Tesi sul tema "V-ATPase"

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.

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.

Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway.

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.

V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane.
Department of Chemistry

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

V-ATPase is a proton pump mainly localized on lysosomes and on plasma membrane of specialized cells. It is responsible of proton translocation and acidification of intra- and extra-cellular environment. Our group demonstrated that the over-expression of the subunit G1 (V1G1) is involved in the maintenance of the stem cell niche in glioblastoma (GBM) and correlates with poor prognosis in GBM patients. In this work we aimed to elucidate the role of V-ATPase in GBM stem cells from a functional perspective. We demonstrated that neurospheres (NS) with higher levels of V1G1 subunit (High-V1G1), compared with NS with lower levels of V1G1 (Low-V1G1), were characterized by increased clonogenicity in vitro and in vivo, invasiveness, lysosomal acidification and ERK pathway activation. Specific inhibition of V-ATPase activity, by Bafilomycin (BafA1), but not of ERK or other lysosomal drugs, induced reactive oxygen species (ROS)-mediated apoptosis only in High-V1G1 NS. In addition, BafA1 treatment affected mitochondria homeostasis only in High-V1G1 NS. Preliminary experiments suggested that a V-ATPase pump might be localized on the mitochondria or it could mediate direct contacts between mitochondria and lysosomes thus causing an imbalance of charges (proton flux) when perturbed. Finally, High-V1G1 and Low-V1G1 NS differed in terms of metabolic behaviours: preferential use of glycolysis by Low-V1G1 NS opposite to use of oxidative metabolism in High-V1G1 NS. V-ATPase block by BafA1 in High-V1G1 NS shifted their metabolism to that of Low-V1G1 NS. On the other hand, the autophagic pathway, that is directly connected with lysosomal function, was blocked by BafA1 only in Low-V1G1 NS. These phenotypes were not modulated by ERK or lysosomal acidification inhibitors alone, indicating a specific role for V-ATPase proton pump in modulating them. Taken together, these results indicate that V-ATPase is crucial for GBM stem cells viability through different mechanisms that include bioenergetics sensing and requiring, mitochondrial homeostasis and ERK signalling activity.

Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war. Wesentliche Ergebnisse dieser Arbeit waren: • Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind. • Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung (NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi. • Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung (Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen waren die Zellen noch in der Lage sich vollständig von einer intrazellullären Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität. • Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der unstimulierten Speicheldrüse von Calliphora vicina. • 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter. • Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei. • Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht werden. Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert.
The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied. Key results were: • pHi measurements in unstimulated glands showed that mainly the V-ATPase and at least one Na+-dependent HCO3--transporter are involved in maintenance of resting pHi. • V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary for the recovery from an intracellular acidication (NH4Cl prepulse). • Recovery from an intracellular alkali load (Na-acetate prepulse) is partially Cl--dependent. • A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi. • 10 nM 5-HT induce an intracellular acidication. This acidication is Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3- -antiporter was suggested. • Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced intracellular acidication. • The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter, a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were determined on mRNA level by RT-PCR. This thesis contributes signicantly to the understanding of pHi regulation in unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by using pHi measurements and molecular biological techniques. Mechanisms which are responsible for the 5-HT-induced intracellular acidication were also clarified. Furthermore a new optical method for measuring O2 consumption in animals cells was established by using the Calliphora salivary glands as a model.

Introduction: Glioblastoma (GBM), the most aggressive form of malignant gliomas, is an incurable tumor of the central nervous system which starts in glial cells, and rapidly diffuses through the brain. Surgical resection, radiotherapy and chemotherapy are the standard of care of GBM, improving the survival rate up to only 14-15 months. The high infiltrating nature of the tumor hampers surgical resection and tumoral cells often develop chemoresistance to the widely-used temozolomide (TMZ). Currently, a targeted therapy is not available. Therefore, the 5 years survival rate of these patients is extremely poor and does not exceed 5%. Most GBMs result from constitutive activation of the epidermal growth factor receptor (EGFR) and mutation in the pro-oncogenic phosphoinositide 3-kinase (PI3K) pathways, which are known regulators of cell growth. These pathways are extremely conserved in fruit fly: Because of this, we used Drosophila melanogaster as a model system to recapitulate in vivo certain tumoral features of GBMs. As the human one, Drosophila glia is constituted of several types of glial cells, which are involved in the formation of blood-brain-barrier and in phagocytosis of neural debris. In Drosophila glial precursor, we over-express a mutated form of the human EGFR and of Dp110, a regulator of PI3K signaling. Such manipulations result in massive hyperplasticity of the larval brain, due to an expansion of the glial compartment. Autophagy is a conserved catabolic process that provides nutrients by degradation of cellular constituents in lysosomes. How autophagy acts in tumor growth is currently unclear and the focus of intense investigation. Aim: We used a Drosophila glioma model to understand in vivo the role of the autophagy-lysosomal pathway in tumor growth and to identify new potential therapeutic targets suitable for clinical translation. Materials and methods: Drosophila 3rd instar larval brains were used to assess tumor growth and to monitor the autophagy-lysosomal pathway. Larvae were grown on standard fly food until the 3rd instar stage and larval brains were isolated and processed for the subsequent experiments. RNA was extracted by Mini-RNeasy kit from Qiagen, while proteins were extracted using RIPA buffer. Disaggregation of larval brains was carried out using Rinaldini’s solution and proteases. For immunofluorescence acquisitions, brains were isolated from larvae and processed with specific antibodies. Down-regulation of a targeted gene was performed using RNAi fly lines. Results: The excessive glial growth affects non-cell autonomously the neural tissue, which appears reduced and morphologically altered. Moreover, tumor growth impairs larval behavior and leads to death. Interestingly, we found that compared to control, the hyperplastic tissue displays a transcriptional and protein up-regulation of the autophagy adapter ref(2)P, the Drosophila p62/SQSTM1. However, canonical regulators of autophagy, controlled by Mitf, the Drosophila TFEB, are not deregulated in the glial tumor, compared to control. This suggests that gliomas might not have the ability to induce the autophagy-lysosomal pathway to supply to their growth needs. Moreover, we observed that the lysosomal compartment is expanded but functional. In contrast, we found that down-regulation of a component of the vacuolar-H+ATPase (V-ATPase), which is required for lysosomal acidification, reduces tumor growth. Surprisingly, ref(2) protein and transcriptional levels are restored back to physiology in tumoral brain in which the component is down-regulated. Importantly, we found that depletion prevents most of glia overgrowth and decreases activity of the PI3K pathway. Conclusion: Collectively, our data indicate that autophagy might be limiting for tumor growth and that components of the V-ATPase proton pump might be novel targets for treatment of GBM.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
O Ãcido abscÃsico (ABA) à um fitohormÃnio que desempenha papÃis crÃticos na regulaÃÃo das respostas das plantas ao crescimento e desenvolvimento. O vacÃolo de plantas superiores à uma organela que ocupa a maior parte da cÃlula. A condiÃÃo de acidez à mantida por duas bombas de prÃtons distintas, V-ATPase e V-PPase. Sabe-se que estas bombas de prÃtons desempenham um papel essencial nas respostas das plantas Ãs mudanÃas ambientais. O gradiente eletroquÃmico promovido por essas enzimas à a forÃa motriz para o acÃmulo de Ãons e outros solutos no vacÃolo, sendo importante para manter a homeostase citosÃlica de Ãons e o metabolismo celular. As enzimas antioxidantes constituem um sistema de defesa contra as espÃcies reativas de oxigÃnio, que podem provocar danos ao desenvolvimento das plantas. O objetivo deste trabalho foi estudar o efeito do Ãcido abscÃsico nos parÃmetros de crescimento e fisiolÃgicos, bem como o efeito na atividade hidrolÃtica e na expressÃo de transcritos das bombas de prÃtons vacuolares e na atividade de enzimas antioxidantes de plantas de Vigna unguiculata. As sementes de V. unguiculata foram germinadas em areia, onde cresceram por 15 dias, com aplicaÃÃo de soluÃÃo nutritiva de Hoagland na ausÃncia (controle) ou presenÃa de ABA 0,1 ÂM no terceiro, no sÃtimo e no dÃcimo dias apÃs a germinaÃÃo. Os efeitos do Ãcido abscÃsico (ABA) sobre o crescimento, a condutÃncia estomÃtica, a transpiraÃÃo, a fotossÃntese, a concentraÃÃo interna de CO2, a atividade e a expressÃo da subunidade A da V-ATPase e da V-PPase e a atividade de enzimas antioxidantes de Vigna unguiculata foram analisados. ABA aumentou o crescimento das plantas, mas nÃo afetou os parÃmetros fisiolÃgicos; induziu um aumento na atividade hidrolÃtica da V-ATPase em folhas e da V- PPase em raÃzes; aumentou os transcritos de VuVHA-A e de VuVHP em folhas e diminuiu os transcritos de VuVHA-A em raÃzes e, por fim, causou aumento na atividade da catalase de folhas e de raÃzes. Esses resultados sugerem que o Ãcido abscÃsico regula a atividade e a expressÃo dos genes das bombas de prÃtons vacuolares, bem como as atividades de enzimas antioxidantes, sendo portanto importantes efetores que regulam o desenvolvimento de plantas de V. unguiculata.
Abscisic acid (ABA) is a phytohormone that plays critical roles in regulating plant responses to growth and development. The vacuole of higher plants is an organelle that occupies a larger part of the cell. The acidic condition is maintained by two distinct proton pumps, VATPase and V-PPase. It is known these proton pumps play essential roles in plant responses to environmental changes. The electrochemical gradient promoted by these enzymes is the driving force for the accumulation of ions and other solutes in the vacuole, being important to maintain cytosolic ion homeostasis and cellular metabolism. Antioxidant enzymes constitute a defense system against reactive oxygen species, which can cause damage to plant development. The aim of this study was to study the effect of the abscisic acid in the growth and physiological parameters, as well as the effect on vacuolar proton pumps and antioxidant enzymes (SOD, CAT and APX) from Vigna unguiculata cv. Pitiuba. The seeds of V. unguiculata were germinated in sand and grown for 15 days, with application of Hoagland solution in the absence (control) or presence of 0.1 ÂM ABA in the third, seventh and tenth days after germination.The effects of the abscisic acid (ABA) on the growth, stomatal conductance, transpiration, photosynthesis, internal CO2 concentration, V-ATPase subunit A and V-PPase activities and expression and antioxidant enzymes activities of Vigna unguiculata were analyzed. ABA increased the plants growth but did not affect the physiological parameters; induced an increase on V-ATPase hidrolytyc activity in leaves and on V-PPase in roots; ABA increased the transcripts of VuVHA-A and VuVHP in leaves and decreased the VuVHA-A transcripts in roots; caused increase in the leaves and roots catalase activity. These results suggest that the abscisic acid regulate the activity and the genes expression of the vacuolar proton pumps, as well as the antioxidant enzymes activity, being thus important effectors that regulate the development of V. unguiculata plants.

