Rozprawy doktorskie na temat „ATPase”

Une banque complète de l'ADN mitochondrial de la lignée HA89 B de tournesol a été construite dans le cosmide pHC79 au site SaII. En utilisant des sondes de mais codant pour les sous-unités α, 6 et 9 du complexe ATPase les gènes de tournesol correspondants ont été identifiés, isolés de la banque et cartographiés. Chez le tournesol, ces trois gènes ne sont présents qu'en une seule copie par génome mitochondrial et respectivement portés par les fragments de restriction SaII de (13,5 et 4,5), 13 et 7 kbp. Dans une première partie, nous avons comparé l'organisation moléculaire et la transcription, au niveau des fleurs, des gènes atpase α, 6 et 9 entre les lignées male-fertile (b) et male-stérile (a) de deux formes (cd et 62) d'un même couple isogénique HA 89. Les deux formes, CD et 62, sont identiques pour les caractères testés. Une recombinaison a été detectée de facon spécifique chez les lignées male-stériles en amont du gène codant pour l'atpase α. Les études de transcription réalisées révèlent, chez les lignées male-stériles, l'existence d'un profil d'ARN codant pour l'atpase α modifiés par rapport à ceux des lignées male-fertiles. Dans une deuxième partie, nous avons déterminé la séquence du gène codant pour l'atpase 9. Le gène de tournesol est actuellement le plus long gène atpase 9 trouvé chez les plantes supèrieures avec 252 nucléotides. La comparaison de cette séquence avec celles obtenues chez 17 autres organismes bactériens, de champignons (gènes nucléaires et mitochondriaux), et de plantes supèrieures (gènes chloroplastiques et mitochondriaux) nous a permis d'ériger un arbre phylogénétique des gènes atpase 9

Le encefalomiopatie mitocondriali rappresentano un gruppo di malattie estremamente eterogeneo ed in continua crescita sia per il sempre maggiore numero di fenotipi clinici descritti che per le conoscenze delle basi molecolari che le sottendono. Esse sono, collettivamente, il risultato di una diminuita capacità da parte dei mitocondri di soddisfare le richieste energetiche della cellula, a causa per lo più della ridotta attività enzimatica della catena respiratoria e la conseguente compromessa produzione di ATP intracellulare. In questo studio sono state indagate le conseguenze patogenetiche delle quattro mutazioni più frequentemente associate alla NARP/MILS - T8993G (L156R), T9176G (L217R), T8993C (L156P) e T9176C (L217P) - nel gene mitocondriale ATPasi 6, che codifica per una subunità del complesso V. La F1F0-ATP sintetasi (complesso V) è un enzima ubiquitario, presente nelle membrane dei batteri, dei cloroplasti e nella membrana interna dei mitocondri dove sintetizza ATP, l' ultimo step della fosforilazione ossidativa (Garcia, 2002). L' enzima è costituito da due subcomplessi F0 e F1. L' attività catalitica dell' enzima è localizzata nella porzione F1, che è estrinseca alla membrana ed utilizza il gradiente protonico per convertire ADP in ATP e viceversa; la subunità F0 si trova nella membrana mitocondriale interna e permette il flusso di protoni dallo spazio intermembrana alla matrice. Valutando la capacità duplicativa delle cellule in terreno standard, abbiamo potuto osservare che i fibroblasti di pazienti con la mutazione T->G, crescono con maggiore difficoltà rispetto a quelli con la mutazione T->C ed ai fibroblasti di controllo. Questa condizione si aggrava ulteriormente quando le cellule vengono sottoposte a stress, come la sostituzione del glucosio con il galattosio, un metabolita che entrando nella glicolisi più a valle, fornisce un numero minore di molecole di ATP e mette in evidenza i danni a carico della catena respiratoria. L' attività del complesso V, in termini di sintesi ed idrolisi, della catena respiratoria è stata valutata sui mitocondri ottenuti sia dai fibroblasti omoplasmici per le quattro mutazioni, che da cloni a diversa percentuale di mutazione. Tali indagini, effettuate mediante metodiche di tipo spettrofotometrico, hanno confermato la gravità delle mutazioni T->G rispetto alle varianti alleliche in entrambi i tipi cellulari. Lâ utilizzo inoltre di cloni a diversa percentuale di mutazione, ha consentito di determinare la soglia del fenotipo patologico per le mutazioni T->G. Inoltre abbiamo studiato lâ effetto dell' oligomicina e della sonicazione sull' attività dell' ATPasi nelle cellule primarie e nei cloni. I nostri risultati suggeriscono che le linee primarie con la mutazione L156R esibiscono una porzione F1 debolmente legata alla membrana poiché l' attività idrolitica non è pienamente sensibile alla oligomicina e poiché a 20â di sonicazione l' ATPasi diventa insensibile all'inibitore, probabilmente per il dovuto rilascio della porzione F1 dalla membrana.
Mitocondrial ATP synthase consists of two functional domains, F1 and F0. F1 protudes in to the matrix, is hydrophilic, and contains five subunits (3α,3β,γ,δ,ε) and inibibitor protein. F0 is hydrophobic, is embedded in the mitochondrial inner membrane, and contains subunits a, b, c, d, e, f, g, F6, OSCP and A6L. F1 is connected to F0 by a stalk that contains the subunits OSCP, F6, b, d. Subunits 6 and A6L are encoded by the mitochondrial genome whereas all of the other subunits are encoded by nuclear genes. ATP synthase deficiency can therefore be due to mutations of either nuclear or mitochondrial genes. Mutations in the ATPase 6 gene, characterized by a profound defect of the activity of the enzyme, are recognized as a cause of maternally-inherited LS. Leighâ s syndrome (LS) is a neurodegenerative disorder of infancy characterized by developmental delay, psychomotor regression, seizures, and symptons of brainstem dysfunction. In addition, biochemical defects in complex I, complex IV and PDHC underlie most of the LS patients. In order to know the different pathogenic mechanism leading to diverse clinical severity, of the mutations in the ATPase 6 gene, we compared cellular ATP production, cell growth and entity of cellular response upon use of metabolic stressors in primary cell cultures obtained from patients harboring either the T8993G, or T8993C, or T9176G, or T9176C and in cybrids clones obtained after fusion of each primary cell lines with Ï 0 cells. Arginine (â â Râ â ) mutations were associated with a much more severe phenotype than Proline ( P ) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both R mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two R mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel.

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni.
The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.

Fighting metastasis is a major challenge in cancer therapy and novel therapeutic targets and drugs are highly appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell-extracellular matrix (ECM) contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is therefore a promising therapeutic approach. The vacuolar H+-ATPase (V-ATPase), a proton pump located at the membrane of acidic organelles, has recently come to focus as an anti-metastatic cancer target. As V-ATPase inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis we proposed that V-ATPase inhibition induces anoikis related pathways in invasive cancer cells. In this study the V-ATPase inhibitor archazolid A was used to investigate the mechanism of anoikis induction in various metastatic cancer cells (T24, MDA-MB-231, 4T1, 5637). Therefore, cells were forced to stay in a detached status to mimic loss of cell-ECM engagement following treatment with archazolid. Indeed, anoikis induction by archazolid was characterized by decreased expression of the caspase-8 inhibitor c-FLIP and caspase-8 activation, thus triggering the extrinsic apoptotic pathway. Interestingly, active integrin β1, which is known to play a major role in anoikis induction and resistance, is reduced on the cell surface of archazolid treated cells. Furthermore, a diminished phosphorylation of the integrin downstream target focal adhesion kinase could be demonstrated. The intrinsic apoptotic pathway was initiated by the pro-apoptotic protein BIM, increasing early after treatment. BIM activates cytochrome C release from the mitochondria consequently leading to cell death and is described as one major inducer of anoikis in non-malignant and anoikis sensitive cancer cells. Of note, we observed that archazolid also induces mechanisms opposing anoikis such as proteasomal degradation of BIM mediated by the pro-survival kinases ERK, c-Src and especially Akt at later time points. Moreover, induction of reactive oxygen species (ROS) influences BIM removal as well, as moderate levels of ROS have second messenger properties amplifying cell survival signals. Thus, to antagonize these anoikis escape strategies a combination of archazolid with proteasome or ROS inhibitors amplified cancer cell death synergistically. Most importantly, intravenous injection of archazolid treated 4T1-Luc2 mouse breast cancer cells in BALB/cByJRj mice resulted in reduced lung metastases in vivo. To summarize this work we propose archazolid as a very potent drug in inducing anoikis pathways in metastatic cancer cells even though having learned that detachment together with treatment triggers multiple resistance mechanisms opposing cell death. Hence, V-ATPase inhibition is not only an interesting option to reduce cancer metastasis but also to better understand anoikis resistance and to find choices to fight against it.

