combinations of one or more key structural features, over a common scaffold. The site features refer to certain residues in the binding site that are seen to most frequently occur at their respective topological positions, a result that was evident upon binding site comparisons and 3-D alignment of sites in the different proteins. In the case of nucleotide ligands, 4,677 structures of protein-nucleotide complexes from PDB, belonging to 145 different structural folds and 394 sequence families were analysed, and our results indicated that the sites from diverse proteins group into 27 site-types and further into nine super-types having a structural motif for each site-type. The identified motifs were highly specific when scanned against the entire PDB. A computational alanine mutagenesis study indicated that residues identified to be highly conserved in the motifs also contribute most to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. Many examples of convergent evolution were identified through this analysis. Next, in order to find the entire set of all binding sites in the proteins that have known 3-D structures, a large-scale analysis of 30457 representative protein structures in PDB was carried out by detecting additional binding sites in them, followed by a site-comparison to recognize unexpected similarities. The pocket-space currently defined in PDB is incomplete, as binding-sites remain uncharacterized in many proteins despite the availability of their structures. To bridge this gap, we computationally detect pockets in the entire PDB and present a comprehensive resource of an ‘augmented pocketome’ consisting of 249,096 pockets (http://proline.biochem.iisc.ernet.in/PocketDB), which shows a 2-fold increase in fold coverage and a 7-fold increase in pocket-space when compared to what is currently known. Possible ligand associations for about 56% of these pockets were deduced. A clustering of the pocketome led to the identification of 2,161 site-types, and the associated ligands into 1,037 ligand-types, which together provided fold-to-site-type-to-ligand-type associations. The resource facilitates a structure-based function annotation, delineation of a structural basis of ligand recognition, and provides functional clues for Domain of Unknown Function (DUFs), allosteric proteins and druggable pockets. Next, it was of interest to study the entire binding site-space in a proteome of a given organism. Mycobacterium tuberculosis (Mtb) is a deadly pathogen being studied from multiple perspectives in the laboratory. Structural models of about 70% of the proteome and a set of 13,858 small molecule binding pockets were already available from a previous study in the laboratory. To systematically identify druggable targets in this category, a genome-wide screen was carried out to comprehensively identify all proteins binding to NTP ligands. Being equipped on one hand, with the knowledge of binding site motifs, and on the other, with the structural models of Mtb proteins at a genome-scale, this exercise was made possible. A total of 1,768 proteins in Mtb (about 43% of the proteome) were predicted to bind NTP ligands, which constitute the NTPome. Using an experimental proteomics approach involving dye-ligand affinity chromatography, 47 different proteins were validated, of which four are hypothetical proteins. This analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes several known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets. Further, 305 proteins in the NTPome were identified as important drug targets. A successful drug discovery program stands on the first principles of good target identification. Previous studies carried out in the laboratory combined with contributions from this thesis work, a promising target, nucleoside diphosphate kinase (NDK), that possesses many of the desirable properties of an ideal target, was identified. Using the NTP motifs previously derived, the nucleotide pocket in NDK was studied for the most important residues that confer binding, thus prioritizing the pocket residues for mediating crucial interactions with the ligand. Next, a screen of the nucleotide pocket of NDK against the drug-binding sites known in PDB resulted in identifying a handful of potential screening candidates that contained several elements of an ideal ligand for the NDK pocket. A thorough biochemical characterization using protein purification and subsequent quantification by a spectrophotometric coupled assay, led to the experimental testing of the identified candidates against NDK enzyme activity. This resulted in identifying enalapril and cloxacillin as NDK inhibitors. Of the two compounds, enalapril was efficient in 100% inhibition of NDK activity, while cloxacillin showed a maximum inhibition of 78%. A test for inhibition of Mtb H37Rv growth resulted in determining cell inhibition for both compounds, of which cloxacillin was known previously. Thus, by using the principles of structure-based drug design, two lead molecules were identified, which are reported for the first time for Mtb NDK. Thus, using a structural bioinformatics approach, a large-scale characterization of small-molecule binding sites has been carried out in this thesis work. The work starts from identifying ligand recognition principles of proteins and understanding the molecular capabilities of ligand binding in diverse proteins, which forms the crux of protein-ligand interaction profiling. Using the sub-structures for recognizing function in uncharacterized proteins, the work carried out here presents a pipeline for function annotation. One goal of understanding the functional capabilities of macromolecules is to design and develop drugs, which have also been dealt with in this thesis work, housing the principles of target identification, druggability and finally, lead identification

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.