Les V-ATPases sont des complexes protéiques des cellules eucaryotes, hautement conservés, associés à la membrane de nombreux organites vésiculaires ou vacuolaires, dont la fonction est d’assurer un niveau approprié d'acidification. Si le mécanisme général du fonctionnement de cette pompe à protons a été bien étudié, on sait par contre relativement peu de choses sur les propriétés spécifiques de la V-ATPase neuronale. Dans les synapses, ce complexe est essentiel pour acidifier les vésicules synaptiques, permettant ainsi aux transporteurs des neurotransmetteurs de les remplir correctement. Notre équipe a identifié une nouvelle protéine essentielle pour la survie de drosophile, dont la séquence la classe dans la famille des protéines associées aux V-ATPases. Plusieurs bases de données suggèrent que cette protéine, que nous avons nommée Lome, puis VhaAC45L, serait exprimée spécifiquement dans le système nerveux. Nos travaux ont confirmé que Lome est spécifique du système nerveux, et ont en outre révélé que sa présence n'est nécessaire que dans les neurones. Sa localisation cellulaire a montré un enrichissement dans les zones synaptiques chez les mouches adultes et les larves. Nous avons donc concentré la suite de notre étude sur la fonction synaptique de Lome, en utilisant la jonction neuromusculaire de la larve comme modèle. En accord avec l’hypothèse d’un dysfonctionnement de la V-ATPase, les larves ayant un niveau de Lome réduit dans les motoneurones présentaient une augmentation anormale du pH interne des vésicules synaptiques, associée à une diminution de la taille quantique, qui est l'amplitude de la réponse postsynaptique à la libération d'une seule vésicule. En conclusion, nos résultats ont permis d’identifier Lome, alias VhaAC45-Like (VhaAC45L) en référence à son plus proche homologue VhaAC45, comme un régulateur spécifique de la V-ATPase neuronale
The V-ATPases are highly conserved protein complexes of eukaryotic cells, associated with the membranes of many vesicular or vacuolar organelles, whose function is to ensure an appropriate level of acidification. While the general functioning mechanism of this proton pump has been well studied, in contrast relatively little is known about the specific properties of neuronal V-ATPase. In synapses, this complex is essential to acidify synaptic vesicles, thus allowing neurotransmitter transporters to properly fill them. Our team identified a novel protein essential for Drosophila survival, predicted from its sequence to belong to the family of V-ATPase-associated proteins. According to several databases, this protein, that we named Lome, and then VhaAC45L, appears to be expressed specifically in the nervous system. Our work confirmed that Lome is specific to the nervous system, and further revealed that its presence is only required in neurons. Its cellular localization showed an enrichment in synaptic areas in both adult flies and larvae. We have therefore focused the next part of our study on the synaptic function of Lome, using the larval neuromuscular junction as a model. Consistent with the hypothesis of a V-ATPase dysfunction, larvae with a decreased level of Lome in motoneurons presented an aberrant increase in the internal pH of synaptic vesicles, associated with a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single vesicle. Overall, our results identified Lome, alias VhaAC45Like (VhaAC45L) in reference to its closest homolog VhaAC45, as a specific regulator of the neuronal V-ATPase

The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.

The aim of the project was to produce stable, soluble multidrug resistance (mdr) ABC. transporters and V-type ATPase subunits suitable for structural studies in order to gain insight into these two families of ATP metabolising membrane proteins. Mdr ABC transporters from a number 'of bacteria were successfully expressed. The best protein in terms of yield of pure protein (5mg.r1) was LmrA from LactococQus factis. Due to seripus aggregation this protein resisted all initial attempts at crystallisation. A novel method was developed, the Ultracentrifugation dispersity sedimentation (UDS) assay, to allow screening a wide range of buffer and detergent conditions to limit the aggregation problem. A small of conditions was identified and LmrA re-submitted to crystallisation trials. Preliminary crystals of LmrA were obtained in the detergents n-dodecylfoscholine (FC-12) and polyoxyethylene(8)dodecyl ether (C12ES). The �·UDS assay has also been used successfully to screen detergents for a range of other membrane proteins. Both the E and G subunits of V-type ATPase from T. thermophilus have been overexpressed and purified. In addition, a method for the reconstitution of the EG complex developed and optimised to produce amounts sufficient for crystallisation trials. Initial crystallisation screens identified several crystallisation leads for subunit E and the EG complex that were further optimised to oqtain diffraction quality crystals. Eventually, both subunit E and the EG complex have yielded diffracting crystals to 3.4 Aand 4.0 Aresolution, respectively. Further work will focus on obtaining better diffracting crystals and phasing of the data.

Alors que s’accumulent des données épidémiologiques qui suggèrent une importance fondamentale des lipides de l’alimentation dans l’homéostasie cellulaire et le développement de nombreuses pathologies humaines, peu d’informations sur leurs fonctions spécifiques sont disponibles à ce jour. Ceci est particulièrement le cas pour la neurosecrétion qui dépend de la fusion d’organites vésiculaires avec la membrane plasmique. Des études récentes ont montré le rôle clé de la compartimentalisation lipidique au niveau des sites d’exocytose et par ailleurs validées la notion de lipides fusogéniques, comme pour l’acide phosphatidique (PA).La V-ATPase, via ses domaines V0 et V1 est à la fois impliquée dans le remplissage en neurotransmetteurs des vésicules, mais aussi dans leur fusion. Nous montrons ici que V0a1 interagit avec le facteur d’échange pour Arf6 ARNO. En bloquant cette interaction, nous avons observé une réduction de l’activation d’Arf6, de l’activité PLD et de l’exocytose, avec une modification de la cinétique des événements unitaires d’exocytose. Nous proposons que la dissociation de V1 de V0 pourrait représenter un signal permettant l’activation de la voie Arf6-ARNO-PLD1 et ainsi promouvoir la synthèse de PA requise à une exocytose efficace dans les cellules neuroendocrines
Lipids play key cellular functions and are involved in many human diseases and little information is available on their exact function. This is especially the case in neurosecretion that relies on the fusion of specific membrane organelle with the plasma membrane for which relatively little attention has been paid to the necessary role of lipids. Recent studies have established the importance of lipid compartmentalization at the exocytotic sites and validated the contribution of fusogenic lipids such as phosphatidic acid (PA) for membrane fusion. The V-ATPase is involved both in the charging of secretory vesicle and the membrane fusion for secretion of vesicle. Indeed, the V1 and V0 subdomains were shown to dissociate during stimulation allowing subunits of the vesicular V0 to interact with different proteins of the secretory machinery. We show here that V0a1 interacts with the exchange factor ARNO and promotes Arf6 activation during exocytosis in neuroendocrine cells. Interfering with the V0a1-ARNO interaction prevented phospholipase D (PLD) activation, phosphatidic acid synthesis during exocytosis, and altered the kinetic parameters of individual fusion events.We suggest that V1 dissociation from V0 could represent the signal that triggers the activation of the ARNO-Arf6-PLD1 pathway and promotes PA synthesis needed for efficient exocytosis in neuroendocrine cells

