Browsing by Author "Loureiro, Cláudia"
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- Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasivenessPublication . Moniz, Sónia; Martinho, Olga; Pinto, Filipe; Sousa, Bárbara; Loureiro, Cláudia; Oliveira, Maria José; Moita, Luís Ferreira; Honavar, Mrinalini; Pinheiro, Célia; Pires, Manuel; Lopes, José Manuel; Jones, Chris; Costello, Joseph F; Paredes, Joana; Reris, Rui Manuel; Jordan, PeterThe gene encoding protein kinase WNK2 was recently identified to be silenced by promoter hypermethylation in gliomas and meningiomas, suggesting a tumour-suppressor role in these brain tumours. Following experimental depletion in cell lines, WNK2 was further found to control GTP-loading of Rac1, a signalling guanosine triphosphatase involved in cell migration and motility. Here we show that WNK2 promoter methylation also occurs in 17.5% (29 out of 166) of adult gliomas, whereas it is infrequent in its paediatric forms (1.6%; 1 out of 66). Re-expression of WNK2 in glioblastoma cells presenting WNK2 gene silencing reduced cell proliferation in vitro, tumour growth in vivo and also cell migration and invasion, an effect correlated with reduced activation of Rac1. In contrast, when endogenous WNK2 was depleted from glioblastoma cells with unmethylated WNK2 promoter, changes in cell morphology, an increase in invasion and activation of Rac1 were observed. Together, these results validate the WNK2 gene as a recurrent target for epigenetic silencing in glia-derived brain tumours and provide first mechanistic evidence for a tumour-suppressing role of WNK2 that is related to Rac1 signalling and tumour cell invasion and proliferation.
- Metabolic tumor cell adaptation: tyrosine phosphorylation modulates cell surface expression of NKCC2 and KCC3Publication . Loureiro, Cláudia; Barros, Patrícia; Matos, Paulo; Jordan, PeterIntroduction: Tumor cells require cellular chloride and potassium transport to adapt to a changing microenvironment, both for cell volume regulation and membrane potential maintenance. Cellular chloride and potassium entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Material and Methods: Cotransporter constructs were transfected into HEK293 cells and the activity of spleen tyrosine kinase (SYK) modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Cotransporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results and Discussions: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that SYK phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Conclusion: We identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings add knowledge on how tumor cells may respond to microenvironmental changes that affect their cell volume or metabolic crosstalk.
- A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpointPublication . Loureiro, Cláudia; Matos, Ana Margarida; Dias-Alves, Ãngela; Pereira, Joana Filipa; Uliyakina, Irina; Barros, Patrícia; Amaral, Margarida; Matos, PauloThe peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.
- Network biology approaches in the identification of novel pharmacological targets – the case of cystic fibrosisPublication . Loureiro, Cláudia; Matos, Ana; Santos, João; Farinha, Carlos; Jordan, Peter; Matos, Paulo; Pinto, FranciscoIn cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide–pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.
- A new WNK4/SYK signaling pathway regulating membrane levels of chloride cotransporters in kidney cellsPublication . Loureiro, Cláudia; Jordan, PeterArterial hypertension affects one third of the Western population and constitutes a remarkable risk factor for cardiovascular disease and stroke. Pseudohypoaldosteronism type II or Gordon's syndrome is a rare familial form of hypertension characterized by increased renal salt reabsorption accompanied by hyperkalemia, and caused by mutations in the WNK1 and WNK4 genes. These encode protein kinases regulating renal electrolyte homeostasis through modulation of several membrane transporters and ion channels but the molecular pathways involved are only partially understood. In a recent study on the regulation of the chloride channel CFTR (Cystic Fibrosis Transmembrane Regulator Condutance), a novel WNK4 signaling pathway was described to regulate the amount of CFTR at the cell surface. The underlying mechanism involves tyrosine kinase Syk (Spleen tyrosine kinase), that phosphorylates CFTR protein and promotes its removal from the plasma membrane whereas this activity is inhibited in the presence of WNK4 [1]. In order to study whether the WNK4/Syk interplay may also operate in the regulation of other chloride co-transporters, their protein sequences were inspected for the presence of a Syk consensus motif. Among 20 different transport proteins, the Syk motif was identified only in the sequence of chloride co-transporters NKCC2 and KCC3, which are important for electrolyte homeostasis in the kidney and blood pressure regulation. In order to investigate whether Syk can phosphorylate these channels in vitro, recombinant NKCC2 and KCC3 fragments were produced and tested as substrates in in vitro phosphorylation assays. Strong phosphorylation of renal channels NKCC2 and KCC3 by Syk was observed. The results of this study are a first step to understand the regulation of these ion channels via the WNK4/Syk pathway and possible identify a new mode regulating electrolyte homeostasis in the kidney, potentially related to hypertension.
