Targeting human aquaporin function : physiological and chemical approaches

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2018Aquaporins (AQPs) are a group of small membrane proteins belonging to a highly conserved family of membrane proteins called MIPs (Major Intrinsic Proteins) that are responsible for the bidirectional transport of wate (orthodox aquaporins) and also small uncharged solutes (aquaglyceroporins) across cell membranes, in response to osmotic or solute gradients. Rapid water flux across membranes is crucial to maintain the water homeostasis in many epithelia and endothelia involved in fluid transport. In addition, due to the unique ability of aquaglyceroporins to transport glycerol in addition to water, they have important roles in glycerol metabolism and skin hydration in non-fluid transporting tissues such as skin, fat and liver. The thesis introduction (Chapter 1) presents an overview of aquaporins structure, their main biological functions and related pathologies, with special emphasis on the so far described mechanisms of regulation. In the first part of this thesis (Chapter 2), we report the discovery of a new role for Aquaporin-5 (AQP5, an orthodox aquaporin) in adipocyte biology, where Aquaporin-7 (AQP7, an aquaglyceroporin) has been the mainly characterized protein in adipose tissue responsible for glycerol efflux. A better understanding of aquaporin regulation and gating would allow manipulation of their activity facilitating the identification of new putative modulators. A cellular model optimized to assess the function of aquaporins and discriminate individually each isoform, instead of mammalian cells where more than one isoform is usually expressed, is a useful tool to study aquaporin regulation. The second part of this thesis (Chapter 3) is dedicated to the functional characterization of different mammalian aquaporin isoforms (AQP3, AQP5, AQP7 and AQP10), using a yeast heterologous expression system devoided of endogenous aquaporins, a background where analysis is unlikely to be compromised by the co-expression of other aquaporin isoforms. Using the stopped-flow technique to evaluate the channel permeability for water and for glycerol, we were able to disclose gating mechanisms of aquaporin isoforms, being given special emphasis to the regulation by pH and phosphorylation. In the third part of this thesis (Chapter 4), a screening of several small gold compounds as inhibitors for Aquaporin-3 (AQP3, an aquaglyceroporin) was performed aiming at identifying new modulators with potential therapeutic use.A água possui um papel crucial para a vida devido às suas propriedades únicas. Todos os processos bioquímicos e fisiológicos de um organismo dependem da presença de água, sendo esta o componente fundamental na manutenção da homeostase celular. Nas células eucarióticas, a água encontra-se distribuída pelos vários compartimentos intracelulares separados entre si por membranas intracelulares e do meio extracelular pela membrana plasmática. Estas membranas de composição bilipídica são normalmente impermeáveis à maioria dos solutos polares e/ou carregados, cuja passagem é facilitada através de canais membranares específicos. No entanto estas membranas são bastante permeáveis à água, sendo então propostas três vias de transporte: por difusão simples, por transporte passivo associado ao transporte de iões e solutos e por canais específicos para a água. Atualmente sabe-se que a maioria das células de um organismo possui proteínas específicas – as aquaporinas – que conferem à membrana uma permeabilidade à água de cerca de 5 a 10 vezes superior às membranas que não possuem estas proteínas. Devido às suas características estruturais, as aquaporinas permitem um rápido transporte bidirecional de água, seletivo e regulado, em resposta a gradientes osmóticos, ao mesmo tempo que previnem a passagem de protões e iões através da membrana plasmática. Em mamíferos, são conhecidas até à data treze isoformas (AQP0-AQP12) que são classificadas em três grupos de acordo com a sua sequência primária, localização celular e seletividade em 1) aquaporinas ortodoxas, primariamente seletivas à água; 2) aquagliceroporinas, para