xii, 45 p. : col. ill.
Regulated acidification of intercellular organelles and vesicles is essential for many cellular processes, from basic metabolism and protein sorting to synapse function and developmental signaling. These diverse processes are driven by spatiotemporal regulation of the V-ATPase, the cellular H + -pump. In yeast and higher eukaryotes V-ATPase localization is directed by the 100-kDa "a" subunit, and many human diseases are linked to mutations in "a". Saccharomyces cerevisiae contains two "a" isoforms, VPH1 and STV1, with all other V-ATPase subunits encoded for by single genes. The V-ATPase contains only one "a" subunit per complex. Complexes that contain Vph1p localize to the vacuole (lysosome), and Stv1p-containing complexes localize to the late Golgi/endosome. Here I present a set of STV1 mutants that are disrupted for Golgi retention but not V-ATPase assembly or enzymatic function. Using a forward genetic screen I defined multiple residues within a 39 amino-acid region of Stv1p that are necessary for Stv1p retention to the Golgi. The residues most strongly affecting Golgi localization are present in a small STV1 -specific insertion of eight residues, suggesting they may bind directly to sorting machinery. However, I also find that Stv1p/Vph1p chimeras containing the STV1 -specific insertion are not sufficient to direct Golgi retention in both minimal (13AA) and expanded (49AA) contexts. I conclude that the Stv1p Golgi retention signal is composed of a complex binding surface, of which the central element is a short peptide rich in amino acids with aromatic side chains.
Committee in charge: Bruce Bowerman, Chairperson; Tom H. Stevens, Advisor; Karen Guillemin, Member; George F. Sprague Jr., Member Kenneth E. Prehoda Outside Member

A digestão nos insetos ocorre no intestino médio de forma compartimentada. A digestão inicial dos polímeros ocorre no interior da membrana peritrófica. Os oligômeros resultantes difundem-se para o espaço luminal exterior à membrana peritrófica onde são atacados por outras enzimas. Na digestão final os dímeros resultantes são hidrolisados por enzimas imobilizadas na superfície do epitélio do intestino médio. Após o processo de digestão final os monômeros são absorvidos pelas células do epitélio intestinal. Os Díptera ditos superiores, incluindo a mosca doméstica, apresentam peculiaridades digestivas que aparentemente resultam de adaptações para digerir uma dieta que consiste principalmente de bactérias. No ventrículo anterior ocorre uma diminuição no conteúdo de amido do bolo alimentar. Na porção seguinte, o bolo alimentar passa para o ventrículo médio onde as bactérias são mortas pela ação combinada de baixo pH, uma lisozima digestiva e uma proteinase tipo catepsina D. O material liberado das bactérias é digerido no ventrículo posterior, como ocorre no ventrículo inteiro da maioria dos insetos de outros grupos taxonômicos. Com o objetivo de compreender a peculiar digestão em Musca domestica, foram utilizadas suas larvas para identificar funcionalmente as regiões absortivas de nutrientes, identificar as moléculas envolvidas na absorção de nutrientes, identificar as moléculas envolvidas com tamponamento e fluxos de fluidos intestinais, sequenciar as enzimas digestivas principais e identificar os seus sítios de secreção. Experimentos fisiológicos de absorção de glicose e análises de atividade enzimática permitiram acessar de maneira direta os aspectos da digestão. Contudo, experimentos de sequenciamento de bibliotecas de cDNA, análise de sequências transcritas e verificação de expressão de genes em diferentes tecidos foram abordagens fundamentais na identificação das moléculas subjacentes aos processos fisiológicos intestinas de Musca domestica. Os indícios de que absorção de glicose no intestino de Musca domestica se dê por transportadores do tipo SGLT, com a possível participação de facilitadores do tipo GLUT, permitem estabelecer um foco para futuros estudos. A descrição de sequências relacionadas ao tamponamento intestinal permitiu ampliar a discussão sobre tal processo. Ao detalhar os sítios de expressão da subunidade a da V-ATPase, do canal de cloreto e do transportador de amônia foi possível testar o modelo de tamponamento proposto anteriormente e propor a participação de outras moléculas no processo. Sequências correspondentes as atividades de carboxipeptidase, maltase e aminopeptidase descritas na literatura foram pesquisadas, gerando sequências candidatas a codificarem as referidas enzimas. Com isso, é possível descrever a digestão de oligômeros e dímeros com base nos genes transcritos e nas sequências de aminoácidos que formam as enzimas digestivas. A descoberta da sequência que transcreve uma metaloproteinase, por sua vez, abre caminhos para a descrição e caracterização de sua atividade proteolítica nos tecidos digestivos da larva de Musca domestica. Essa análise permitiu também elucidar a localização dos sítios de expressão e, portanto, as zonas de secreção de enzimas. De maneira geral, este estudo contribuiu para a compreensão de diversos aspectos da digestão de Musca domestica, elucidando questões da particular fisiologia digestiva desse inseto.
Digestion in insects occurs in the midgut in a compartmentalized way. Initial digestion takes place inside the peritrophic membrane. The resulting oligomers diffuse into the luminal space outside the peritrophic membrane where they are hydrolyzed by other enzymes. In the final digestion, the resulting dimers are hydrolyzed by enzymes immobilized on the midgut epithelium. After the final digestion, the monomers are absorbed by intestinal epithelial cells. The so-called higher Diptera, including the house fly, have digestive peculiarities apparently resulting of adaptations to digest a diet consisting mainly of bacteria. In the anterior midgut there is a decrease in the starch content of the food bolus. The bolus now passes into the middle midgut, where bacteria are killed by the combined action of low pH, a special lysozyme and a cathepsin D-like proteinase. Finally, the material released by bacteria is digested in the posterior midgut, as is observed in the whole midgut of insects of other taxa. In order to understand the peculiar digestion in Musca domestica, the larvae were used to identify (a) the functionally the nutrient absorptive regions, (b) the molecules involved in the absorption of nutrients, (c) the molecules involved in buffering and fluid flows, (d) the cDNA sequences corresponding to intestinal digestive enzymes, (e) the main sites of secretion. Physiological experiments of glucose absorption and enzyme activity analysis allowed a direct access to aspects of digestion. Otherwise, cDNA library sequencing followed by sequence annotation and tissue-specific expression analysis were fundamental approaches in the understanding of intestinal physiology of Musca domestica. Evidence that glucose absorption in the gut of Musca domestica occurs through SGLT-like transporters, with the possible participation of facilitators GLUT-like, allowed us to establish a focus for future studies. The description of cDNA sequences corresponding to proteins putatively responsible for intestinal buffering widened the discussion of this process. The finding of the expression sites of V-ATPase subunits, chloride channel, and ammonia transporter led to revising the present buffering model and the inclusion of other molecules in the process. The cDNA sequences corresponding to the activities of carboxypeptidase, aminopeptidase and maltase described in the literature were searched for as candidate sequences to encode those enzymes. This made it possible to describe the digestion of oligomers and dimers based on transcribed genes and enzyme amino acid sequences. The discovery of the metalloproteinase transcribing sequence opened a new research line: the description and characterization of its proteolytic activity in the midgut of the Musca domestica larvae. This study also allowed elucidating the location of digestive enzyme expression sites and, therefore, the putative zones of enzyme secretion. Overall, this study contributed to understanding many aspects of digestion of Musca domestica, clarifying aspects of the peculiar digestive physiology of this insect.