- Uma nova via de regulação do transportador renal NKCC2 envolvido na hipertensão arterialPublication . Loureiro, Cláudia; Jordan, PeterA hipertensão arterial afeta um terço da população adulta é um fator de risco para a doença cardiovascular ou o acidente vascular cerebral. O pseudohipoaldosteronismo tipo II, ou síndroma de Gordon, é uma forma rara de hipertensão familiar causada por mutações nos genes WNK1 e WNK4. Esta síndrome envolve a desregulação da homeostase renal dependente da aldosterona, principalmente nos túbulos distais do nefrónio onde as proteínas cinases WNK1 e WNK4 modulam a atividade de diversos canais de transporte iónico, incluindo os alvos de fármacos diuréticos NCC e NKCC2. É conhecido que a WNK1 e WNK4 ativam as cinases OSR1 e SPAK, que por sua vez fosforilam diretamente as proteínas da família SLC12 (NCC, NKCC1 e NKCC2), ativando o transporte iónico. Aqui apresentamos evidência para uma segunda via de regulação do NKCC2 pela cinase WNK4. O mecanismo envolve um antagonismo entre WNK4 e a tirosina cinase Syk (spleen tyrosine kinase), que fosforila diretamente as proteínas NKCC2 e KCC3. NKCC2 transporta sódio, potássio e cloreto para resorção enquanto KCC3 compensa com o efluxo de potássio e cloreto. Ambos são importantes para a homeostase eletrolítica nos rins e na manutenção da tensão arterial. Foram produzidos os fragmentos recombinantes das regiões N-terminais de NKCC2 e KCC3 que contêm os motivos de reconhecimento da Syk. Após a sua purificação, estas proteínas recombinantes foram utilizados como substratos em ensaios de proteína cinase in vitro e complementados com Syk imunoprecipitada a partir de células HEK 293T transfectadas. Estes ensaios demostraram a fosforilação de NKCC2 e KCC3 pelaSyk. Estão em curso os ensaios em células renais para verificar o efeito da Syk e WNK4 sobre a quantidade de NKCC2 e KCC3 presentes na membrana plasmática. O trabalho contribui para a compreensão da regulação dos referidos canais renais e visa identificar novos alvos terapêuticos no tratamento da hipertensão arterial.
- Regulação dos níveis de transportadores de cloreto na membrana plasmática de células renais pela nova via de sinalização WNK4/SykPublication . Loureiro, Cláudia; Jordan, Peter; Farinha, CarlosA hipertensão arterial afeta, em países ocidentais, quase um terço da população adulta, sendo um fator de alto risco para a doença cardiovascular ou o acidente vascular cerebral. O pseudohipoaldosteronismo tipo II, ou síndroma de Gordon, é uma forma rara de hipertensão familiar, caracterizada pela ocorrência de hipertensão acompanhada por hipercalemia, e causado por mutações nos genes WNK1 e WNK4. Os mecanismos moleculares subjacentes a esta condição envolvem a regulação da homeostase renal de eletrólitos, principalmente nos túbulos distais do nefrónio onde ocorre a modulação de diversos canais iónicos e transportadores através da cooperação das proteínas cinases WNK, como do canal de potássio ROMK e do cotransportador de sódio e cloreto NCC. A WNK1 e WNK4 ativam as cinases OSR1 e SPAK, que por sua vez fosforilam diretamente as proteínas da família SLC12 (NCC, NKCC1 e NKCC2), ativando o transporte iónico. A subfamília das proteínas cinases WNK (with-no-lysine [K]) pode regular a atividade dos vários canais iónicos envolvidos na homeostase de sódio, potássio e cloreto através da sua fosforilação como também o número de canais proteicos expressos na membrana plasmática (MP). Num estudo realizado recentemente sobre a regulação do canal de cloreto CFTR (Cystic Fibrosis Transmembrane Condutance Regulator), verificou-se que a presença de WNK4 aumenta também a quantidade de CFTR na MP. O mecanismo é diferente e envolve um papel antagonista ao da tirosina cinase Syk (spleen tyrosine kinase), que fosforila a proteína CFTR e promove a sua remoção da MP, uma atividade inibida pela WNK4. O alvo da regulação é o resíduo tirosina 512 da CFTR, localizado num motivo de consenso da cinase Syk. Este motivo peptídico especifico foi, por sua vez encontrado na sequência primária de mais dois canais iónicos: NKCC2 e KCC3. O primeiro é um cotransportador renal de sódio, potássio e cloreto que facilita o influxo de sódio e cloreto nas células enquanto o segundo compensa com o efluxo de potássio e cloreto e tem expressão renal e neuronal. Ambos são importantes para a homeostase eletrolítica nos rins e na manutenção da tensão arterial. O presente estudo teve como objetivo verificar experimentalmente o envolvimento da via WNK4/Syk na regulação dos canais renais NKCC2 e KCC3, nomeadamente se a Syk fosforila os canais in vitro, e realizou-se em três fases. Numa primeira fase, foram analisadas as sequências dos canais renais NKCC2 e KCC3 no sentido de selecionar fragmentos dos respetivos domínios N-terminais que contêm os VIII motivos de fosforilação pela Syk e que apresentavam uma previsão computacional de gerarem proteínas recombinantes solúveis. Estes