Thesis (Ph.D.)--University of Toledo, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Document formatted into pages: iii, 156 p. Title from title page of PDF document. Bibliography: pages 64-67, 97-100, 116-117, 125-155.

Thesis (Ph.D.)--University of Toledo, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

Purification of the DNA gyrase B protein consistently led to two contaminating bands of 47kDa and 43kDa molecular masses. These were found to be the C- and N-terminal fragments of GyrB. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA. This was due to about 90% of GyrB being in an uncoupled form. The uncoupled GyrB was found to have a relatively high ATPase activity, therefore an in-depth kinetic study on DNA gyrase was not possible. Kinetic studies were carried out on the 43kDa protein, which is a cloned N-terminal fragment of GyrB. The 43kDa protein was found to hydrolyse ATP at a relatively low rate, with 10 muM 43kDa having and apparent kcat of 0.01 s-1, and 20 muM a kcat of 0.02 s-1. A greater than first order dependence of rate upon 43kDa concentration was observed in the concentration range of 2-40 muM. Hyperbolic type kinetics were observed at constant 43kDa concentration (5, 10, 20 and 40 ?M) for the rate with respect to ATP concentration. A model which was found to be consistent with molecular weight studies and the kinetic data has been proposed. The 43kDa monomer can bind ATP but is not competent to hydrolyse ATP. Hydrolysis can only occur in the context of a 43kDa2ATP2 dimer, which leads to the collapse of the dimer into monomers and release of products. The rate limiting step at the protein concentration range used is the dimerisation step. Novobiocin and coumermycin inhibit the ATPase reaction, with novobiocin binding at a stoichiometry of 1 novobiocin molecule to 1 43kDa monomer and coumermycin binding with a stoichiometry of 1 molecule to two molecules of 43kDa protein. The inhibition by coumarin drugs appears non-competitive. ADPNP binding to the 43kDa protein was found to be slow, with a second order rate constant of 0.86 M-1s-1 to 9.9 M-1s-1. ADPNP seems to bind with stoichiometries varying from 2 per 43kDa dimer to 1 per 43kDa dimer. ATP and ADP inhibit the amount of ADPNP bound, with ATP having no effect on the rate of ADPNP binding and ADP decreasing the rate of ADPNP binding. Novobiocin and coumermycin inhibit ADPNP binding to the 43kDa protein. ADPNP dissociates from the 43kDa protein at a very slow rate, with a half life of about 8 days. ATP and ADP have little effect on this rate. However high concentrations of novobiocin (10-100 mM) dramatically increase the rate of ADPNP dissociation from the 43kDa protein, indicating different coumarin and nucleotide binding sites.

La Na/K ATPase est impliquée dans la physiopathologie de la neuropathie diabétique. Cette activité enzymatique est basse chez le patient diabétique de type 1, particulièrement chez ceux atteints de neuropathie, et chez certains sujets prédisposés à la neuropathie en cas de diabète. Les facteurs génétiques et environnementaux pouvant influencer l'activité Na/K ATPase ont été analysés. A cet effet, l'érythrocyte a été utilisé comme source enzymatique. Il semble que l'activité de la Na/K ATPase dans le globule rouge reflète celle dans le nerf. En effet, les activités Na/K ATPase nerveuses et érythrocytaires sont étroitement corrélées entre elles et aux vitesses de conduction nerveuses. D'autre part, la même isoforme al de la Na/K ATPase est exprimée dans ces deux types cellulaires. L'activité Na/K ATPase érythrocytaire est diminuée chez les sujets diabétiques de type 1 par rapport aux sujets contrôles et diabétiques de type 2. Cette activité est indépendamment corrélée au taux circulant de peptide C. L'activité Na/K ATPase est abaissée chez les sujets contrôles et diabétiques d'origine nord africaine. Les interventions thérapeutiques montrent que l'insuline et le peptide C, non sécrétés en cas de diabète insulinoprive, stimulent l'activitcNa/K ATPase. Une activité Na/K ATPase basse semble être un marqueur de neuropathie diabétique. Il pourrait exister une prédisposition génétique à développer une neuropathie. C'est pourquoi un polymorphisme de restriction, par l'enzyme Bgl Il, du gène ATPlAl codant pour !'isoforme al de la Na/K ATPase a été recherché. L'allèle clivé [R] du gène ATPlAl est associé à une activité Na/K ATPase abaissée, une diminution d'expression de al ainsi qu'à une prévalence accrue de la neuropathie. Cette influence du fond génétique n'est observable que chez les sujets insulinoprives. Ces faits suggèrent une interaction entre les facteurs génétiques (présence de l'allèle clivé [R]) et les facteurs environnementaux (absence de sécrétion de peptide C).