xvii, 167 p. : ill. (some col.)
The vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit enzyme complex responsible for acidification of cellular organelles. The V-ATPase hydrolyzes ATP to pump protons across membranes to create an electrochemical gradient. Acidification of vesicular compartments is critical in numerous biological processes including protein trafficking, endocytosis, and ion homeostasis; defects in V-ATPase function can also lead to human diseases. While the function of the V-ATPase enzyme is highly conserved across eukaryotes, the molecular architecture of this protein complex has undergone unique structural changes through evolutionary time. The goal of this work is to investigate the assembly, transport, and evolution of this critical molecular machine in the model organism Saccharomyces cerevisiae. A series of genetic screens was performed in budding yeast to identify factors and pathways that are involved in promoting full V-ATPase function. I utilized several "assembly factor" alleles to serve as sensitized genetic backgrounds to partially reduce enzyme function; this work implicated sphingolipid composition in promoting full vacuolar ATPase enzyme function. I also used ancestral gene reconstruction to analyze the two isoforms of subunit a of the V₀ subdomain (Vph1p and Stv1p) by recreating the most recent common ancestral subunit (Anc.a). Characterization of Anc.a demonstrated that this ancient subunit was able to properly assemble and function within a hybrid V-ATPase complex. While the Vph1p-containing complex localized to the vacuole membrane and the Stv1p-containing complex was present on the Golgi/endosome, incorporation of Anc.a caused the V-ATPase to localize to both types of cellular compartments. Finally, I used ancestral reconstruction to investigate the lineage-specific gene duplication of one of the proteolipid subunits of the V₀ subcomplex that occurred within the fungal clade. I demonstrate that inclusion of a third proteolipid subunit within fungi (as compared to two subunits within metazoans) could have occurred via neutral processes by asymmetric degeneration of subunit-subunit interfaces that "ratcheted" the duplicated subunit with the V₀ ring. These results present a model that describes how macromolecular machines can increase in complexity through evolutionary time. This dissertation includes previously published co-authored material and unpublished co-authored material.
Committee in charge: George Sprague, Chairperson; Tom H. Stevens, Advisor; Victoria Herman, Member; Bruce Bowerman, Member; Ken Prehoda, Outside Member

The aims of this thesis were to investigate the effects of insulin on the cellular distribution and activity of the Na+/It exchanger (NHEI) and vacuolar proton pumps (VATPases) in cardiomyocytes and to evaluate their association with GLUT4 vesicles. To address these issues, a highly specific anti-NHEI antibody was raised, which enabled immunofluorescence, immunoprecipitation and co-immunoprecipitation studies into the effects of insulin on NHE1 distribution to be carried out. These studies provided evidence for an insulin-stimulated increase in NHEl at the plasma membrane and a decrease in intracellular NHEI, even though the overall amount cellular NHEI remained constant. A cell free acidity assay (CFAA) was developed to measure NHEI activity within subcellular fractions. An insulin-stimulated increase in plasma membrane NHEI activity was identified in accordance with the increase in plasma membrane NHEI. The CFAA also identified an inhibitable portion of V-ATPase activity within the low density microsomes (LDM) from basal cardiomyocytes, which was absent from LDM of insulinstimulated cells. The insulin-stimulated decrease in LDM V-ATPase activity implied that in the presence of insulin, V-ATPases translocate, as do GLUT4 vesicles. Structural associations between two complexes of V-ATPase (namely complexes Vo and VI) with GLUT4 vesicles were identified by co-immunoprecipitation of GLUT4 vesicles and subsequent Western blotting for the V-ATPase protein. The studies generated data that was consistent with the hypothesis that the insulin-stimulated change in V-ATPase activity in LDM samples may partly occur as a result of the insulinstimulated decrease in general vesicle trafficking and fusion and in part to GLUT4 vesicle translocation and fusion. The thesis identifies further investigations, that are needed to determine whether (a) V-ATPases are necessary for vesicle acidification to occur prior to fusion; (b) whether V-ATPases are involved in the formation of a Yo-Yo transcomplex necessary for docking ofthe GLUT4 vesicles with the PM and finally (c) whether insulin affects the distribution ofNHEI either by stimulating NHEI trafficking or their turnover.

Recent evidences highlighted that glioblastomas (GBM) secreted microvesicles (EVs), particularly exosomes (Exo) and large oncosomes (LO), play a major role in the cross-talk between tumor cell and non-neoplastic parenchyma. Recent work from our group has identified the vacuolar pump H+-ATPase (V-ATPase) as an important effector of GBM growth and glioma stem cells (GSC) maintenance. Additionally, in ExoCarta database V-ATPase subunits have been described in Exo from different cancer cell types. Taken together, these data identify V-ATPase as an important driver of gliomagenesis, and a novel, actionable therapeutic target for disease intervention. However, the role of V-ATPase in reprogramming the GBM microenvironment has not been previously investigated. The aim of this project was investigate production, biological effect and content of extracellular vesicles according to proton pump activity in glioma stem cells. Our data show that GSC are able to produce different types of EVs, which are internalized by recipient cells of different histology, such as non-neoplastic brain tumor margins, primary GBM monolayers (both differentiated and undifferentiated), and commercial glioma cultures. Exo and LO from GSC induces in recipient cells distinct effects. In particular, Exo significantly increased cell growth and cell motility, and these effects were stronger with Exo produced by NS with higher V-ATPase expression (V1G1HIGH NS). On the other hand, LO were able to strongly induce the sphere formation ability of primary GBM cultures. This effect lasted up 90 days after co-culture. In both situations, the block of V-ATPase activity by Bafilomycin A1 in NS-producing EVs completely reverted the effects. Interestingly, exosomes are able to vehiculate on their surface the V-ATPase G1 subunit, and its protein level increased in recipient cells after co-culture with EVs. At the molecular level, profiling of Exo-derived miRNAs distinguishes V1G1HIGH NS from V1G1LOW cultures. In silico analysis and annotation of miRNA target genes showed an enrichment of cancer, cell cycle and MAPK/Erk pathways. Regarding signaling pathway modulation by Exo in recipient cells, exosomes from V1G1HIGH NS activated the MAPK/Erk pathway. Altogether, these data point toward the central role of different EV types in GBM communication and suggest a role of the V-ATPase proton pump in regulating exosomes contents.