fragmentos foram clonados no plasmídeo pET-28a e as respetivas proteínas recombinantes expressas em bactérias. Após a lise das bactérias por sonicação, verificou-se que a maioria das proteínas se encontrou na fração solúvel, confirmando experimentalmente a previsão bioinformática. De seguida, procedeu-se à purificação das proteínas recombinantes a partir do lisado bacteriano. Na segunda fase, foi avaliado em ensaios de fosforilação in vitro se os substratos recombinantes produzidos anteriormente são substratos da cinase Syk. Estes ensaios foram realizados com a cinase imunoprecipitada a partir de células HEK 293T transfectadas com os construtos Syk-wt, Syk-kd e com o vetor não recombinante. Para além dos fragmentos recombinantes das proteínas NKCC2 wt e KCC3 wt foram também utilizados os fragmentos mutantes, NKCC2 Y45F e KCC3 Y63F. Estes mutantes possuem uma substituição no resíduo de tirosina, local de fosforilação pela cinase Syk. Nestes ensaios, observou-se claramente a fosforilação dos canais renais NKCC2 wt e KCC3 wt por parte da cinase Syk-wt. Para além disso, observou-se também uma fosforilação residual dos canais mutantes pela Syk-wt. Numa terceira fase, identificou-se a expressão da proteína NKCC2 endógena numa linha celular renal, indicando assim um modelo celular para o estudo do canal in vivo. Esta fase tem como objetivo determinar o efeito que a transfecção de células renais com Syk e WNK4 terá sobre a quantidade na membrana plasmática dos dois canais renais em estudo. O presente trabalho contribuiu para a compreensão da regulação dos referidos canais iónicos a partir da via Syk/WNK4. A validação da fosforilação dos dois canais justifica agora um estudo mais alargado que visa caracterizar mais um modo de atuação de WNK4 sobre a homeostase eletrolítica nos rins, como também a contribuição desta via para a hipertensão. Assim, este estudo poderá identificar novos alvos terapêuticos no tratamento da hipertensão arterial.
- Regulação dos níveis de transportadores de cloreto na membrana plasmática de células renais pela nova via de sinalização WNK4/SYKPublication . Loureiro, Cláudia; Jordan, Peter
- Regulation of epithelial chloride transport by phospho-tyrosine-initiated protein networksPublication . Loureiro, Cláudia; Jordan, Peter; Clarke, LukaIon transport is crucial for cell volume regulation by compensating variations in extracellular tonicity, playing an important role in maintaining the structural integrity and intracellular milieu in cells. These functions require a dynamic, spatio-temporally coordinated regulation of ion transport, which occurs in cells by two mechanisms: first, the amount of channel or cotransporter inserted into the plasma membrane (PM) from a pool of endosomal storage vesicles, and second, the ion transport activity regulated by post-translational modifications such as phosphorylation of channel or transporter proteins. Previous results from the host laboratory showed that phosphorylation by spleen tyrosine kinase (SYK) of Tyr512 in the NBD1 domain regulates PM abundance of CFTR, the chloride channel involved in cystic fibrosis. The main objective of this PhD project was to identify phospho-tyrosine-binding proteins involved in the regulation of chloride transport proteins and the underlying molecular mechanism. First, it was found that besides CFTR two further renal ion cotransporters, NKCC2 and KCC3, are phosphorylated by SYK in vitro on an N-terminal tyrosine residue and that experimental manipulation of either SYK expression levels or its catalytic activity affect the cell surface abundance of these cotransporters. Interestingly, the very same phosphorylation pathway leads to a decrease in NKCC2 but to an increase in KCC3 PM levels. Second, the underlying biochemical mechanism was identified using peptide pulldown assays followed by mass spectrometry and revealed that the adaptor protein SHC1 binds to phospho-tyrosine in NKCC2, KCC3 and CFTR through its PTB domain. Upon depletion of endogenous SHC1 expression, KCC3 decreased at the PM, whereas NKCC2 and CFTR levels increased. In the case of phosphorylated NKCC2, SNX27 and NCK1 were identified as additional binding partners. Lastly, SHC1 was shown to form a complex with CFTR following activation of protein kinase SYK, but does not affect the PM level of the most frequent mutant F508del-CFTR. The results described in this work identified a novel SYK/SHC1 pathway that regulates the cotransporters NKCC2 and KCC3 and the chloride channel CFTR and have potential biomedical implications for the identification of new therapeutic targets in diseases like hypertension or cystic fibrosis, or those involving regulation of cell volume.
- Regulation of epithelial chloride transport by phospho-tyrosine-initiated protein networksPublication . Loureiro, Cláudia; Jordan, PeterRegulation of epithelial chloride transport by phospho-tyrosine-initiated protein networks