碩士
國立清華大學
生命科學系
91
Crystal Structure of Nucleosie Diphosphate Kinase in Rice Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms (eukaryotes and prokaryotes) and cell type. It catalyses the transfer of the tpphosphoryl group from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP). NDK is involved and required for coleoptile elongation in rice. The level of the enzyme changes during seed germination and the early stages of seedling growth. Although several NDK structures from bacteria and animals have previously been available, the structures from plants are not yet determined. Crystals of the rice NDK have been obtained and several sets of data were collected at SPring-8 BL12B2 in Japan. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell parameters a = 70.98 Å, b = 182.26 Å, c = 188.30 Å, and diffract to 2.5 Å. The structure is determined by molecular replacement method and shows the overall folding with four -sheets surrounded by six -helices. The model contains 149 residues of a total 152 residues and averaged 18 water molecules. Hexameric molecular packing in both crystals and solution implies the rice NDKs function as hexamers. In this study, we have determined the first rice NDK structure, which provides the detailed structural-functional information to understand the functional significance of this enzyme during plants growth and development in rice as well as plants.

碩士
國立臺灣大學
分子醫學研究所
107
The fission and fusion dynamics of mitochondria are crucial for their role in physiological processes and are precisely regulated. As protein machineries governing mitochondrial dynamics have been fully discovered, how lipids affect mitohcondrial morphology remains comparatively unclear. Highly conserved nucleoside diphosphate kinases are previously discovered as GTP fueling enzymes for dynamin superfamily proteins during membrane remodeling processes. Here we demonstrated that NME3 is required for mitochondrial morphology maintenance that depletion of NME3 caused mitochondrial fragmentation in C2C12 myoblast as well as in myotube. Surprisingly, a kinase-dead NME3 could still rescued this phenotype, yet the N-terminus truncated NME3 could not. Utilizing biochemical reconstitution experiments, we found NME3 directly binds to phosphatidic acid (PA) and functions as a mitochondrial tethering protein through the amphipathic helix formation and oligomeric architecture. Together my study proposes a function of amphipathic helix in organelle integrity maintenance and provides a mechanistic explanation for the role of PA in mitofusin-mediated mitochondrial fusion.

博士
國立臺灣大學
生物化學暨分子生物學研究所
103
Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. Ribonucleotide reducatse (RNR) is a rate-limiting enzyme for dNTP synthesis required for DNA repair, by reduction of four ribonucleoside diphosphates to generate corresponding dNDPs. In my thesis, the functional importance of UMP/CMP kinase (CMPK), an enzyme catalyzes (d)CDP formation and nucleoside diphosphate kinase 3 (NME3), one of ten isoforms of NME that converts (d)NDPs to (d)NTPs in DNA repair are demonstrated. Knockdown of CMPK delayed DNA repair after UV-irradiation in serum-deprived cells but had no effect on proliferating cells, while knockdown of NME3 declined DNA repair rate after doxorubicin exposure in proliferating cells. In addition, these two enzymes could form a complex with RNR and Tip60, a histone acetyltransferase by their N-terminal regions and recruited to DNA damages sites in a Tip60-depedent manner. The mutants of CMPK and NME3 with their N-terminal sequences deletion to eliminate their interaction with Tip60 also show the functional deficiency in DNA repair as compared to wild-type enzymes. I propose that the site-specific multienzyme complex formation provides a means for local synthesis of dNTPs to facilitate DNA repair in serum-deprived cells, which contain low cellular dNTP supply from cytosol. In addition to Tip60/RNR interaction for dNTPs supply at DNA damage sites, another function of NME3 in proliferating cells for DNA repair may involve the local production of ribonucleoside triphosphates (rNTPs).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1991.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 202-218).

Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below. N1TP + NDK ↔N1DP+ −NDK-His* (1) N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M. smegmatis, being a saprophytic, fast growing and non-pathogenic mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism. The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans. Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from Mycobacterium smegmatis mc 155 The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M. smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK. Chapter 3. Characterisation of Oligomerisation Property of M. smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat and SDS. Chemical cross-linking experiments revealed that MsNDK forms dimer, tetramer and hexamer. Homology modeling of MsNDK on the MtNDK crystal structure supported the existence of hexamer as three homodimers. Gln 17, Ser 24 and Glu 27 were found to be positioned at the dimer interface. Mutations on these residues did not abolish the stability of the respective mutant dimers in the presence of SDS and heat. Modeled structure of MsNDK revealed the existence of hydrophobic interactions at the dimer interface. In silico approach helped in mapping the existence of hydrophobic interactions at the dimer interface as two consecutive β-strands. Exposure of hydrophobic residues, using organic solvent methanol, abolished the dimer completely, indicating the vital role of hydrophobic interactions in the dimer stability. In solution, the native MsNDK was found to be a hexamer. Chapter 4. Mycobacterial Nucleoside Diphosphate Kinase Functions as GTPase Activating Protein for Mycobacterial Cytokinetic Protein FtsZ In Vitro Mammalian, plant, and bacterial NDKs can function as GTPase activating protein (GAP) for small G proteins namely, p21 Ras, Rad, and Rho-GTPases in animals and Pra1, Pra2, and GPA1 in Arabidopsis thaliana in vitro. We examined whether NDK of M. tuberculosis (MtNDK) can function as GAP in vitro for the cytokinetic protein FtsZ of Mycobacterium tuberculosis (MtFtsZ), which is a protein with a classical G-protein fold, possessing GTP-binding and GTPase activities (like G proteins). Both MtNDK and MsNDK could function as GAP for MtFtsZ and FtsZ of M. smegmatis (MsFtsZ) respectively in vitro. Similarly, MtNDK could function as GAP for MsFtsZ and reciprocally MsNDK could function as GAP from MtFtsZ. Interaction of NDK with respective FtsZ could be observed. Physiological implications of GAP activity of NDK on FtsZ are discussed. Chapter 5. Transcriptional Analyses of Nucleoside Diphosphate Kinase Gene of Mycobacterium smegmatis mc 155 Although there are studies on the structural and functional aspects of NDK, there are not many studies available on the transcriptional analysis of nucleoside diphosphate kinase (NDK) gene expression in general and nothing in particular in mycobacterial systems. Therefore we studied the transcriptional analysis of expression of Msndk gene, in order to map the Transcriptional Start Site (TSS), identification of promoter elements, and elucidated of transcriptional activity of the promoters. Expression of Msndk gene was analysed in exponential growth phase and under two different stress conditions wherein DNA replication gets arrested. Hydroxy Urea (HU), which reduce dNTP pools by inhibiting ribonucleotide reductase and Phenethyl Alcohol (PEA), which affects membrane structure resulting in DNA replication arrest, were used. Two transcripts and their promoter elements were mapped and their promoter activities were demonstrated. The profile of transcripts was found to be identical under the three different conditions examined.

Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below. N1TP + NDK ↔N1DP+ −NDK-His* (1) N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M. smegmatis, being a saprophytic, fast growing and non-pathogenic mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism. The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans. Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from Mycobacterium smegmatis mc 155 The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M. smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK. Chapter 3. Characterisation of Oligomerisation Property of M. smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat and SDS. Chemical cross-linking experiments revealed that MsNDK forms dimer, tetramer and hexamer. Homology modeling of MsNDK on the MtNDK crystal structure supported the existence of hexamer as three homodimers. Gln 17, Ser 24 and Glu 27 were found to be positioned at the dimer interface. Mutations on these residues did not abolish the stability of the respective mutant dimers in the presence of SDS and heat. Modeled structure of MsNDK revealed the existence of hydrophobic interactions at the dimer interface. In silico approach helped in mapping the existence of hydrophobic interactions at the dimer interface as two consecutive β-strands. Exposure of hydrophobic residues, using organic solvent methanol, abolished the dimer completely, indicating the vital role of hydrophobic interactions in the dimer stability. In solution, the native MsNDK was found to be a hexamer. Chapter 4. Mycobacterial Nucleoside Diphosphate Kinase Functions as GTPase Activating Protein for Mycobacterial Cytokinetic Protein FtsZ In Vitro Mammalian, plant, and bacterial NDKs can function as GTPase activating protein (GAP) for small G proteins namely, p21 Ras, Rad, and Rho-GTPases in animals and Pra1, Pra2, and GPA1 in Arabidopsis thaliana in vitro. We examined whether NDK of M. tuberculosis (MtNDK) can function as GAP in vitro for the cytokinetic protein FtsZ of Mycobacterium tuberculosis (MtFtsZ), which is a protein with a classical G-protein fold, possessing GTP-binding and GTPase activities (like G proteins). Both MtNDK and MsNDK could function as GAP for MtFtsZ and FtsZ of M. smegmatis (MsFtsZ) respectively in vitro. Similarly, MtNDK could function as GAP for MsFtsZ and reciprocally MsNDK could function as GAP from MtFtsZ. Interaction of NDK with respective FtsZ could be observed. Physiological implications of GAP activity of NDK on FtsZ are discussed. Chapter 5. Transcriptional Analyses of Nucleoside Diphosphate Kinase Gene of Mycobacterium smegmatis mc 155 Although there are studies on the structural and functional aspects of NDK, there are not many studies available on the transcriptional analysis of nucleoside diphosphate kinase (NDK) gene expression in general and nothing in particular in mycobacterial systems. Therefore we studied the transcriptional analysis of expression of Msndk gene, in order to map the Transcriptional Start Site (TSS), identification of promoter elements, and elucidated of transcriptional activity of the promoters. Expression of Msndk gene was analysed in exponential growth phase and under two different stress conditions wherein DNA replication gets arrested. Hydroxy Urea (HU), which reduce dNTP pools by inhibiting ribonucleotide reductase and Phenethyl Alcohol (PEA), which affects membrane structure resulting in DNA replication arrest, were used. Two transcripts and their promoter elements were mapped and their promoter activities were demonstrated. The profile of transcripts was found to be identical under the three different conditions examined.