Alveolar surfactant is the key physiological structure that every mammal utilizes to live while breathing air. It is a mixture of proteins and lipids that lines the external part of the alveoli and allows a water-based organism to use air as source of oxygen. Disorders in surfactant’s metabolism are known to be involved in life-threatening diseases like the acute respiratory distress syndrome and cystic fibrosis. In this thesis we describe the kinetics of two main surfactant’s phospholipids (phosphatidylglycerol and disaturated-phosphatidylcholine) and of its specific protein B, in vivo in humans, all obtained with stable isotopes infusion protocols in children (phospholipids) and adults (protein SP-B). In the last part we tried to use the model of renal intercalated cells to approach the role of pH in the secretion of surfactant.
Il surfattante alveolare è la struttura fisiologica che viene utilizzata da ogni mammifero per vivere e respirare in atmosfera. Si tratta di un complesso di proteine e lipidi che si trova nella parte esterna degli alveoli, dove svolge la sua funzione fisiologica permettendo lo scambio gassoso tra organismo (principalmente acquoso) e aria. Alterazioni del metabolismo del surfattante sono note in numerose e gravi malattie, come la sindrome da distress respiratorio e la fibrosi cistica. In questa tesi sono descritte le cinetiche di due principali fosfolipidi (fosfatidilglicerolo e fosfatidilcolina disatura) e di una proteina specifica del surfattante (SP-B), misurati in vivo nell’uomo attraverso l’infusione di isotopi stabili. Nell’ultima parte è descritto il tentativo di utilizzare il modello delle cellule intercalate del rene per studiare il ruolo delle variazioni di pH nella secrezione del surfattante.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2015.
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RNA de interferência (RNAi) é um processo bioquímico potente e específico de silenciamento de genes, que ocorre em uma variedade de organismos como mamíferos, fungos e plantas. O mecanismo atua a nível pós-transcricional, e pode resultar na degradação ou não tradução de RNAs mensageiros (mRNA). Esse mecanismo foi utilizado com o objetivo de silenciar o gene v-ATPase da mosca branca (Bemisia tabaci), que é considerada uma importante peste da agricultura em regiões tropicais e sub-tropicais em todo o mundo. Nesse estudo, um plasmídeo contendo um cassete de interferência para um fragmento de v-ATPase foi desenvolvido e utilizado para transformar cotilédones de alface (Lactuca sativa). Um total de 25 linhagens transgênicas foram geradas, das quais sete foram avaliadas para estudos moleculares. Análise de progênie confirmou a preseça do inserto na geração T1 das sete linhagens assim como a análise de Northern, que permitiu a detecção de siRNAs correspondentes ao gene de v-ATPase. Um estudo de silenciamento da v-ATPase foi feito por meio de um bioensaio no qual plantas das diferentes linhagens foram submetidas à presença de 20 moscas brancas, e a taxa de mortalidade, bem como a alteração de desenvolvimento em diferentes estágios do ciclo de vida da mosca foram avaliados, durante um período de 32 dias. A análise da mortalidade de insetos que se alimentaram em plantas transgênicas demonstrou que, em três dias de alimentação, uma queda de aproximadamente 75% nessa população pôde ser observada quando comparado ao controle (p<0,05). Alterações significativas no ciclo de desenvolvimento de insetos se alimentando em plantas transgênicas também foram observadas (p<0,05). Dessa forma, dados apresentados nesse trabalho funcionam como prova de conceito no desenvolvimento de tolerância à mosca branca mediado por RNAi. ______________________________________________________________________________ ABSTRACT
RNA interference (RNAi) is a potent biochemical phenomenon that targets and silences specific genes in different life forms like mammals, fungi and plants. The process of silencing takes place at post-transcriptional level leading to the degradation of messenger RNAs (mRNAs) or inhibition of their translation. RNAi has been demonstrated to be useful in silencing v-ATPase gene of whitefly (Bemisia tabaci), an important agricultural pest in the tropics and sub-tropic regions of the world. Here, a plasmid containing an interferent cassette designed to generate siRNA molecules that target v-ATPase gene transcript was cloned in a binary vector, which was used to transform cut-pieces of cotyledons from germinating lettuce (Lactuca sativa). A total of 25 transgenic lines were generated, of which seven were selected for further molecular analysis. Progeny analysis confirmed that these lines have passed the inserted character to the next generation (T1). Northern blot analysis detected siRNAs corresponding to the v-ATPase insert. The lines were used to perform a bioassay in order to evaluate the silencing effect of v-ATPase. Plants from these lines were infested with 20 whiteflies and the mortality as well as alterations in growth and development of different stages of the whitefly life cycle was monitored over a period of 32 days. Analysis of mortality showed that within three days of feeding, insects on transgenic plants showed a mortality rate of about 75% higher than those on control plants (p<0.05). Significant alterations in the development of the insects on transgenic plants were also observed (p<0.05). Data presented in this work may function as a proof of concept in the development of plants with tolerance to whitefly via RNAi.

The abnormal regulation of H+ ions, leading to a reversed pH gradient in cancer cells when compared to normal cells, is considered to be one of the most distinctive features of cancer. However, this characteristic has yet to be fully exploited as a therapeutic target in cancer. This project assessed whether targeting pH regulating proteins, which permit cancer cells to survive in the hostile hypoxic and acidic tumour microenvironment, could produce an effective therapeutic response in breast cancer. The pH regulating proteins carbonic anhydrase IX (CAIX), Na+/H+ exchanger 1 (NHE1) and vacuolar (H+)-ATPase (V-ATPase) were the focus of this thesis. Initial experiments were conducted in 2D tissue culture before progressing into 3D, using models that more faithfully re-create the in vivo tumour microenvironment. Expression analysis conducted with MCF-7, MDA-MB-231 and HBL-100 human breast cancer cell lines cultured in 2D, and in 3D as multicellular tumour spheroids, showed that protein and mRNA levels of CAIX were very responsive to lower O2 concentrations. Both MDA-MB-231 and HBL-100 cells displayed large increases in CAIX expression levels in hypoxia, with the HBL-100 cell line exhibiting the largest change in CAIX mRNA (42-fold increase) and protein (78-fold increase) levels in 0.5% O2 conditions. Hypoxia inducible factor 1-α (HIF-1α) controls the expression of CAIX, but the induction of CAIX in hypoxic MCF-7 cells was lower in comparison to the other cell lines, despite the presence of similar levels of HIF-1α. The differences in CAIX expression observed between the cell lines was consistent with a varying activity of factor inhibiting HIF-1 (FIH-1), an oxygen sensor that controls signalling through HIF-1α, as siRNA targeting FIH-1 led to increased levels of CAIX in hypoxic MCF-7 cells. While NHE1 protein levels did increase in hypoxic conditions in MCF-7 cells in 3D, overall, the expression levels of both NHE1 and V-ATPase were not as responsive to changes in O2 concentrations when compared to CAIX across differing O2 conditions in each of the cell lines. Inhibitors targeting CAIX, NHE1 and V-ATPase were all shown to reduce cancer cell number in 2D culture conditions. Differing O2 conditions changed the sensitivity of these cell lines to CAIX inhibition. Cells cultured in 20% O2 conditions were responsive to CAIX inhibition, while acute hypoxic cells cultured in 0.5% O2 displayed an increased resistance to drug treatment. Chronically hypoxic cells, which had spent over 10 weeks in 0.5% O2 before treatment, exhibited a re-sensitisation to CAIX inhibition. 3D invasion assays demonstrated that CAIX inhibition significantly reduced the invasion of cells from MDA-MB-231 spheroids into collagen type 1 in both 20% O2 and 0.5% O2 conditions, while drugs targeting either NHE1 or V-ATPase had no such inhibitory effects. Preliminary clonogenic assays, used to assess radiation sensitivity and performed with MDA-MB-231 spheroids, indicated that inhibitors targeting CAIX and NHE1 led to a significant decrease in colony formation when combined with irradiation, compared to either drug treatment or irradiation alone. Further invasion assays, carried out with primary tissue derived from human patients, showed that drugs targeting CAIX retained their inhibitory effects when tested on heterogeneous cancer material of varying tumour subtypes. CAIX inhibition also exhibited anti-cancer effects in vivo on mouse MDA-MB-231 xenografts, significantly reducing the proliferation and growth of tumours within mice. Together, this work demonstrates that inhibitors targeting the pH regulation mechanisms of cancer cells have potential in the treatment of breast cancer, highlighted by their capacity to affect cancer cell number, cancer cell invasion, and their ability to combine with irradiation. Of the 3 pH regulatory molecules studied, CAIX appears to be the target with the most therapeutic potential.

The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronomia, 2018.
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O tomateiro (Solanum lycopersicum L.) apresenta importância econômica e alimentar, sendo uma das principais hortaliças produzidas no Brasil. O alto custo de produção e grandes perdas econômicas da cultura do tomateiro estão fortemente relacionados a problemas fitossanitários, dentre eles, a infestação de mosca branca Bemisia tabaci que além de causar danos como inseto sugador, atua como vetor de diferentes gêneros virais, o que torna fundamental manter populações baixas desse inseto vetor no campo. A mosca branca é responsável por causar grandes danos à agricultura, por possuir desenvolvimento rápido e ser adaptada a diversos hospedeiros. A natureza invasora da mosca branca e sua alta taxa de reprodução dificultam o controle e favorecem o desenvolvimento de resistência aos inseticidas utilizados no seu combate, evidenciando a necessidade de alternativas mais eficientes de controle. O silenciamento gênico pós-transcricional via RNA interferente (RNAi) é uma das estratégias da engenharia genética, que pode ser utilizada na busca da obtenção de plantas resistentes a pragas com maior especificidade. A estratégia de RNAi foi utilizada com o objetivo de obter plantas de tomateiro resistentes à mosca branca, pelo silenciamento do gene da v-ATPase de Bemisia tabaci, devido à expressão de siRNAs. Explantes cotiledonares de tomateiro da variedade Micro-Tom foram transformados via Agrobacterium tumefaciens EHA 105 contendo um plasmídeo, que possui um cassete de supressão para o silenciamento do gene da v-ATPase de mosca branca. Foram geradas 13 linhagens transgênicas, confirmadas por PCR pela detecção do transgene de 576 pb correspondente à região do gene da v-ATPase. A inserção do transgene no genoma em alguns eventos foi confirmada por meio da análise de Southern blot. Análise da progênie confirmou a presença do inserto na geração T1, segregando em proporção mendeliana 3:1 e 15:1. Um bioensaio realizado com folhas destacadas das plantas T0 mostrou que a mortalidade de moscas brancas pode chegar próximo a 100% em cinco dias de interação com algumas linhagens.
The tomato plant (Solanum lycopersicum L.) is economically important, being one of the main vegetables produced in Brazil. The high cost of production and huge economical losses in tomato cultivation are strongly related to phytosanitary problems, among them the whitefly Bemisia tabaci, besides causing damage as a sucking insect, acts as a vector of different viral genus, which needs keeping low populations in rural areas. The whitefly is responsible for a great damage to agriculture due its quick development and adaptation to several hosts. The invasive nature of the whitefly and its high reproduction rate hinder the control and favor the development of resistance to insecticides, which evidences the need for more efficient alternatives control. The post-transcriptional gene silencing via interfering RNA (RNAi) is one of the genetic engineering’s strategies that should be used for obtaining resistant plants to plagues with greater specificity. The RNAi mechanism was utilized to obtaining tomato resistant plants to the whitefly by silencing the v-ATPase gene from Bemisia tabaci. Cotyledonary explants from Micro-Tom variety were transformed via Agrobacterium tumefaciens EHA 105 containing a suppression cassette for the whitefly’s v-ATPase gene silencing. Thirteen transgenic lines were generated, confirmed by PCR, through the detection of a 576 pb transgene fragment corresponding to the v-ATPase gene. The transgene insertion into de genome of some events was confirmed by Southern blot analysis. Progeny analysis confirmed the presence of the transgene into the T1 progeny, segregating in Mendelian proportion 3:1 e 15:1. A preliminary bioassay performed with detached leaves from T0 plants showed a whiteflies’ mortality around 100% in some lines after five days of interaction.

