Academic literature on the topic 'V-ATPase'

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&#8240; ou ainda aclimatados por 10 dias a diferentes salinidades (5-21&#8240;), 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 &#945; 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 (&#8776; 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 (&#8776; 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&#8240;. 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&#8240;, 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&#8240;. 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&#8240; mostraram também uma diminuição acentuada da atividade V-ATPase em resposta ao aumento da salinidade. Entretanto, com exceção da salinidade mais baixa (5&#8240;) 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+.<br>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.<br>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.<br>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.