além de água também transportam pequenos solutos neutros, como glicerol e ureia; e 3) super-aquaporinas, que são encontradas em membranas intracelulares e possuem menor homologia. No entanto, a lista de substâncias que são capazes de permear as diferentes aquaporinas tem aumentado ao longo do tempo. Recentemente, para além de água e glicerol, foi também descrito o transporte facilitado através de algumas isoformas de arsenito, amoníaco e peróxido de hidrogénio. Devido à grande diversidade de tecidos onde são encontradas as aquaporinas, o seu papel de facilitar o transporte de água e/ou solutos através das membranas plasmáticas é importante em vários processos fisiológicos, tais como: secreção de fluido glandular, mecanismo de concentração urinária, excitabilidade neuronal, metabolismo dos lípidos, hidratação epidérmica e balanço de água no cérebro. A observação do fenótipo de ratinhos geneticamente modificados com knock-out de determinadas aquaporinas revelou funções fisiológicas muito importantes no aparecimento e desenvolvimento de várias patologias, como epilepsia, edema cerebral, glaucoma, cancro e obesidade. No Capítulo 1 é apresentada uma introdução geral que visa proporcionar um conhecimento abrangente sobre as principais funções das aquaporinas humanas e patologias associadas, dando especial atenção aos diferentes mecanismos de regulação já conhecidos. Na primeira parte dos resultados desta tese (Capítulo 2), através da construção de linhas celulares de pré adipócitos de ratinho 3T3-L1 com diferentes níveis de expressão da Aquaporin-5 (cenário de ganho e perda de função) foi possível estabelecer um novo e determinante papel desta aquaporina na diferenciação dos adipócitos. Na segunda parte dos resultados desta tese (Capítulo 3), pretendeu-se usar um sistema de expressão heteróloga em Saccharomyces cerevisiae (S. cerevisiae) para permitir avaliar de forma individual a função de cada aquaporina. A levedura S. cerevisiae é considerada um valioso sistema de expressão heteróloga para estudar inúmeras proteínas devido à elevada homologia funcional entre esta e os eucariontes superiores, incluindo mamíferos. Pelo facto de existir uma grande biblioteca de estirpes disponíveis, ser de fácil manipulação genética, ser pouco dispendioso em comparação com as culturas de células animais e poderem ser testadas uma variedade de condições externas, este sistema oferece condições experimentais ótimas para estudar a especificidade e regulação das aquaporinas. Após a expressão e confirmação da sua localização celular, procedeu-se à caracterização da função de cada isoforma, utilizando a técnica de interrupção brusca de fluxo, seguindo a variação de volume celular por fluorescência quando se introduz uma perturbação no meio extracelular. Os fluxos de água através da membrana celular causados por gradientes de pressão osmótica (de solutos impermeantes ou permeantes) provocam alterações transitórias de volume, até se atingir um novo volume final de equilíbrio osmótico. A velocidade com que as alterações de volume ocorrem e o tempo que a célula leva a re-estabelecer o seu novo equilíbrio osmótico dependem diretamente das características intrínsecas de transporte da membrana, em particular da quantidade de canais específicos para a água e para o soluto em questão. No Capítulo 3 foram estudadas quatro isoformas diferentes (AQP3, AQP5, AQP7 e AQP10) e os seus mecanismos de regulação por pH e fosforilação foram revelados pela primeira vez. Vários esforços têm vindo a ser feitos com o intuito de desenvolver possíveis fármacos para tratamento das aquaporinopatias, mas até agora nenhum composto se revelou qualificado para estudos in vivo, quer pela sua fraca solubilidade quer pela sua baixa capacidade de inibição. Na terceira parte dos resultados desta tese (Capítulo 4), deu-se especial atenção à descoberta de novos compostos organometálicos, inibidores da função da Aquaporina-3, que poderão ser usados para benefício clínico na prevenção ou tratamento das várias patologias associadas.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52384/2013, Programa de Doutoramento Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD

The mechanism of aquaporin inhibition by gold compounds elucidated by biophysical and computational methods

The inhibition of water and glycerol permeation via human aquaglyceroporin-3 (AQP3) by gold(III) complexes has been studied by stopped-flow spectroscopy and, for the first time, its mechanism has been described using molecular dynamics (MD), combined with density functional theory (DFT) and electrochemical studies. The obtained MD results showed that the most effective gold-based inhibitor, anchored to Cys40 in AQP3, is able to induce shrinkage of pores preventing glycerol and water permeation. Moreover, the good correlation between the affinity of the Au(III) complex to Cys binding and AQP3 inhibition effects was highlighted, while no influence of the different oxidative character of the complexes could be observed

SARS-CoV-2 decreases malaria severity in co-infected rodent models

Coronavirus disease 2019 (COVID-19) and malaria, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Plasmodium parasites, respectively, share geographical distribution in regions where the latter disease is endemic, leading to the emergence of co-infections between the two pathogens. Thus far, epidemiologic studies and case reports have yielded insufficient data on the reciprocal impact of the two pathogens on either infection and related diseases. We established novel co-infection models to address this issue experimentally, employing either human angiotensin-converting enzyme 2 (hACE2)-expressing or wild-type mice, in combination with human- or mouse-infective variants of SARS-CoV-2, and the P. berghei rodent malaria parasite. We now show that a primary infection by a viral variant that causes a severe disease phenotype partially impairs a subsequent liver infection by the malaria parasite. Additionally, exposure to an attenuated viral variant modulates subsequent immune responses and provides protection from severe malaria-associated outcomes when a blood stage P. berghei infection was established. Our findings unveil a hitherto unknown host-mediated virus-parasite interaction that could have relevant implications for disease management and control in malaria-endemic regions. This work may contribute to the development of other models of concomitant infection between Plasmodium and respiratory viruses, expediting further research on co-infections that lead to complex disease presentations.info:eu-repo/semantics/publishedVersio

Construçao do masculino na Curitiba das décadas de 1940 e 1950 tornar-se homem

Orientadora : Maria Luiza AndreazzaAutor não autorizou a divulgação do arquivo digitalDissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Humanas, Letras e ArtesInclui referência

Sweet Cherry (Prunus avium L.) PaPIP1;4 is a functional aquaporin upregulated by pre-harvest calcium treatments that prevent cracking

The involvement of aquaporins in rain-induced sweet cherry (Prunus avium L.) fruit cracking is an important research topic with potential agricultural applications. In the present study, we performed the functional characterization of PaPIP1;4, the most expressed aquaporin in sweet cherry fruit. Field experiments focused on the pre-harvest exogenous application to sweet cherry trees, cultivar Skeena, with a solution of 0.5% CaCl2, which is the most common treatment to prevent cracking. Results show that PaPIP1;4 was mostly expressed in the fruit peduncle, but its steady-state transcript levels were higher in fruits from CaCl2-treated plants than in controls. The transient expression of PaPIP1;4-GFP in tobacco epidermal cells and the overexpression of PaPIP1;4 in YSH1172 yeast mutation showed that PaPIP1;4 is a plasma membrane protein able to transport water and hydrogen peroxide. In this study, we characterized for the first time a plasma membrane sweet cherry aquaporin able to transport water and H2O2 that is upregulated by the pre-harvest exogenous application of CaCl2 supplements.This work was supported by the “Contrato-Programa” UIDB/04050/2020 and UIDB/04033/2020 funded by national funds through the FCT I.P. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research projects MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341), BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165) and CherryCrackLess (PTDC/AGR-PRO/7028/2014). R.B. was supported with a PhD student grant (PD/BD/113616/2015) under the Agrichains Doctoral Program (PD/00122/2012) funded by FCT. H.N. was supported by an FCT postdoctoral grant (SFRH/BPD/115518/2016) and A.C. was supported by a contract in the MitiVineDrought project.info:eu-repo/semantics/publishedVersio

Human aquaporin-5 facilitates hydrogen peroxide permeation affecting adaption to oxidative stress and cancer cell migration