L’exocytose des neurotransmetteurs repose sur la fusion des vésicules synaptiques avec la membrane plasmique en réponse à l’influx calcique qui accompagne les potentiels d’action. La fusion nécessite l’assemblage des protéines SNAREs de la vésicule (la VAMP2) et de la membrane plasmique (la syntaxine 1 et la SNAP25) à l’interface des deux bicouches lipidiques, et elle est déclenchée par la synaptotagmine, un senseur de Ca2+ aux propriétés fusogènes. Des senseurs calciques additionnels semblent participer au déclenchement de la libération et à la dynamique du pore de fusion, en particulier la calmoduline dont le rôle au cours de l’exocytose demeure obscur. Au moyen d’une technique dérivée de la FRET, nous démontrons ici que la Ca2+/calmoduline inhibe l’assemblage du complexe SNARE et la fusion membranaire SNARE-dépendante in vitro en se liant aux domaines juxtamembranaires de la VAMP2 et de la syntaxine 1. De plus des données récentes indiquent que le secteur V0 de la pompe à protons V-ATPase peut participer à la fusion membranaire, et plus particulièrement qu’un hexamère formé de ses sous-unités c constitue un pore capable de libérer de l’acétylcholine en présence de calmoduline. Au moyen des techniques du double-hybride de levure et de SPR notamment, nous avons identifié un lien direct entre boucle 3-4 de la sous-unité c et VAMP2, impliquant le domaine de liaison à la calmoduline de cette dernière. La perturbation de cette interaction in vivo, par injection d’un peptide interférant, inhibe la neurotransmission, suggérant que l’interaction de la machinerie d’exocytose avec ce pore protéique potentiel est impliquée dans la libération des neurotransmetteurs
Action potential-evoked neurotransmitter release relies on Ca2+-driven synaptic vesicle fusion with the plasma membrane. Fusion involves assembly of the vesicular protein VAMP2 (a v-SNARE) with the plasma membrane proteins syntaxin 1 and SNAP25 (t-SNAREs) into a tight trans complex at the interface between the bilayers, and is triggered by synaptotagmin, a fusogenic Ca2+-sensor. Additional Ca2+-sensors are likely to participate in release triggering and fusion pore dynamics, and notably our understanding of calmodulins actions in exocytosis remains elusive. By means of a FRET-derived method, we demonstrate that Ca2+/calmodulin inhibits SNARE complex assembly and SNAREdependent membrane fusion in vitro, by binding to the juxtamembrane regions of VAMP2 and syntaxin 1. The newly-identified calmodulin binding site on syntaxin overlaps with the synaptotagmin- interacting region, and the two interactions are mutually exclusive, suggesting antagonistic roles for the two sensors in membrane fusion. Moreover, recent data point to the involvement of the V0 sector of the proton pump V-ATPase in various membrane fusion events. They indicate that pore-forming c-subunits hexamers confer Ca2+- dependent release of acetylcholine to synthetic liposomes in the presence of calmodulin. Using yeasttwo- hybrid and SPR, we have identified a direct link between the c-subunit loop 3-4 and the v-SNARE VAMP2, involving the calmodulin-binding domain of the latter. Disturbing this interaction in vivo by acute injection of an interfering peptide inhibited neurotransmission, suggesting that association of the exocytotic machinery with the putative proteic pore is involved in neurotransmitter release

La pompe protonique V-ATPase est un complexe impliqué dans l'acidification intracellulaire. Elle est formée par le secteur V0 pour le transport des protons et le secteur V1 pour l'hydrolyse de l'ATP. Des études pionnières chez S. cerevisiae montrent que l'assemblage du secteur V0 commence dans le réticulum endoplasmique (ER) grâce au contrôle de 5 facteurs d'assemblage: Vma21p, Vma12p, Vma22p, Pkr1p et Voa1p. Une fois assemblé, le secteur V0 est transporté par Vma21p jusqu'au cis-Golgi, où, avec le secteur V1, constitue un holoenzyme fonctionnel. Ce processus chez l'homme n¿est pas encore clair. Il a été récemment démontré que tous les facteurs d'assemblage, à l'exception de Pkr1p, sont conservés chez l'homme et que des mutations chez trois d'entre eux, ATP6AP1, TMEM199 et CCDC115, sont responsables d'un nouveau sous-groupe d'anomalies congénitales de la glycosylation (CDG). Dans la première partie de ma thèse, j'ai identifié des mutations faux-sens dans ATP6AP2, qui provoquent une CDG similaire, avec des anomalies de la glycosylation, une stéatose du foie et des anomalies cognitives et immunitaires. Auparavant, des mutations exon-skipping dans ATP6AP2 avaient été associées à une maladie cérébrale d'apparition tardive, avec du parkinsonisme et de l'épilepsie. Nos travaux ont révélé que les mutations faux-sens dans ATP6AP2 conduisent à une activité défectueuse de la V-ATPase, avec réduction de l'acidification organellaire, de la dégradation lysosomale et du flux autophagique. De ce fait, la clairance des gouttelettes lipidiques ne peut pas se produire dans les autolysosomes, produisant une stéatose dans le foie des patients. Conformément au phénotype clinique similaire, nous avons constaté que ATP6AP2 interagissait avec les facteurs d'assemblage du secteur V0, tandis que les mutations faux-sens en réduisaient l'interaction, suggérant un assemblage compromis chez les patients. En revanche, chez les patients présentant une mutation exon-skipping, nous avons trouvé une glycosylation normale des protéines sériques et aucun effet sur l'interaction entre ATP6AP2 et ATP6AP1, ce qui suggère que les mutations faux-sens ont un impact plus fort sur la fonction globale de ATP6AP2 que celles exon-skipping. Ces résultats révèlent des nouveaux aspects de l'assemblage de la V-ATPase dans l'ER et suggèrent que ATP6AP2 est un membre supplémentaire des facteurs d'assemblage chez l'homme. Dans la deuxième partie de ma thèse, j'ai démontré que des mutations dans VMA21 sont également à l'origine d'une CDG associée à une maladie du foie, avec un phénotype similaire aux autres déficits en facteur d'assemblage de la V-ATPase. Auparavant, des mutations en VMA21 étaient associées à une myopathie X-linked avec une autophagie excessive (XMEA), une vacuolisation progressive et une atrophie du muscle squelettique. En utilisant des fibroblastes de patients, nous avons testé l'impact fonctionnel de ces mutations sur l'assemblage et la fonction de la V-ATPase. Les mutations de VMA21 sont hypomorphes, réduisant l'expression de l'ARNm et de la protéine, et provoquent des défauts de l'autophagie avec une moindre dégradation des gouttelettes lipidiques, similaires à ce qui a été observé dans la déficience en ATP6AP2. Enfin, les fibroblastes des patients montrent une accumulation de cholestérol non estérifié dans des vésicules, similaire à celle observée dans la maladie de surcharge lysosomale Niemann-Pick de type C (NPC). La séquestration du cholestérol dans les lysosomes active la lipogenèse via la protéine SREBP, ce qui conduit à une hypercholestérolémie chez les patients. Globalement, nos résultats montrent que les déficiences de la V-ATPase constituent un nouveau groupe de syndromes métaboliques qui affectent l'homéostasie lysosomale et autophagique. L'étude de ces maladies rares permettra de mieux comprendre la pathogénie de la stéatose hépatique non alcoolique (NAFLD), un problème fréquent dans le syndrome métabolique
The V-ATPase is a large complex involved in the acidification of intracellular organelles. It is formed by a proton pore (V0 sector) and ATP hydrolysis domain (V1 sector). Pioneering studies in S. cerevisiae have shown that the assembly of the V0 sector occurs in the endoplasmic reticulum (ER) under the guidance of 5 assembly factors: Vma21p, Vma12p, Vma22p, Pkr1p and Voa1p. The newly assembled V0 sector is then escorted by Vma21p to the cis-Golgi, where it will bind the V1 sector to constitute a functional holoenzyme. How the assembly works in mammalian systems is currently still unclear. Yet, it was recently shown that all the assembly factors except Pkr1p are conserved in mammals and that mutations in three of them, ATP6AP1, TMEM199 and CCDC115, were identified to cause a new subgroup of congenital disorders of glycosylation (CDG). Using human genetics and functional validation, I identify in the first part of my thesis a novel mammalian assembly factor called ATP6AP2 that causes a similar CDG-like disease when mutated. Apart from glycosylation defects, patients with missense mutations in ATP6AP2 show steatotic liver disease, cognitive defects and immune defects. Previously exon-skipping mutations in ATP6AP2 had been associated with a late onset cerebral disease, with Parkinsonism and epilepsy. Our work revealed that ATP6AP2 missense mutations lead to defective V-ATPase activity, subsequently causing reduced organellar acidification, lysosomal degradation and autophagic flux. Because of this, clearance of lipid droplets cannot take place in the autolysosomes, giving rise to the steatotic phenotype in the patients hepatocytes. Consistent with the similar clinical phenotype, we found that ATP6AP2 interacts with V0 assembly factors, while the missense mutations reduced the interaction, suggesting a compromised V-ATPase assembly in the patients. By contrast, in patients with exon-skipping mutation we found normal glycosylation of serum proteins and no effect on the interaction between ATP6AP2 and ATP6AP1, suggesting that the missense mutations have a stronger impact on overall ATP6AP2 function than the exon-skipping ones. These results shed light on the V-ATPase assembly in the ER and suggest that ATP6AP2 is an additional mammalian member of the assembly factors. In the second part of my thesis, I demonstrate that mutations in VMA21 also cause CDG with liver disease. Previously, mutations of VMA21 had been associated with an X-linked myopathy with excessive autophagy (XMEA), characterized by progressive vacuolation and atrophy of skeletal muscle. Yet, we were able to identify VMA21 mutations with a similar clinical phenotype compared to the other V-ATPase assembly factor deficiencies. Using patient fibroblasts, we tested the functional impact of the newly identified VMA21 mutations on V-ATPase assembly and function, with the attempt to highlight the differences between with the XMEA ones. First, we could show that VMA21 mutations are hypomorphic and reduce both Vma21 mRNA and protein expression. Second, VMA21 mutations cause autophagic defects with decreased lipid droplet degradation, similar to those observed in ATP6AP2-deficient cells. Finally, VMA21 fibroblasts showed an accumulation of unesterified cholesterol in vesicular structures, similar to what has been reported for the lysosomal storage disease Niemann-Pick type C (NPC). The sequestration of cholesterol in lysosomes triggers lipogenic pathways mediated by the sterol response element-binding protein (SREBP), most likely leading to hypercholesterolemia in the patients. Altogether, our results show that V-ATPase deficiencies are a novel group of metabolic syndromes that affect lysosomal/autophagic homeostasis. Studying these rare V-ATPase assembly disorders that are featured by liver steatosis as a unifying pathology may lead to a better understanding of the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is a common problem in metabolic syndrome