碩士
國立陽明大學
生化暨分子生物研究所
102
Deoxyribonucleic acid (DNA) is composed of four deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP). The adequate intracellular dNTPs supply is crucial for DNA replication and repair. It has been reported that the rate-limiting enzyme of de novo pathway, ribonucleotide reductase(RNR) interacts with histone acetyltransferase 5 (Tip60) through R1 subunit, by which RNR is recruited to DNA damage sites. DNA repair needs dNTPs for material. dNDPs are generated by RNR, which required the coupling of nucleoside diphosphate kinase (NME) to complete dNTPs synthesis. There are 8 NME isoforms in human cells. In this study, I aimed to address whether NMEs are involved in DNA repair by forming complex with Tip60 and RNR. By using immunoprecipation, I identified NME3,NME4 and NME6 capable of interacting with Tip60. NME3 and NME4 complexes contain R2 subunit of endougenous RNR. Moreover, NME3 can form a complex with CMPK. Deletion analysis indicated that N-terminal extension fragment of NME3 is required for Tip60 complex formation, but not required for CMPK. By in vitro binding assay, I found that NME3 can directly interact with Tip60. Knockdown of NME3 reduces DNA repair efficiency during recovery from doxorubicin-induced DNA damage. Re-expression of wild type but not N-terminal deletion of NME3 restores the efficiency in DNA repair in NME3 knockdown cells. I proposed that complex formation of NME3 and Tip60 for dNTPs synthesis is critical for DNA repair.

Les peroxyrédoxines (PRXs) forment une famille de peroxydases communes à tous les organismes vivants et ubiquitaires dans la cellule. Leur particularité provient d’un ou deux résidus cystéines accomplissant un cycle d’oxydo-réduction à l’aide d’un donneur d’électron. Ces protéines thiols sensibles au potentiel redox sont impliquées dans le mécanisme de détoxification du H2O2, une molécule oxydante induite lors de situations de stress. Les PRXs pourraient être induites par le stress et régulées par phosphorylation. En effet, des expérimentations in vitro ont démontré que la nucléoside diphosphate kinase 1 (NDPK1) a la capacité de phosphoryler une PRX cytosolique de pomme de terre. Ce mémoire décrit les travaux expérimentaux effectués pour caractériser la fonction de la PRX. Pour cela, le clonage d’une isoforme a été effectué, suivi d’une caractérisation biochimique et d’une étude d’expression de la protéine. Les données de séquençage révèlent qu’il s’agit d’une PRX de type II phylogénétiquement liée aux PRXs cytosoliques. L’ADNc codant pour cette peroxyrédoxine (PRX1) a été cloné chez Solanum chacoense. Une protéine recombinante portant une étiquette (6xHis) en N-terminale a été produite. Des essais enzymatiques ont confirmé la fonction antioxydante de la protéine recombinante et un anticorps polyclonal a été généré chez le lapin puis utilisé en conjonction avec un anticorps anti-NDPK1 pour déterminer les patrons d’expression généraux de ces protéines chez Solanum lycopersicum et Solanum tuberosum lors de situations de stress. Les données démontrent que les deux protéines sont généralement co-exprimées mais pas co-régulées et que la PRX1 est induite en certaines situations de stress.
The peroxiredoxins (PRXs) are a recently discovered family of peroxidases found in all organisms and ubiquitous in the cell. An important particularity of these proteins is the presence of one or two active cysteines that accomplish an oxydo-reduction cycle with an electron donor. The PRXs are sensitive to the redox potential and are implicated in the detoxification of the H2O2, an oxidante molecule induced in stress situations. The PRXs should be induced in stress situations and regulated by phosphorylation. Indeed, in vitro experimentations have shown that the NDPK1 can phosphorylate a cytosolic PRX isoform of the potato. This dissertation describes the experimentation made to acquire a preliminary understanding of the function of the PRX. For this purpose, we cloned a PRX isoform, followed by a biochemical characterization and expression studies of the protein. The sequencing data shown a type II PRX phylogenetically related to the cytosolic isoforms. The cDNA of this peroxiredoxin (PRX1) has been cloned in Solanum chacoense. The recombinant protein produced had a N-terminal (6xHis) tag. Enzymatic assays confirmed the antioxidant activity of the recombinant protein and a polyclonal antibody has been generated from the rabbit. This antibody was used in conjunction with an antibody anti-NDPK1 to determine the general expression patterns of those proteins during stresses in Solanum lycopersicum and Solanum tuberosum. The results obtained showed that the two proteins are generally co-expressed but not co-regulated. Obvious experimental facts displayed an induction of the PRX1 in biotic and abiotic stresses situations.