Os crustáceos são originariamente marinhos; ao longo da evolução, diversas espécies invadiram ambientes de salinidades menores, chegando à água doce. A capacidade dos crustáceos colonizarem com sucesso o ambiente dulcícola depende do desenvolvimento de mecanismos eficientes de hiperosmorregulação. A osmolalidade e a composição iônica da hemolinfa de um crustáceo, em meios diluídos, refletem o equilíbrio dinâmico entre a perda de íons por difusão e pela urina e sua reabsorção do meio externo, através das brânquias. A (Na,K)-ATPase branquial desempenha um papel chave no processo de captura de Na+ a partir de ambientes diluídos e suas características cinéticas vem sendo investigadas recentemente, embora as enzimas de caranguejos dulcícolas sejam pouco conhecidas. Segundo o modelo atual, a afinidade por Na+ é o parâmetro cinético mais variável entre as enzimas de diferentes espécies, refletindo a salinidade do habitat do animal, de modo que enzimas de espécies bem adaptadas à água doce apresentam afinidades maiores por Na+. Entretanto, vários resultados conflitantes têm sido relatados nos últimos anos. Recentemente, foi proposto que uma V-ATPase também desempenha papel essencial na captação de Na+ através das brânquias dos crustáceos dulcícolas. Esta enzima ainda é praticamente desconhecida: suas características cinéticas não foram estudadas e a relação entre a magnitude da sua atividade e a salinidade do meio externo não está estabelecida. Este projeto teve por objetivo a caracterização das enzimas (Na,K)-ATPase e V-ATPase das brânquias posteriores do caranguejo hololimnético Dilocarcinus pagei, considerado um antigo invasor da água doce. A (Na,K)-ATPase foi caracterizada em animais mantidos em água doce, a fim de comparar suas propriedades cinéticas com aquelas das enzimas de outras espécies de caranguejos, habitantes de meios mais salinos, visando melhorar o entendimento das adaptações bioquímicas associadas à invasão da água doce. A V-ATPase foi caracterizada em animais mantidos em água doce ou expostos por diferentes intervalos de tempo à salinidade de 21‰ ou ainda aclimatados por 10 dias a diferentes salinidades (5-21‰), visando estabelecer uma relação entre a magnitude da atividade e a salinidade do meio, além de investigar os mecanismos de regulação da atividade da enzima. A análise da fração microsomal branquial de D. pagei mantido em água doce em gradiente contínuo de sacarose mostrou dois picos protéicos (25-35% e 35-45% de sacarose), ambos com atividades K+-fosfatase, (Na,K)-ATPase e V-ATPase. Estes resultados indicam a presença de frações de membrana com densidades distintas, apresentando, em ambos os casos, as principais bombas de íons envolvidas na captação de Na+. Estas membranas podem ser originárias de locais distintos do epitélio branquial posterior assimétrico deste caranguejo. A análise por Western blotting revelou duas bandas imunoespecíficas (Mr 116 kDa e 105 kDa) correspondentes à subunidade α da (Na,K)-ATPase, sugerindo a presença de duas isoformas nas brânquias posteriores do animal. A estimulação da atividade K+-fosfatase da (Na,K)-ATPase pelo PNFF envolveu interações sítio-sítio (nH= 1,4), com V= 43,4 ± 2,2 U mg-1 e K0,5= 1,13 ± 0,06 mmol L-1. A estimulação da atividade da enzima por K+ (V= 39,9 ± 1,9 U mg-1 e K0,5= 4,2 ± 0,2 mmol L-1), Mg2+ (V= 45,0 ± 2,2 U mg-1, K0,5= 0,82 ± 0,04 mmol L-1) e NH4+ (V= 31,7 ± 1,6 U mg-1, K0,5= 19,0 ± 0,9 mmol L-1) também ocorreu por meio de interações sítio-sítio. A afinidade aparente da enzima pelo PNFF e Mg2+ foi similar às relatadas para enzimas de outros crustáceos, incluindo caranguejos habitantes de meios mais salinos. Entretanto, a enzima de D. pagei apresentou menor afinidade aparente por íons K+ que as outras espécies já estudadas. A atividade K+-fosfatase da (Na,K)-ATPase branquial de D. pagei mantido em água doce foi estimulada sinergicamente por K+ e NH4+ sugerindo a presença de dois sítios de ligação para estes íons na molécula da enzima. Ouabaína (4 mmol L-1) inibiu a atividade PNFFase total da preparação (≈ 89%), por meio de uma curva monofásica (KI= 225,6, ± 11,3 µ mol L-1), sugerindo que, se presentes na fração microsomal, as duas isoenzimas da (Na,K)-ATPase apresentam sensibilidades próximas para o inibidor. Ortovanadato (1µmol L-1) inibiu 95% da atividade PNFFase total por meio de uma curva bifásica, reforçando a sugestão da presença de duas isoenzimas na preparação. A hidrólise do ATP pela (Na,K)-ATPase branquial de D. pagei mantido em água doce ocorreu em sítios de alta (V= 6,4 ± 0,32 U mg-1 e K0,5 = 0,34 ± 0,02 µmol L-1) e baixa afinidade (V= 127,1 ± 6,2 U mg-1e KM = 84 ± 4,1 µmol L-1). Não foi encontrada uma correlação direta entre a afinidade pelo ATP e o habitat de diferentes espécies de caranguejos. A atividade (Na,K)-ATPase específica de D. pagei mantido em água doce foi cerca de 3 vezes menor que relatada para Potamon edulis, única espécie de caranguejo dulcícola para a qual este parâmetro foi relatado. Atividades específicas muito maiores foram encontradas para caranguejos estuarinos, particularmente quando aclimatados a salinidades baixas. A baixa atividade específica determinada para D. pagei pode ser atribuída ao baixo gradiente osmoiônico que este animal mantém entre a hemolinfa e o meio externo, comparado a outros caranguejos dulcícolas, que o caracteriza como uma espécie particularmente bem adaptada ao ambiente dulcícola. A estimulação da atividade da enzima por íons Na+ (V = 133,8 ± 7,3 U mg-1e K0,5= 4,7 ± 0,3 mmol L-1), Mg2+ (V= 136,5 ± 8,0 U mg-1, K0,5= 0,62 ± 0,04 mmol L-1), K+ (V = 131,7± 7,9 U mg-1 e K0,5= 0,47 ± 0,03 mmol L-1) e NH4+ (V= 125,6 ± 6,3 U mg-1, K0,5= 1,90 ± 0,09 mmol L-1) ocorreu por meio de interações sítio-sítio. A afinidade aparente por Na+ da enzima de D. pagei é baixa, se comparada às relatadas para outros animais dulcícolas, e similar às encontradas para espécies estuarino/marinhas. Em contraste, a afinidade aparente por K+ é 2,5 a 5 vezes maior que as determinadas para espécies habitantes de meios mais salinos e aparentemente está mais relacionada ao habitat do animal que a afinidade por Na+. Esta possibilidade é coerente com o fato da (Na,K)-ATPase branquial dos crustáceos apresentar os sítios de ligação de K+ expostos para a hemolinfa, o que possibilita a modulação da atividade da enzima pela concentração de K+ na hemolinfa. Ao contrário do observado para várias outras espécies de caranguejos, a atividade (Na,K)-ATPase branquial de D. pagei não foi estimulada sinergisticamente por K+ e NH4+. Entretanto, a presença de um dos íons no meio reacional provoca o aumento da afinidade aparente da enzima pelo outro em cerca de 3 vezes. Fisiologicamente, esta característica cinética pode ser importante para garantir o transporte de ambos os íons pela enzima, mesmo em presença de concentrações relativamente elevadas do outro. Ouabaína (3 mmol L-1) inibiu a atividade ATPase total (≈ 78%) por meio de uma curva bifásica (KI= 6,21 ± 0,32 µmol L-1 e 101,2 ± 5,1 µmol L-1), reforçando os resultados anteriores no sentido de demonstrar a existência de duas isoenzimas da (Na,K)-ATPase nas brânquias posteriores de D. pagei. Observou-se também uma inibição bifásica por ortovanadato (10 µmol L-1), que inibiu a atividade ATPase total em 85%. O pH ótimo para a atividade V-ATPase branquial de D. pagei foi de 7,5. A modulação da atividade V-ATPase do animal mantido em água doce por ATP (V= 26,5 ± 1,3 U mg-1; K0,5= 3,9 ± 0,2 mmol L-1) e Mg2+ (V = 27,9 ± 1,4 U mg-1; K0,5 =0,80 ± 0,04 mmol L-1) ocorreu por meio de interações cooperativas. Já a inibição da atividade ATPase insensível ao ortovanadato por bafilomicina A1 ocorreu segundo uma curva monofásica (KI= 55,0 ± 2,8 nmol L-1). Cerca de 44 % da atividade ATPase total foi inibida, correspondendo à V-ATPase. A atividade V-ATPase branquial de D. pagei diminuiu acentuadamente em resposta à exposição à salinidade de 21‰. Após 1h de exposição, a atividade diminuiu cerca de 3 vezes, chegando a 4 vezes após 24h, o que indica a atuação de mecanismos eficientes de regulação a curto prazo. Curiosamente, a atividade V-ATPase foi cerca de 2 vezes maior para um tempo de aclimatação de 120h a 21‰, comparado a 24 h, embora 2 vezes menor que a estimada em água doce. Passadas 240 h, a atividade voltou aos baixos níveis observados entre 1h e 24h, o que indica a ação de mecanismos de regulação a longo prazo. Além da diminuição da atividade específica também foi observado aumento da afinidade da enzima por ATP (12 vezes) e Mg2+ (3 vezes) em resposta à exposição dos animais a 21‰. Similarmente, ocorreu um aumento de até 190 vezes na afinidade da enzima por bafilomicina A1. Propõe-se que, em resposta à alteração de salinidade, ocorrem mudanças conformacionais tanto em V1 (onde se encontram os sítios de ligação de ATP e Mg2+) quanto V0 (onde se localiza o sítio de ligação de bafilomicina), resultando numa maior exposição do sítio para o inibidor e no aumento da afinidade por Mg2+ e ATP. Como os aumentos de afinidade são observados já após 1h de exposição, este mecanismo parece ser independente da expressão protéica e, portanto, não estaria relacionado à expressão de isoformas diferentes de alguma das subunidades da enzima. A diminuição da atividade V-ATPase branquial de D. pagei em resposta à exposição a uma salinidade elevada é compatível com os mecanismos propostos para a atuação desta enzima no processo de captura ativa de Na+ em crustáceos dulcícolas. Após 10 dias de aclimatação ainda se tem atividade V-ATPase detectável nas frações microsomais das brânquias posteriores do animal, possivelmente envolvida nas funções de regulação ácido-base e excreção de amônia. Os resultados obtidos para a aclimatação de D. pagei por um período de 10 dias a salinidades entre 5 e 21‰ mostraram também uma diminuição acentuada da atividade V-ATPase em resposta ao aumento da salinidade. Entretanto, com exceção da salinidade mais baixa (5‰) não se observou aumento da afinidade da enzima por bafilomicina, sugerindo que esta alteração seja limitada a tempos de aclimatação mais curtos. Entretanto, também se verificou um aumento acentuado da afinidade da enzima por ATP e Mg2+.
Crustacean arose in the sea but, during evolution, several species invaded lower salinity biotopes, reaching fresh water. The ability of crustaceans to successfully colonize the freshwater biotope depends on efficient mechanisms of hyperosmoregulation. In dilute media, crustaceans\' hemolymph osmolality and ionic composition reflect a balance between diffusive and urinary ion losses, and active ion capture through the gills. The gill (Na,K)- ATPase plays a pivotal role in Na+ capture from dilute environments and its kinetic characteristics are under investigation in recent years, although freshwater crab enzymes are poorly known. According to the most recent model, the apparent affinity for Na+ is the most variable kinetic parameter among gill enzymes from different species, and reflects the salinity of the species\' habitat. Thus, enzymes from species which are well adapted to freshwater usually present higher affinities for Na+. However, several recent results are incompatible with this model. On the other hand, it has been proposed that a V-ATPase is also involved in Na+ capture through the gills of hololimnetic crustaceans. This enzyme is almost completely unknown: its kinetic characteristics have not been studied yet and the relationship between the magnitude of its activity in the gills and the external medium salinity has not been established. This work aimed to characterize the (Na,K)-ATPase and V-ATPase from the posterior gill from the holimnetic crab Dilocarcinus pagei, considered an old fresh water colonizer. The (Na,K)- ATPase was characterized in animals maintained in fresh water, in order to establish a comparison of its kinetic properties with those of enzymes from other crab species that inhabit more saline media. This comparison may enhance our understanding of the biochemical adaptations associated to fresh water invasion. V-ATPase was characterized in animals kept in fresh water or exposed for varying time intervals to a medium of 21? salinity, or else acclimated for 10 days to media of different salinities (5-21?), aiming to establish a relationship between the enzyme specific activity in the gill tissue and the external salinity, and also investigate the mechanisms involved in enzyme activity regulation. The analysis of D. pagei gill microsomes in a continuous-density sucrose gradient revealed two protein peaks (25-35% and 35-45% sucrose), both showing K+-phosphatase, (Na,K)-ATPase and V-ATPase activities. These results indicate the presence of membrane fractions of distinct densities, both presenting the main ion pumps involved in Na+ capture. These membranes may originate from different places in the asymmetric posterior gill epithelium from this crab. Western compared to those reported for other freshwater animals, but similar to those found for estuarine/marine species. In contrast, the apparent affinity for K+ is 2.5 to 5-fold higher than those estimated for species that inhabit more saline media, and is apparently more related to the animals\' habitat than Na+ affinity. This possibility is consistent with the location of the (Na,K)-ATPase in crabs gill tissue, with K+ binding sites exposed to the hemolymph, allowing the direct modulation of enzyme activity by hemolymph K+ concentration. In contrast to data reported for other crab species, D. pagei gill (Na,K)-ATPase activity was not synergistically stimulated by K+ and NH4 +. However, the presence of one of these ions in the reaction medium results in an increase of about 3-fold in the apparent affinity of the enzyme for the other. This kinetic characteristic may be physiologically relevant to assure the transport of both ions, even in the presence of elevated concentrations of the other. Ouabain (3 mmol L-1) inhibited total ATPase activity (? 78%) through a biphasic curve (KI= 6.21 ± 0.32 mol L-1 and 101.2 ± 5.1 mol L-1) reinforcing previous results suggesting the presence of two isoenzymes in the microsomal preparations. A biphasic inhibition by orthovanadate (10 mol L-1) to about 15% residual activity was also observed. Optimal pH for D. pagei gill V-ATPase activity was 7.5. The modulation of enzyme activity of the animal kept in fresh water by ATP (V= 26.5 ± 1.3 U mg-1; K0.5= 3.9 ± 0.2 mmol L-1) and Mg2+ (V = 27.9 ± 1.4 U mg-1; K0.5 =0.80 ± 0.04 mmol L-1) occurred with positive cooperativity. The inhibition of the orthovanadate insensitive ATPase activity by bafilomycin A1 followed a monophasic curve (KI= 55.0 ± 2.8 nmol L-1). About 44 % of total ATPase activity was inhibited, corresponding to the V-ATPase. Dilocarcinus pagei gill V-ATPase activity substantially decreased in response to animal\'s exposure to 21? salinity. After 1h exposure, the activity diminished about 3-fold, reaching 4- fold after 24h, indicating the action of efficient short-time regulation mechanisms. Interestingly, V-ATPase activity was about 2-fold higher after 120h exposure, compared to 24h, although 2- fold lower compared to that estimated in fresh water. After 240h, the activity returned to the low levels observed for 1 and 24 h, indicating efficient long-term regulation. Besides the decrease in specific activity, it was also observed an increase in enzyme\'s apparent affinity for ATP (12 fold) and Mg2+ (3 fold) in response to animal\'s exposure to 21? salinity. Simultaneously, the enzyme\'s affinity for bafilomycin A1 increased up to 190-fold. We propose that, in response to salinity alteration, conformational changes take place both in V1 (in which the ATP and Mg2+ binding sites are located) and V0 (which contains the bafilomycin A1 bindind site), resulting in higher exposition of the inhibitor binding site and also higher affinity for Mg2+ and ATP. As the affinity increases are observed after just 1h exposure, this regulatory mechanism seems to be independent of protein expression and, thus, should not be related to the expression of distinct isoforms of some enzyme subunit. The lowering of gill V-ATPase activity in D. pagei in response to exposure to an elevated salinity is consistent with the mechanisms proposed for the role of this enzyme in active Na+ capture in hololimnetic crustaceans. After 10 days at 21, the gill microsomal fractions still show a little V-ATPase activity, possibly related to acid-base regulation and ammonia excretion processes. The results obtained for the acclimation of D. pagei for 10 days at salinities in the range 5 to 21? also showed a substantial decrease of V-ATPase activity in response to the increase in medium salinity. However, except for 5?, it was not observed an increase of enzyme\'s affinity for bafilomycin, suggesting that this alteration is limited to shorter periods of exposure. However, a significant increase in the enzyme\'s affinity for ATP and Mg2+ was also observed.

La ségrégation, ou partition, des chromosomes et des plasmides bactériens est l'étape fondamentale du cycle cellulaire qui assure la transmission de l'ensemble du génome aux cellules filles. C'est l'équivalent procaryote de la mitose. Des systèmes de ségrégation, appelés les loci par, ont été identifiés sur les plasmides à bas nombre de copies, et des homologues de ces systèmes de partition sont présents sur la majorité des chromosomes bactériens. Le système code deux protéines, une ATPase et une protéine qui se fixe spécifiquement sur une région centromérique. Ces deux protéines interagissent entre elles, permettent la localisation subcellulaire des réplicons et assurent ainsi leur maintien dans les générations futures. Au laboratoire, nous étudions l'un des systèmes modèles majeurs, le système de partition du plasmide F d'Escherichia coli, afin de déterminer le mécanisme moléculaire assurant le processus de ségrégation et son contrôle pendant le cycle cellulaire. La stabilité du plasmide F est assurée par le système de partition sopABC. Après la réplication du plasmide, la protéine SopB s'assemble sur le centromère sopC pour former un complexe de partition qui permet aux copies du plasmide d'être positionnés au centre de la cellule. Avant la division cellulaire les plasmides migrent aux positions 1/4 et 3/4 de la cellule et assurent ainsi l'héritage des réplicons dans les futures cellules filles. L'ATPase SopA est essentielle dans le processus de partition, mais son rôle n'est pas bien défini. SopA pourrait être impliquée dans les étapes de positionnement et/ou de déplacement des plasmides de part et d'autre de la cellule. SopA possède plusieurs activités. In vivo, SopA agit comme autorépresseur de l'opéron sopAB en se fixant sur la région promotrice. De plus elle interagit avec le complexe de partition et forme des polymères en présence d'ATP. Nous avons montré que cette activité est régulée par SopB et par l'ADN. L'activité ATPase de SopA est essentielle pour la partition. Elle est légèrement stimulée par SopB et par l'ADN, mais lorsque ces deux facteurs sont présents, elle est fortement stimulée. Nous avons entrepris de caractériser les interactions existantes entre ces trois protagonistes. Ainsi, nous avons démontré que cette stimulation nécessite une interaction de SopA avec SopB d'une part et avec l'ADN d'autre part. Nous avons également montré que le site centromérique sopC potentialise la stimulation de l'activité ATPase par l'intermédiaire de SopB. Nous nous sommes intéressés ensuite à l'interaction SopA-SopB, et nous avons mis en évidence que SopB stimule l'activité ATPase de SopA via un motif arginine finger. Pour finir, nous avons montré que in vivo, la stimulation de l'activité ATPase de SopA joue un rôle dans la régulation de l'opéron sopAB mais aussi dans la partition du plasmide F
Mitotic segregation of chromosomes and plasmids, termed partition in bacteria, is a fundamental step of the cell cycle that ensures the transmission of the whole genome to daughter cells. It is governed by specific genetic loci named par, first identified in low copy number plasmids and later found to be present as homologues in most bacterial chromosomes. Par loci encode two proteins, an ATPase and a DNA binding protein, and include a cis-acting centromeric site. These components interact with each other to direct the subcellular localization that ensures stability of their replicons. To determine the molecular mechanisms of the partition process and its control during the cell cycle, we study the Sop partition system of the Escherichia coli plasmid, F. Sop is one of the best-known partition systems. After F plasmid replication, SopB protein binds to the sopC centromeric site to form a partition complex. The complex on each plasmid copy interacts with SopA, an ATPase, and activates it to move the plasmid molecules towards the two cell poles. SopA ATPase is essential to the segregation process but its role is not defined. SopA has many activities. In vivo it represses its own operon by binding to the sopAB promoter. Moreover, in addition to its interaction with the partition complex it polymerizes in the presence of ATP. We have shown that SopB and DNA regulate this activity. Although the ATP-binding site on SopA is essential for partition, ATP hydrolysis by SopA is very weak. It is stimulated modestly by DNA and by SopB and strongly in the presence of both. We have characterized the interactions necessary for stimulation of ATP hydrolysis. First we found that the SopB-sopC partition complex is required for maximal stimulation. Then we showed that SopB and DNA contact SopA by two distinct interactions to fully activate ATPase activity. We also found that SopB activates SopA ATPase through an arginine finger motif. Finally, we have shown that in vivo, stimulation of the ATPase activity is necessary for both regulation of the sopAB operon and partition of plasmid F to be efficient

The endogenous Mg²⁺ -ATPase activity of isolated intact turnip and mung bean mitochondria is very low relative to that of animal mitochondria, and is not stimulated by uncouplers. Results indicate that the endogenous ATPase activity is not low due to either the existence of a permeability barrier to ATP, or to a reduced content of the ATPase enzyme in turnip mitochondria. The absence of uncoupler-stimulation with mung bean mitochondria is partly due to the transport and/or permeability characteristics of the membrane. However, in both cases, an (additional) 'ATPase-inhibitory' factor is implicated. The ATPase activity of turnip mitochondria and membrane particles can be 'activated' (up to 50-fold) kinetically, and in a time-dependent manner, evidence which points to the existence of a proteinaceous ATPase-inhibitor, like that in mammalian mitochondria, which is responsible for the low ATPase activity and lack of uncoupler-stimulation in turnip mitochondria. However, alternate inhibitory ligands cannot be dismissed at present. The 'activated' ATPase activity is similar to that of the F_OF₁-ATPase with respect to inhibitor sensitivity, optimum pH, bivalent cation requirement, and sensitivity to 'activating anions'. The F₁ sector has been solubilized from turnip mitochondria by dichloromethane-extraction, and further purified to yield a preparation containing the five polypeptides (α, β, γ, δ, ε) characteristic of MF_1, BF_1 and CF_1, plus an additional sixth polypeptide (δ') which has been found in certain plant (and animal) MF₁ preparations, but whose identity has yet to be determined. The M_r values of the δ and ε polypeptides are closer to those of BF₁ and CF₁ than MF₁. The specific activity of the purified enzyme is higher than previously reported for a plant F₁-ATPase. Other notable features include apparent negative cooperativity for ATP hydrolysis, even with 'activating anions' present, reduced specificity for adenine nucleotides, high Ca²⁺ -ATPase activity, and slight stimulation by the chloride anion.

Molluscan adductor muscles display thick filament regulation. A calcium binding site (regulatory domain) near the neck of the myosin molecule is responsible for controlling the ATPase activity, hence the rate of contraction. In the absence of calcium, the ATPase activity is highly suppressed. When calcium binds to the regulatory domain the inhibition is relieved allowing contraction to occur. Steady-state measurements are insufficient to characterise the ATPase activity in detail because of the dominant contribution from unregulated myosin molecules. Therefore spectoscopic techniques, allied to transient kinetic analysis were used to determine the effect of calcium on the various steps of the HMM ATPase mechanism. ATP, ADP and calcium binding to HMM caused small, but measurable, enhancements (upto 8%) in the proteins tryptophan fluorescence. Stopped-flow fluorescence spectroscopy allowed the kinetics of binding and dissociation to be elucidated. Calcium bound to, and dissociated from, HMM rapidly (108 M-1 S-1 and 25 S-1 respectively). When calcium was bound to the regulatory domain the affinity of the active site for nucleotide was reduced, an effect seen as an increase in the rate constant for nucleotide dissociation. Fluorescent ATP analogues, based on formycin were synthesised. These nucleotides displayed large fluorescence enhancements on binding to HMM (upto 500%). Turnover of FTP by HMM was suppressed 100-fold by the removal of calcium, as determined by transient kinetic measurements. The large fluorescence enhancements seen on binding of various formycin nucleotides allowd the effect of calcium on the association and dissociation processes to be examined in great detail. Binding was found to be a complex, multistep process in which the presence of calcium increased the rate of interconversion of the various HMM/nucleotide complexes by several orders of magnitude.

S.cerevisiae mutants were isolated showing nuclear-coded resistance to the antibiotic venturicidin, a known F0ATPase binding antibiotic. Two types of mutant were identified, one of which had cross-resistance to a variety of antibiotics and appeared linked to the leu1 locus on chromosome VII, and one which was cross-resistant to chloramphenicol only and not linked to leu1. The level of resistance to venturicidin was not increased in isolated mitochondria, therefore resistance shown in both groups is believed to be due to a decrease in plasma membrane permeability to these antibiotics. The fluorescence properties of several organotin compounds, derivatives of the substituted flavones 3-hydroxyflavone (hof) and penta-hydroxyflavone (morin), were investigated on incubation with rat liver mitochondria. The compound Bu2SnBr(of) was found to show fluorescence enhancement when added to mitochondrial preparations, which could be lowered by addition of the non-fluorescent compound Bu3SnAc. Addition of Bu2SnBr(of) did not affect mitochondrial membrane potential, and conversely the energetic state of the mitochondrial inner membrane had no effect on Bu2SnBr(of) fluorescence. This compound was shown to be an inhibitor of mitochondrial ATPase, and is thought to have its binding site on the F0 moiety of that enzyme complex. The nuclear gene causing respiratory deficiency in the complementation group G57 was cloned and sequenced. This gene (PET57) encoded a protein of 36 Kdal which did not show significant homology with any known protein. The mutant strain was deficient in mitochondrial ATPase activity, but the major F1ATPase subunits were detected in mutant mitochondria, although in reduced amounts. Mutant F1 showed abnormal membrane binding and could not be isolated by standard methods. The protein encoded by the gene PET57 is transported into mitochondria and is thought to contribute to processing or assembly of one or more of the cytoplasmic subunits of the F1F0ATPase complex.

The mitochondrial F-ATPases make about 90% of cellular ATP. They are multi-protein assemblies with a membrane extrinsic catalytic domain attached to a membrane embedded sector. They operate by a mechanical rotary mechanism powered by an electro-chemical gradient, generated across the inner mitochondrial membrane by respiration. A detailed molecular description has been provided by X-ray crystallographic studies and "single molecule" observations of the mechanism of the F1 catalytic domain. Details are known also of the architecture of the peripheral stalk of part of the stator and the membrane embedded region of the rotor. However, knowledge of the detailed structure of the rest of the membrane domain, and the detailed mechanism of generation of rotation is lacking. Recently, studies of the intact mitochondrial F-ATPases, determined by cryo-electron microscopy (cryo-em), have provided structural information at intermediate levels of resolution. Whilst these structures have given insights into the mechanism of generation of rotation, the information required for a molecular understanding of this mechanism is still lacking. Moreover, the locations and roles of six supernumerary membrane subunits are unclear. Some of them are likely to be involved in the formation of dimers of the enzyme which line the edges of mitochondrial cristae. Therefore, in this thesis, a procedure is described for the purification of dimers of the bovine and yeast F-ATPases. The structure of the bovine dimer has been determined by cryo-em at a resolution of ca. 6.9 Angstrom. This structure confirms features concerning the trans-membrane spans of the a-, A6L- and b-subunits observed in the monomeric complex. In addition, the single trans-membrane a-helix of the f-subunit has been located, and the subunit appears to mediate dimer formation. The structure of A6L has been extended, and the a-helices of subunits e- and g- have been located. Another novel feature has been assigned to the DAPIT subunit, and may provide links between dimers in forming larger oligomers. Further improvement in the resolution of the structure is hampered by the extreme conformational heterogeneity of the F-ATPase. To this end, the simpler Fo membrane domain has been isolated and characterized initially by electron microscopy in negative stain.

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Quassia amara L. (Simaroubaceae) is a small tree, widely distributed in the Amazon, known as ―pau-amargo‖. The tea from its leaves and bark are used in popular medicine for gastric disorders and malaria. β-carboline and indolic alkaloids, steroids and quassinoids (quassin and neoquassin) were isolated from this plant and evaluated in animal models of malaria with promising results, but still preliminary. Previous pharmacological studies performed with non-standardized apolar extracts, reported sedative, analgesic, anti-inflammatory, emollient and anti-ulcer activities. In view of the reputation of Q. amara in folk medicine and scarce consistent results in the scientific literature, this work studied the systemic actions of the standardized extracts from the native plant collected in the region of Manaus. Semi-purified extracts and high purity fractions were obtained from the chemical standardization and were used to study the mechanisms of actions detected experimentally. In these studies, the popular use of Q. amara in malaria led us also to investigate the pharmacological actions of its extracts and purified fractions in mammalian P-ATPases (H+-K+-ATPase and Ca+2-ATPase) that have high structural homology with isoenzymes essential to the Plasmodium survival. The standardized extract was obtained by the partition of Q. amara AE in buthanol originating the buthanolic fraction (BuF) with the same pharmacological activity and fivefold more concentrated than AE. The purification of BuF by high performance liquid chromatography (HPLC) yielded 18 high purity fractions (F1 to F18), still in identification process. The pharmacological screening of AE and BuF did show remarkable action in the CNS. AE showed anti-inflammatory effect, apparently associated with the inhibition of pro-inflammatory mediators histamine and serotonin, but no analgesic action was observed. AE increased gastrointestinal motility and inhibited the gastric ulcers induced by stress and ethanol. BuF at tenfold lower doses inhibited the secretion and gastric acidity in vivo and the intermediate dose inhibited the total acidity stimulated by histamine, but did not when induced by bethanechol (muscarinic agonist), indicating a possible selective action in the histamine/cAMP pathway. The BuF and fractions isolated by HPLC F10, F14 and F15 potentiated the direct elicited twitches in the rat diaphragm muscle. In the Ca+2-ATPase isolated from skeletal muscle, the BuF and fractions F05, F06, F08, F09, F10, F13, F14, F15,F16 inhibited its activity and this effect may explain the potentiation of the diaphragm contraction. The FBut and fractions F11, F12, F13 and F16 inhibited the H+-K+-ATPase activity from the gastric mucosa in vitro; this effect may explain the anti-secretory and anti-ulcer activity observed in vivo, effects related to the mainly popular use of Q. amara.
A Quassia amara L. (Simaroubaceae) é árvore de pequeno porte, de ampla distribuição amazônica, conhecida como pau-amargo; o chá das folhas e cascas é utilizado na medicina popular para distúrbios gástricos e na malária. Da planta foram isolados alcalóides indólicos e β-carbolínicos, esteróides e quassinóides (quassina e neoquassina) avaliados em modelos do parasitismo animal com resultados promissores, mas ainda iniciais. Estudos farmacológicos anteriores realizados com extratos apolares sem padronização, descrevem atividades sedativa, analgésica, anti-inflamatória, emoliente e anti-úlcera gástrica. Em vista da reputação da Q. amara na medicina popular e da pouca consistência científica dos resultados disponíveis na literatura, este trabalho retomou o estudo das ações sistêmicas do extrato aquoso padronizado da planta nativa coletada na região de Manaus. Da padronização química foram obtidos extratos semi-purificados e frações de alto grau de pureza, que serviram também ao estudo do mecanismo das ações detectadas experimentalmente. Nesses estudos, o uso popular na malária nos levou também a investigar as ações farmacológicas dos extratos purificadas e frações nas H+-K+-ATPase e Ca+2-ATPase de mamíferos, P-ATPases essas que guardam elevada homologia estrutural com as isoenzimas do Plasmodium sp. essenciais à sua sobrevida. A padronização química do EA da Q. amara foi obtida após partição em butanol dando origem à fração butanólica (FBut) de mesma atividade e 5 vezes mais concentrada. A purificação da FBut por cromatografia líquida de alta eficiência (CLAE) originou 18 frações de elevado grau de pureza (F1 a F18) que encontram-se em processo de identificação. A triagem farmacológica do EA e da FBut mostrou ação pouco marcada no SNC. O EA mostrou ação anti-inflamatória, aparentemente associada à inibição dos mediadores pró-inflamatórios histamina e serotonina, mas não mostrou efeito analgésico. O EA aumentou a motilidade gastrintestinal e inibiu as úlceras gástricas induzidas por estresse e por etanol. A FBut, em doses 10 vezes menores, inibiu a secreção e a acidez gástrica in vivo, a dose intermediária inibiu a acidez total estimulada pela histamina, mas não alterou quando induzida por betanecol (agonista muscarínico), indicando provável ação seletiva na via da histamina/AMPc. A FBut e as frações isoladas em CLAE F10, F14 e F15 potenciaram a contração do músculo diafragma de rato sob estímulo elétrico direto. A FBut e frações F05, F06, F08, F09, F10, F13, F14, F15 e F16 inibiram a atividade da Ca2+-ATPase de músculo esquelético; este efeito pode explicar a potenciação da contração do músculo diafragma. A FBut e as frações F11, F12, F13 e F16 inibiram a atividade da H+-K+-ATPase da mucosa gástrica in vitro; este efeito pode explicar a atividade antissecretora ácida e a atividade anti-úlcera observadas in vivo, provavelmente relacionadas com o uso popular mais freqüente.

Les ATPases F1F0 sont présentes chez la majorité des bactéries, notamment les mycoplasmes qui sont caractérisés par un génome réduit et un mode de vie parasitaire. En plus de l’opéron codant l’ATPase F1F0, des clusters apparentés de sept gènes ont été identifiés dans le génome de nombreux mycoplasmes. Au cours de cette thèse, nous avons cherché à caractériser l’évolution et la fonction de ces clusters supplémentaires. Quatre des protéines codées par ces clusters présentent des similarités structurales avec les sous-unités α, β,  et ε de l’ATPase F1F0, résultant en une potentielle structure F1-like. Les trois autres protéines ne présentent aucune similarité avec des protéines connues. Une localisation transmembranaire est prédite pour deux d’entre elles. Deux types d’ATPase F1-like, Type 2 et Type 3, ont été identifiés. Les clusters de Type 2 et de Type 3 pourraient être originaires du groupe phylogénétique Hominis, les clusters de Type 3 ayant vraisemblablement été disséminés par des transferts horizontaux de gènes entre mycoplasmes colonisant le même hôte. Les gènes du cluster de Type 3 de Mycoplasma mycoides subsp. mycoides sont organisés en opéron et exprimés en milieu axénique. Des études de mutagénèse et de complémentation démontrent que le cluster de Type 3 est associé à une activité ATPase majeure des fractions membranaires. Des analyses biochimiques suggèrent que l’activité ATPase du cluster est sensible au ∆pH mais pas au ∆Ψ. Ces analyses suggèrent que le sodium et le potassium ne sont pas impliqués dans le fonctionnement de l’ATPase F1-likeX0. Les sous-unités des ATPases F1-likeX0 et F1F0 présentent un comportement différent en présence de détergents. L’ensemble de ces expériences suggèrent que l’ATPase F1-likeX0 est un complexe plus fragile que l’ATPase F1F0. Nos résultats montrent qu’en dépit d’une tendance à la réduction de génome, les mycoplasmes ont développé et échangé des ATPases sans équivalent chez d’autres bactéries. Nous proposons un modèle dans lequel une structure F1-like est associée avec un domaine hypothétique X0, enchâssé dans la membrane des mycoplasmes
F1F0 ATPases have been found in most bacteria, including mycoplasmas that are characterized by drastically reduced genomes and a parasitic lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase, related clusters of seven genes were identified in many mycoplasmas. In this work, we investigated the evolution and the function of these supplementary clusters. Four proteins encoded by these clusters present structural similarities with subunits α, β,  and ε of F1F0 ATPases, resulting in potential F1-like structures. The three other encoded proteins did not show any similarity to known proteins. Transmembrane helices were predicted for two of them, suggesting a membrane localisation. Two types of F1-like ATPases, Type 2 and Type 3, were identified. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. Further spreading of Type 3 ATPases towards other phylogenetic groups by horizontal gene transfers in between mycoplasmas sharing a same host was proposed on the basis of phylogenetic trees and genomic context. Functional analyses indicated that genes of Type 3 cluster in the ruminant pathogen Mycoplasma mycoides subsp. mycoides were organized as an operon. Proteomic analyses indicated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation assays demonstrated that Type 3 cluster was associated with a major ATPase activity of membrane fractions. Biochemical analyses indicated that this ATPase activity was sensitive to ΔpH but not to ΔΨ. These analyses suggested that Na+ and K+ were not involved in the F1-likeX0 functioning. Our results indicated a behaviour of F1-likeX0 ATPase subunits that is different to that of F1F0 ATPase subunits in presence of detergents. Altogether, these analyses suggest that the F1-likeX0 complex could be more fragile than the F1F0 complex. Our results showed that despite their tendency to genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model in which the F1-like structure is associated with a hypothetical X0 sector embedded in the membrane of mycoplasmal cells

Le carcinome hépatocellulaire (CHC) est le principal cancer primitif du foie et est associé à un très mauvais pronostic. Notre équipe a mis en évidence que la Reptine et la Pontine, des AAA+ ATPases homologues, sont surexprimées dans le CHC par rapport au foie non tumoral. Au cours de ce travail de thèse, j’ai contribué à démontrer que l’extinction de la Reptine par l’induction de shRNA suffit à arrêter la croissance de tumeurs déjà établies, et même à induire leur régression dans des xénogreffes chez la souris. Ces résultats encourageants suggèrent que la Reptine pourrait être une cible thérapeutique dans le CHC. L’utilisation de siRNA en thérapeutique n’étant pas envisageable actuellement, il parait plus pertinent de tenter de cibler la Reptine via son activité ATPase. Le principal objectif de ma thèse était donc de déterminer l’implication de l’activité ATPase de la Reptine pour ses propriétés oncogéniques dans le CHC. Nos résultats ont montré que des mutants inactifs de la Reptine (D299N et E300G) ont un effet dominant négatif et ne sont pas capables de complémenter l’absence de la Reptine endogène, ce qui conduit à une diminution significative de la croissance des cellules HuH7 et Hep3B, et à une induction de l’apoptose. Ceci indique que l’activité ATPase de la Reptine est nécessaire pour la croissance et la survie des cellules de CHC. Enfin, grâce à une étude transcriptomique, nous avons identifié de nouveaux gènes dont l’expression est régulée par la Reptine et/ou la Pontine. Parmi ces gènes, certains pourraient être impliqués dans les fonctions oncogéniques de la Reptine et/ou de la Pontine dans le CHC. Finalement, ce travail a permis de mettre en évidence l’implication de l’activité ATPase de la Reptine, et d’apporter des éléments permettant de mieux comprendre le mécanisme d’action de la Reptine dans le CHC
Hepatocellular carcinoma (HCC) is the main primary cancer of the liver and is often associated with poor prognosis. Our team has demonstrated that Reptin and Pontin, two AA+ ATPases, are overexpressed in HCC compared to normal liver. Moreover this overexpression is also associated with poor prognosis. In the course of my PhD, I demonstrated that shRNA-mediated silencing of Reptin is sufficient to inhibit tumor growth and even can promote their regression in xenografted mice. These encouraging results suggest that Reptin might represent a novel therapeutic target in HCC. As the use of siRNA as therapeutic tools is still debated, the targeting of Reptin enzymatic activity might represent a more relevant approach to impair its functions. To this end I first proposed to determine the involvement of Reptin ATPase activity in HCC oncogenesis. My results show that ATPase inactive Reptin mutants (D299N and E300G) play dominant negative roles toward Reptin functions and are unable to complement for the depletion of endogenous Reptin, thereby leading to a significant decrease of cell growth and to a significant increase of apoptosis in HuH7 and Hep3B cells. These results show that Reptin’s ATPase activity is necessary for HCC cell growth and survival. Moreover, using a transcriptomic approach that compared gene expression upon siRNA-mediated Reptin or Pontin silencing, we identified specific genes whose expression is under the control of those proteins and whose functions might provide mechanistic explanation to Reptin’s involvement in HCC. Collectively, the results obtained during my PhD thesis have characterized the contribution of Reptin ATPase activity to HCC growth and development and might represent a founding step in the understanding of Reptin’s biology in cancer development

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A capacidade de bactérias endofíticas em solubilizar fosfato inorgânico é alvo de grande interesse por parte dos microbiologistas, uma vez que as fosfatases são responsáveis por hidrolisar compostos orgânicos produzindo fósforo solúvel. Dessa forma, a fosfatase ácida ligada à membrana (MBAP) foi obtida a partir de Enterobacter sp. isolada de raízes de Cattleya walkeriana (Orchidaceae) e identificada pelo seqüenciamento do gene 16S rRNA. A expressão da enzima mostrou-se estritamente regulada pelo fósforo (expressão ideal em 7 mm). O pH ótimo aparente (3,5) não foi afetado pela concentração de p-nitrofenilfosfato. Em pH 3,5, a enzima é uma fosfomonidrolase inespecífica capaz de hidrolisar os substratos PNPP (61,2 U/mg), ATP (19,7 U/mg), e o pirofosfato (29,7 U/mg), com K0.5 de 0,06 mM, 0,11 mM e 0,08 mM, respectivamente. A enzima exibi cinética Michaelina para o pNPP (n=1,2). Para o ATP e o pirofosfato interações sítio-sítio foram observadas com n=1,6 e 2,3, respectivamente. Os íons de magnésio foram potentes estimuladores (K0.5=2,2 mM), enquanto o arsenato e o fosfato foram potentes inibidores competitivos. A atividade PNPPase foi inibida pelo EDTA, mas não pelo cálcio, levamisol, zinco, cobalto e phidroximercuribenzoato. A entalpia de inativação térmica foi da ordem de 77,5 kcal.mol- 1. Os resultados sugerem que a produção da fosfatase ácida ligada à membrana representa um mecanismo de solubilização do fosfato mineral aumentando a disponibilidade de nutrientes para as plantas
The ability of endophytic bacteria in solubilizing inorganic phosphate is of great interest by microbiologists since phosphatases are responsible for catalyzing the hydrolysis of organic compounds producing soluble phosphorus. Thus, the membranebound acid phosphatase (MBAP) was obtained from Enterobacter sp. isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis. The enzyme expression was demonstrated to be strictly regulated by phosphorus (optimal expression at 7 mM). The enzyme was obtained by centrifugation at 100.000g for 1 h at 4ºC. The apparent optimal pH (3.5) was not affect by p-Nitrophenyl phosphate concentration. At pH 3.5, the enzyme showed a broad substrate specificity hydrolyzing different substrates such as PNPP (61.2 U/mg), ATP (19.7 U/mg), and pyrophosphate (29.7 U/mg), with K0.5 values of 0.06 mM, 0.11 mM and 0.08 mM, respectively. The hydrolysis of PNPP by the enzyme exhibited Michaelian kinetics with n= 1.2. For ATP and pyrophosphate site-site interactions were observed with n= 1.6 and 2.3, respectively. Although magnesium ions were stimulatory (K0.5= 2.2 mM), arsenate and phosphate were a powerful competitive inhibitor. The PNPPase activity was inhibited EDTA but not by calcium, levamisole, zinc, cobalt and phydroxymercurybenzoate. The ΔH for thermal inactivation was 77.5 kcal.mol-1. Our results suggest that the production of a membrane-bound acid phosphatase might be one mechanism of mineral phosphate solubilization turn it´s nutrients availability to plants

Die vakuoläre Protonen-ATPase, kurz V-ATPase, ist ein multimerer Enzymkomplex, der in fast jeder eukaryotischen Zelle zu finden ist und den aktiven elektrogenen Transport von Protonen über Membranen katalysiert. Die Aktivität der V-ATPase ist essentiell für eine Vielzahl physiologischer Prozesse. Ein grundlegender Mechanismus zur Regulation der V-ATPase-Aktivität ist die reversible Dissoziation des Holoenzyms in den integralen VO-Komplex, der als Protonenkanal dient, und den cytosolischen V1-Komplex, der ATP hydrolysiert und somit den Protonentransport energetisiert. Die Untereinheit C, die im dissoziierten Zustand der V-ATPase als einzige Untereinheit isoliert im Cytoplasma vorliegt, scheint bei der Bildung des aktiven Holoenzyms eine Schlüsselrolle zu übernehmen. In den Speicheldrüsen der Schmeißfliege Calliphora vicina ist die V-ATPase an der Speichelsekretion beteiligt. In den sekretorischen Zellen wird die Bildung des V-ATPase-Holoenzyms in der apikalen Plasmamembran durch das Neurohormon Serotonin (5-HT) stimuliert. Der Effekt von 5-HT auf die V-ATPase wird intrazellulär durch die Proteinkinase A (PKA) vermittelt und hält nur für die Dauer der Stimulierung an. In der vorliegenden Arbeit wurde mittels Phosphoproteinfärbungen und 2D-Elektrophorese nachgewiesen, dass infolge einer Stimulierung der Drüsenzellen mit 5-HT die Untereinheit C der V-ATPase durch die PKA reversibel phosphoryliert wird. Die Phosphorylierung geht einher mit einer Umverteilung der Untereinheit C aus dem Cytoplasma zur apikalen Plasmamembran und der Bildung des aktiven Holoenzyms. Immuncytochemische Untersuchungen zeigten, dass die katalytische Untereinheit der PKA ebenfalls umverteilt wird und in stimulierten Zellen im Bereich der apikalen Plasmamembran konzentriert vorliegt. Um herauszufinden welche Proteinphosphatase der PKA entgegenwirkt, wurden luminale pH-Messungen durchgeführt und der Effekt von spezifischen Proteinphosphatase-Inhibitoren und veresterten Komplexbildnern zweiwertiger Kationen auf die V-ATPase-Aktivität untersucht. Diese Messungen führten zu der Schlussfolgerung, dass eine Proteinphosphatase des Typs 2C an der Inaktivierung der V-ATPase beteiligt ist. Mit weiteren Phosphoproteinfärbungen konnte gezeigt werden, dass die Dephosphorylierung der Untereinheit C ebenfalls durch eine Proteinphosphatase 2C katalysiert wird und dies vermutlich die Dissoziation des VO- und V1-Komplexes begünstigt. Darüber hinaus konnte durch luminale pH-Messungen und ergänzende biochemische Untersuchungen eine Calcineurin-vermittelte Modulation des cAMP/PKA-Signalweges durch den parallel aktivierten IP3/Ca2+-Signalweg und damit einhergehend eine Beeinflussung der V-ATPase-Aktivität durch den [Ca2+]-Spiegel nachgewiesen werden.
The vacuolar-type H+-ATPase (V-ATPase) is a multimeric enzyme that can be found in nearly every eukaryotic cell. It catalyses the active electrogenic transport of protons across membranes and is essential for a multitude of physiological processes. A fundamental mechanism to regulate V-ATPase activity is the reversible dissociation of the holoenzyme into an integral proton conducting VO-complex and a cytosolic V1-complex that hydrolyses ATP and thus energises proton translocation. Subunit C occurs isolated in the cytoplasm upon dissociation of the V-ATPase complexes and seems to be critical for the formation of active holoenzymes. In the salivary glands of the blowfly Calliphora vicina the V-ATPase is involved in fluid secretion. In secretory cells, formation of the V-ATPase holoenzyme is stimulated by the hormone serotonin (5-HT). The effect of 5-HT on V-ATPase activity is mediated by protein kinase A (PKA) and persists for the duration of the 5-HT stimulus. In this study, it was shown by phosphoprotein stainings and two-dimensional electrophoresis that subunit C of the V-ATPase becomes phosphorylated by PKA upon exposure of blowfly salivary glands to 5-HT. Parallel to the phosphorylation event, subunit C translocates from the cytoplasm to the apical plasma membrane for the assembly of active V-ATPase holoenzymes. Using immunofluorescence staining, it could be shown that PKA catalytic subunit translocates as well to the apical membrane upon 5-HT stimulation. To examine which protein phosphatase counteracts PKA, luminal pH-measurements were carried out. Based on the results with protein phosphatase inhibitors and esterified chelating agents of bivalent cations, it may be concluded that a protein phosphatase 2C is involved in the process leading to V-ATPase inactivation. Phosphoprotein stainings revealed that dephosphorylation of subunit C is likewise catalysed by a protein phosphatase 2C. Therefore the dephosphorylation of subunit C seems to promote dissociation of VO- and V1-complexes. Finally, luminal pH-measurements and supplemental biochemical experiments revealed a Ca2+/calcineurin-mediated modulation of the cAMP/PKA signalling cascade and an influence of intracellular calcium on the V-ATPase activity.

This study concerns the isoform-specific effects of activation of protein kinase C (PKC) and protein kinase A (PKA) on sodium pump activity of HeLa cells transfected with the individual isoform subunits $ alpha$1, $ alpha$2$ sp*$ and $ alpha$3$ sp*$ of the rat Na/K-ATPase, and a truncated mutant of the rat $ alpha$1 isoform, $ alpha$1M32. The asterisks denote forms mutated to be ouabain resistant as described by Jewell, E. A. and Lingrel, J. (1991) J. Biol. Chem. 266, 16925-16930. After activation of PKC it was observed that (i) the activity of the endogenous (human) $ alpha$1, the rat $ alpha$1, $ alpha$2$ sp*$ and $ alpha$3$ sp*$ isoforms, as well as the $ alpha$1M32 truncated mutant were inhibited by $ sim$20%; (ii) no change in the K$ sp+$ response profiles of Na$ sp+$-ATPase activity at low (1 $ mu$M) ATP concentration of the $ alpha$1, $ alpha$2$ sp*$ and $ alpha$3$ sp*$ could be detected and (iii) $ sp{32}$P-labeling of the $ alpha$1 subunit could not be detected. After activation of PKA, it was observed that activity the transfected rat $ alpha$1, $ alpha$2$ sp*$ and $ alpha$3$ sp*$ isoforms was inhibited by $ sim$20%, the rat $ alpha$3$ sp*$ isoform has a reduced apparent affinity for intracellular sodium and the rat $ alpha$2$ sp*$ isoform has a small decrease in apparent affinity for extracellular potassium.