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

ABSTRACT During my Ph.D., I have been involved in two different projects: 1. The study of the role of ESCRT-0 in Notch signaling and tumor suppression. 2. The study of the regulation of V-ATPase in a subset of Notch-dependent developmental processes. 1. Abstract Project 1 Sorting and degradation of ubiquitylated cargoes depends on the endosomal sorting required for transport (ESCRT) machinery. The ESCRT machinery is composed of four multi-subunit ESCRT complexes (ESCRT-0, -I, -II, -III), which act in a sequential fashion to deliver endocytic cargoes into the internal luminal vesicles (ILVs) of the multivescicular endosome (MVE) for subsequent degradation. ESCRTs sort a number of transmembrane proteins including Notch and the JAK/STAT signaling receptor Domeless. In Drosophila epithelial tissue, mutation in ESCRT –I, -II, -III components results in misregulation of several signaling pathways, loss of epithelial polarity and unrestrained proliferation, suggesting that ESCRT genes act as tumor suppressors. Unexpectedly, Drosophila Hrs, one of the two components of the ESCRT-0 complex that acts upstream of the other ESCRT complexes have been found to be dispensable for tumor suppression. Thus, when I started my Ph.D. it was unclear whether ESCRT-0 had a tumor suppressive function. In my first project, I have found that mutation of Stam, a second ESCRT-0 component or of both Hrs and Stam result in accumulation of ubiquitinated proteins and of the signaling receptors Notch and Domeless. Nevertheless, mutant tissue displays normal tissue architecture, proliferation and Notch signaling activation. Overall, our in vivo data indicate that the ESCRT-0 complex does not play a crucial role in tumor suppression. 2 Abstract Project 2 In mammals, the Transcription Factor EB (TFEB) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether and how these two processes are interconnected. In Drosophila, the Microphthalmia-associated Transcription Factor (Mitf) is the unique homolog of the TFEB family. In my second project I have found that Mitf acts similar to its mammalian counterparts as transcription factor shuttling from lysosomes to the nucleus to regulate V-ATPase expression and lysosomal biogenesis. Interestingly, I found that V-ATPase subunits display diverse expression patterns in the wing imaginal disc, suggesting complex regulation of V-ATPase during development. Remarkably, I could show that Mitf cooperates to regulate expression of a key component of the V-ATPase during differentiation of proneural clusters (PNCs), a process that specifies cells with neuronal identity. In addition, I have observed that the PNCs possess a distinctive endo-lysosomal compartment and Notch localization. Finally, I have determined that modulation of V-ATPase and Mitf in the disc alters endo-lysosomal function and PNC development. Overall my in vivo analysis indicates that lysosomal-associated functions regulated by V-ATPase/Mitf axis might play a role in tissue patterning during Drosophila development. In addition to the work described above, I co-wrote a chapter on immunohistochemical tools and techniques to visualize Notch in Drosophila, in Methods in Molecular Biology Vol. 1187 (Tognon & Vaccari, 2014)

Abstract: Notch signaling is prominently involved in cell fate decision and growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. The Vacuolar H+ ATPase (V-ATPase) is responsible for acidification of endocytic organelles and recently it has been shown that mutants in V-ATPase subunit encoding genes in model organisms display loss of Notch signaling phenotypes. In the first part of my graduate studies, we aimed at discovering whether pharmacologic reduction of V-ATPase activity affected Notch signaling. We found that administration of BafilomycinA1 (BafA1), a highly specific V-ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we have found that BafA1 treatment leads to accumulation of Notch in the endo-lysosomal system, and reduces its processing and signaling activity. In Notch-addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, V-ATPase inhibition reduces growth of leukemia cells, without affecting Notch activating cleavage. However, consistent with the emerging roles of V-ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1-treated breast cancer cells. Our data support V-ATPase inhibition as a novel therapeutic approach to counteract tumor growth sustained by signaling pathways regulated at the endo-lysosomal level. The functions of Notch throughout the life of an individual are varied and complex. This complexity is not sufficiently accounted for by the limited core of known Notch signaling components and a growing body of evidence attributes it to additional factors that determine whether, when and how Notch functions within a given context. Considering this, in the second part of my graduate work, we sought to identify novel genes that might influence Notch. Thus, we performed a high content immunofluorescence-based RNA interference screen of a pharmacologically-relevant subset of the human genome. To this end, we monitored how knockdown of specific genes perturbs the localization of the Notch-1 receptor in human breast cells under resting and signaling conditions. Here we present the screen setup, the primary screen results and the candidate follow-up strategy.