Reactive oxygen species (ROS), including H2O2, contribute to oxidative stress and may cause cancer initiation and progression. However, at low concentrations, H2O2 can regulate signaling pathways modulating cell growth, differentiation, and migration. A few mammalian aquaporins (AQPs) facilitate H2O2 diffusion across membranes and participate in tumorigenesis. AQP3 and AQP5 are strongly expressed in cancer tissues and AQP3-mediated H2O2 transport has been related to breast cancer cell migration, but studies with human AQP5 are lacking. Here, we report that, in addition to its established water permeation capacity, human AQP5 facilitates transmembrane H2O2 diffusion and modulates cell growth of AQP5-transformed yeast cells in response to oxidative stress. Mutagenesis studies revealed that residue His173 located in the selective filter is crucial for AQP5 permeability, and interactions with phosphorylated Ser183 may regulate permeation through pore blockage. Moreover, in human pancreatic cancer cells, the measured AQP5-mediated H2O2 influx rate indicates the presence of a highly efficient peroxiporin activity. Cell migration was similarly suppressed by AQP3 or AQP5 gene silencing and could be recovered by external oxidative stimuli. Altogether, these results unveiled a major role for AQP5 in dynamic fine-tuning of the intracellular H2O2 concentration, and consequently in activating signaling networks related to cell survival and cancer progression, highlighting AQP5 as a promising drug target for cancer therapies

The aquaporin-3-inhibiting potential of polyoxotungstates

Polyoxometalates (POMs) are of increasing interest due to their proven anticancer activities. Aquaporins (AQPs) were found to be overexpressed in tumors bringing particular attention to their inhibitors as anticancer drugs. Herein, we report for the first time the ability of polyoxotungstates (POTs), such as of Wells-Dawson P2W18, P2W12, and P2W15, and Preyssler P5W30 structures, to affect aquaporin-3 (AQP3) activity and impair melanoma cell migration. The tested POTs were revealed to inhibit AQP3 function with different effects, with P2W18, P2W12, and P5W30 being the most potent (50% inhibitory concentration (IC50) = 0.8, 2.8, and 3.2 µM), and P2W15 being the weakest (IC50 > 100 µM). The selectivity of P2W18 toward AQP3 was confirmed in yeast cells transformed with human aquaglyceroporins. The effect of P2W12 and P2W18 on melanoma cells that highly express AQP3 revealed an impairment of cell migration between 55% and 65% after 24 h, indicating that the anticancer properties of these compounds may in part be due to the blockage of AQP3-mediated permeability. Altogether, our data revealed that P2W18 strongly affects AQP3 activity and cancer cell growth, unveiling its potential as an anticancer drug against tumors where AQP3 is highly expressed.Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BTM-SAL/28977/2017, PTDC/MED-QUI/31721/2017, UID/DTP/04138/2019, UIDB/04326/2020, SFRH/BD/117586/2016. Austrian Science Fund (FWF) P27534, M2203info:eu-repo/semantics/publishedVersio

Insights into the mechanisms of aquaporin-3 inhibition by gold(III) complexes: the importance of non-coordinative adduct formation

A series of six new Au(III) coordination compounds with phenanthroline ligands have been synthesized and studied for the inhibition of the water and glycerol channel aquaporin-3 (AQP3). From a combination of different experimental and computational approaches, further insights into the mechanisms of AQP3 inhibition by gold compounds at a molecular level have been gained. The results evidence the importance of noncoordinative adduct formation, prior to “covalent” protein binding, to achieve selective AQP3 inhibition

Molecular Basis of Aquaporin-7 Permeability Regulation by pH

The aquaglyceroporin AQP7, a family member of aquaporin membrane channels, facilitates the permeation of water and glycerol through cell membranes and is crucial for body lipid and energy homeostasis. Regulation of glycerol permeability via AQP7 is considered a promising therapeutic strategy towards fat-related metabolic complications. Here, we used a yeast aqy-null strain for heterologous expression and functional analysis of human AQP7 and investigated its regulation by pH. Using a combination of in vitro and in silico approaches, we found that AQP7 changes from fully permeable to virtually closed at acidic pH, and that Tyr135 and His165 facing the extracellular environment are crucial residues for channel permeability. Moreover, instead of reducing the pore size, the protonation of key residues changes AQP7’s protein surface electrostatic charges, which, in turn, may decrease glycerol’s binding affinity to the pore, resulting in decreased permeability. In addition, since some pH-sensitive residues are located at the monomer-monomer interface, decreased permeability may result from cooperativity between AQP7’s monomers. Considering the importance of glycerol permeation via AQP7 in multiple pathophysiological conditions, this mechanism of hAQP7 pH-regulation may help the design of selective modulators targeting aquaglyceroporin-related disorders