La v-ATPase est une pompe à protons. Elle permet l’acidification d’organelles, ce qui est indispensable à de nombreux processus cellulaires. Cette enzyme est composée de 14 sous-unités différentes, organisées en deux domaines, le domaine catalytique V1 et le domaine membranaire V0. La sous-unité a du domaine V0 est essentielle au transport des protons. Il en existe 4 isoformes (a1 à a4) et des variants d’épissage (a1-I à a1-IV pour a1) permettant à la v-ATPase d’être adressée vers différents compartiments et donc d’être impliquée dans différents processus. Deux projets visant à étudier cette sous-unité ont été réalisés.En plus de son rôle dans le transport des protons, il a été montré que le domaine V0 de la v-ATPase est impliqué dans des évènements de trafic membranaire, tel que l’exocytose de vésicules de sécrétion. Ce rôle semble nécessiter des interactions avec les protéines SNAREs. J’ai montré, pendant la première partie de ma thèse, que les sous-unités flag-a1-I et flag-a1-IV sont toutes deux adressées aux granules de sécrétion de cellules neurosécrétrices et interagissent avec les protéines SNAREs VAMP2 et syntaxine-1. De façon intéressante la syntaxine-1 semble interagir préférentiellement avec la sous-unité a1-I qui dans les neurones est l’isoforme adressée aux terminaisons nerveuses. Les sous-unités a1-IV ne diffèrent d’a1-I que par l’ajout de 7 acides aminés dans sa moitié N-terminale. Le domaine d’interaction de la sous-unité a avec la syntaxine-1 semble donc être localisé dans cette région.Dans la deuxième partie de ma thèse, j’ai mis en évidence et étudié l’expression de la sous-unité rénale a4 dans des gliomes humains. Ces tumeurs sont les tumeurs cérébrales les plus fréquentes et sont en général associées à un mauvais pronostic. L’OMS distingue, en fonction de paramètres histologiques, les astrocytomes (de grade I à IV), les oligodendrogliomes et les gliomes mixtes (chacun de grade II ou III). Cette classification est controversée, notamment à cause de son manque de reproductibilité, et la prise en compte de marqueurs moléculaires semble s’imposer comme une solution pour la renforcer.J’ai quantifié par RT-PCR quantitative l’expression du gène ATP6V0A4 (codant la sous-unité a4) dans 188 prélèvements de gliomes humains. Nous avons ainsi montré que l’expression de la sous-unité a4 peut être utilisée comme marqueur diagnostique des oligodendrogliomes anaplasiques (35 % l’expriment). Dans un prélèvement, la présence de la codélétion 1p/19q et l’expression de a4, tout deux marqueurs indépendants des oligodendrogliomes, permettra le renforcement du diagnostique oligodendrogliome anaplasique. De plus a4 est fréquemment exprimée par les astrocytomes pilocytiques (70%), où elle est associée à la duplication en tandem de la région chromosomique 7q34 située à proximité directe du gène ATP6V0A4. Enfin une observation prometteuse est que l’expression de a4 pourrait être un marqueur de mauvais pronostic pour les patients atteints d’oligodendrogliome anaplasique ne présentant pas la co-délétion 1p/19q, observation qui devra être confirmée sur une plus grande cohorte de patients
Vacuolar type H+-ATPase is a proton pump, which acidifies numerous organelles, crucial for many cellular processes. This enzyme is composed of 14 different subunits organized in two domains, a catalytic V1 domain and a V0 membrane domain. The a-subunit of V0 is essential for proton transport. There are 4 isoforms of a (a1 to a4) and splicing variants (a1-I to a1-IV for the a1 subunit). v-ATPases containing different a-subunit isoforms are localized in different compartments allowing v-ATPase to participate in different processes. The a-subunits were studied in this work in two distinct projects.Besides its role in proton pumping, V0 domain of v-ATPase is implicated in organelles trafficking events, like vesicles exocytosis. This role seems to require interactions of V0 with SNARE proteins. During my thesis work, I showed that flag-a1-I and flag-a1-IV are both targeted to secretion granules in PC12 neurosecretory cells. These subunits interact with the SNARE proteins VAMP2 and syntaxin-1. Interestingly, syntaxin-1 seems to preferentially interact with the a1-I subunit, isoform which in neurons is sorted to nerve terminals. The only difference between a1-I and a1-IV subunits is the addition of 7 amino acids in the N-terminal half of a1-IV. So syntaxin-1 probably interacts with a1-I at this location. In a second project, I studied the expression of the renal a4-subunit in human gliomas. These tumors are the most frequent brain tumors and are generally associated with a poor prognosis. Based on histological parameters,WHO distinguishes, astrocytomas (grade I to IV), oligodendrogliomas and oligoastrogliomas (each of grade II or III). This classification suffers of a lack of reproducibility, which could be overcome by the identification of specific molecular markers.In the present work, by real time quantitative PCR, ATP6V0A4 gene (encoding the renal a4) expression was quantified in 188 human glioma biopsies. We established a4 expression as a new marker of grade III oligodendrogliomas (35 % express it), independent of the 1p/19q codeletion, an established marker of oligodendrogliomas. Moreover, a4 is expressed in 70% of pilocytic astrocytomas, in which it is associated with the tandem duplication of 7q34, localized at direct proximity of the ATP6V0A4 gene. Of promising interest is the observation that a4 expression could be considered as a bad prognostic marker for patients with 1p/19q non-deleted oligodendrogliomas, an observation that should be confirmed on larger cohorts of patients

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O acÃmulo de Na+ no vacÃolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulaÃÃo dos volumes e conteÃdos dos vacÃolos de cÃlulas vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prÃtons (V-ATPase e V-PPase), aquoporinas e vÃrios sistemas de transportes ativos e/ou secundÃrios, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prÃtons transmembranares funcionam como sistemas de transporte primÃrio nas cÃlulas vegetais e ambas as enzimas geram uma diferenÃa de potencial eletroquÃmico de prÃtons atravÃs da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquÃmico de prÃtons gerado pelos transportadores primÃrios para transportar Na+ para dentro do vacÃolo. No presente trabalho inicialmente foram determinados os conteÃdos de Ãons Na+ e K+ em raÃzes, hipocÃtilos e folhas e em seguida a anÃlise da expressÃo dos genes das bombas de prÃtons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plÃntulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmÃtico. As plÃntulas foram crescidas em meio nutritivo na ausÃncia de NaCl e PEG (controle), na presenÃa de 100 mM de NaCl (estresse salino) ou na presenÃa de 200,67 g/L de PEG (estresse osmÃtico). O conteÃdo de Ãons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteÃdo de Ãons K+ diminuiu na mesma condiÃÃo. A expressÃo dos genes das bombas de prÃtons e dos contra-transportadores vacuolares de folhas e de raÃzes no estresse salino aumentou em todas as condiÃÃes estudadas, porÃm o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulaÃÃo paralela entre esses genes. JÃ no estresse osmÃtico, os resultados para as folhas mostraram que a expressÃo dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes nÃo sofreram mudanÃas significativas. Nossos resultados sugerem que o estresse salino e o estresse osmÃtico induziram uma regulaÃÃo diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmÃtico.
The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress

SOBREIRA, Alana Cecília de Menezes. Estudo da expressão dos genes das bombas de prótons (V-ATPase e V-PPase) e dos contra-transportadores vacuolares (NHX) de Vigna unguiculata (L.) Walp submetidos a estresses abióticos.2009 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, 2009.
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The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress.
O acúmulo de Na+ no vacúolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulação dos volumes e conteúdos dos vacúolos de células vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prótons (V-ATPase e V-PPase), aquoporinas e vários sistemas de transportes ativos e/ou secundários, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prótons transmembranares funcionam como sistemas de transporte primário nas células vegetais e ambas as enzimas geram uma diferença de potencial eletroquímico de prótons através da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquímico de prótons gerado pelos transportadores primários para transportar Na+ para dentro do vacúolo. No presente trabalho inicialmente foram determinados os conteúdos de íons Na+ e K+ em raízes, hipocótilos e folhas e em seguida a análise da expressão dos genes das bombas de prótons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plântulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmótico. As plântulas foram crescidas em meio nutritivo na ausência de NaCl e PEG (controle), na presença de 100 mM de NaCl (estresse salino) ou na presença de 200,67 g/L de PEG (estresse osmótico). O conteúdo de íons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteúdo de íons K+ diminuiu na mesma condição. A expressão dos genes das bombas de prótons e dos contra-transportadores vacuolares de folhas e de raízes no estresse salino aumentou em todas as condições estudadas, porém o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulação paralela entre esses genes. Já no estresse osmótico, os resultados para as folhas mostraram que a expressão dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes não sofreram mudanças significativas. Nossos resultados sugerem que o estresse salino e o estresse osmótico induziram uma regulação diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmótico.