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- Analysis of translation of 5’ untranslated regions in cancerPublication . Silva, Joana; Romão, Luísa; Luchessi, AugustoShort upstream open reading frames (uORFs) are cis-acting elements located within the 5'- leader sequence of transcripts. Recent genome-wide ribosome profiling (RiboSeq) studies have demonstrated the widespread presence of uORFs in the transcriptome and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream main open reading frame (mORF) by triggering messenger RNA (mRNA) decay or by regulating translation. Thus, disruption, elimination or creation of uORFs can elicit the development of several genetic diseases, such as cancer. The ATP-binding cassette subfamily E member 1 (ABCE1) gene, belongs to the ABC gene family of transporters. However, it does not behave as a drug transporter like the other members of this family. ABCE1 actively participates in the different stages of the translation process and is involved in cell proliferation and anti-apoptotic signaling processes, associating ABCE1 to a potential oncogenic function. RiboSeq occupancy profiles of the ABCE1 mRNA 5’-leader sequence indicate an active translation associated with the presence of uORFs, which is suggestive of a high translational regulation. Our aim was to study the translational regulation mediated by the five AUG and five non- AUG uORFs present in the human ABCE1 5’-leader sequence in colorectal cancer. With this purpose, we constructed a set of Firefly luciferase (FLuc) reporter vectors derived from the wild-type one containing the native configuration of the human ABCE1 5’-leader sequence upstream of the FLuc ORF, and transiently transfected colorectal cancer HCT116 cells. Here we show that ABCE1 mORF expression is regulated by its uORFs. Our results are consistent with a model wherein uORF1 recruits ribosomes onto the mRNA, behaving like a ribosomal barrier. The ribosomes that efficiently bypass uORF1 and/or uORF2, must probably reinitiate at uORF3 and/or uORF5, while uORF4 is greatly bypassed. uORF3 and uORF5 function as repressive uORFs that may cooperate to reach a maximum repression of the mORF. Thus, both bypass and reinitiation events of the AUG uORFs within ABCE1 5’-leader sequence contribute for the translational control of the mORF. In constrast, the non-AUG uORFs seem to be devoided of a significant inhibitory activity. The AUG uORF-mediated translational control is maintained in normal and in endoplasmic reticulum (ER) stress conditions, which keeps the expression level of ABCE1 at a minimum, showing that ABCE1 is a resistant transcript whose functions are equally essential in normal and in coditions of global translation impairment. In addition, we show that ABCE1 uORF-mediated translational regulation is preserved in non-tumorigenic and cancerous cells, which is consistent with a lack of an oncogenic function by the uORFs, as well as ABCE1 himself, in the colorectal cancer cell line tested. This study contributes with an additional example of how uORF-mediated translational regulation can occur. In addition, it reveals how important is to screen the 5’-leader sequence of the transcripts in search for potential disease-related variants. This information might be relevant for the implementation of new diagnostic and/or therapeutic tools for diseases associated with the deregulation of uORF-mediated translational control.
- In vitro genotoxic and epigenotoxic effects of occupational exposure to nanofibresPublication . Ventura, Célia; Uva, António Neves Pires de Sousa; Silva, Maria JoãoIn recent years, human exposure to nanofibres (i.e., fibres with diameters < 100 nm) has considerably increased due to their incorporation in a wide range of consumer products. As such, there is a growing concern that the distinctive physicochemical characteristics of nanofibres may lead to adverse human health effects, mainly by their inhalation in occupational settings. Several toxicological studies have indicated that some nanofibres, such as the MWCNT-7, display in vitro toxicity and induce pulmonary inflammation, fibrosis, granulomas and carcinogenesis in vivo. Others, like cellulose nanofibrills (CNF), seem biocompatible and promising for biomedical applications. Thus, the genotoxic effects of nanofibres must be deeply studied to identify their possible hazard, and new “omics” methodologies can uncover their underlying mechanisms of action. Moreover, distinctive genomic or epigenomic expression profiles may be biomarkers of occupational exposure to nanofibres. In this work, the two above-mentioned nanofibres are analyzed in vitro, and the toxic effects of MWCNT-7 compared to those of crocidolite asbestos. Cytotoxicity and genotoxicity were investigated through conventional assays, and the differentially expressed microRNA (DE miRNA) in alveolar epithelial cells exposed to MWCNT-7 or crocidolite identified by next-generation sequencing. The overall results demonstrate that MWCNT-7 is cytotoxic, genotoxic and immunotoxic. Notably, it induced nucleoplasmic bridges in alveolar cells, possibly due to its resemblance with the microtubules and physical interference with the mitotic spindle. Different viabilities and micronucleus frequencies were observed in alveolar cells when using a conventional monoculture or a co-culture of these cells with macrophages, which may be related to their epithelial-mesenchymal transition and consequent increase of cell resistance to apoptosis. Regarding CNF, at low concentrations it stimulates cell proliferation, whereas at higher ones it is moderately toxic. Although no immunotoxicity and no significant DNA damage were detected, low CNF doses induced micronucleus. Concerning the epigenotoxic study, several DE miRNA were identified in alveolar cells exposed to MWCNT-7 or crocidolite, and a unique set was identified for each exposure. Both materials caused common changes in pathways related to cell metabolism, cell growth and death, cell-to-cell communication, protein processing, and signal transduction. Other functional pathways were distinctively identified for each material that sugest particular mechanisms of action. Since most are cancer related, a network of DE miRNA and target cancer genes was constructed, highlighting the carcinogenic potential of both materials.
- Mitochondrial dysfunction in fatty acid β-oxidation disordersPublication . Rocha, Hugo; Vilarinho, Laura; Amado, Francisco Manuel Lemos; Ferreira, Rita Maria Pinho[PT] A mitocôndria desempenha um papel fundamental na regulação de vários processos celulares, com particular relevância na produção de energia, sendo a β-oxidação mitocondrial dos ácidos gordos uma das vias metabólicas que tem lugar neste organelo. Os défices da β-oxidação mitocondrial dos ácidos gordos estão entre os grupos de doenças metabólicas mais estudados, existindo contudo, algumas questões que continuam por esclarecer, como a sua prevalência ao nascimento em determinadas regiões da Europa e quais e de que forma os vários determinantes patofisiológicos interatuam para produzir um determinado fenótipo. A análise dos dados de programas de rastreio neonatal da península Ibérica possibilitou estimar a prevalência ao nascimento dos défices da β-oxidação mitocondrial, tendo-se observado um dos valores mais elevados (particularmente em Portugal) no âmbito das regiões europeias, fundamentalmente devido à grande prevalência ao nascimento dos défices da desidrogenase dos ácidos gordos de cadeia média. Estes resultados realçam o impacto deste grupo de doenças genéticas nesta região europeia. A caracterização do proteoma mitocondrial, a partir de fibroblastos em cultura, de doentes com défices da β-oxidação mitocondrial (especificamente défice múltiplo das desidogenases (MADD) e défice da desidrogenase dos ácidos 3- hidroxilados de cadeia longa (LCHADD)) permitiu obter uma perspetiva geral sobre a plasticidade do proteoma mitocondrial nestas doenças assim como avaliar quais as principais vias metabólicas envolvidas na sua patogénese. Em formas severas de MADD foi observada uma sobre-expressão de chaperones, enzimas antioxidantes e proteínas associadas à apoptose. Nestas células foi igualmente observada a sobre-expressão de enzimas glicolíticas, como adaptação ao bloqueio da β-oxidação. A análise de amostras de doentes com LCHADD também evidenciou uma sobre-expressão de enzimas glicolíticas, assim como de proteínas relacionadas com a apoptose, e a modulação do sistema de defesa antioxidante. O doente com uma forma severa de LCHADD apresentou níveis de stress oxidativo elevados, associados a uma sobreexpressão da MnSOD, enquanto o doente com uma forma moderada apresentou níveis mais baixos de stress oxidativo e uma sub-expressão da MnSOD. Estes resultados são provavelmente o reflexo do papel da MnSOD na regulação dos níveis de ROS, mantendo-os em níveis que não provoquem danos, mas que permitam iniciar processos de sinalização com vista à manutenção celular. A comparação de forma moderadas com severas de MADD não revelou diferenças significativas, muito provavelmente porque os níveis de stress oxidativo são suficientemente altos para despoletar uma resposta semelhante às formas severas. Os presentes resultados realçam as diferenças na modulação do sistema de defesa antioxidante no espectro dos défices da β-oxidação mitocondrial. No seu conjunto os resultados obtidos revelam as principais vias moduladas nos défices da β-oxidação mitocondrial e a importância do stress oxidativo e sistema de defesa antioxidante para o fenótipo. Ao permitem compreender melhor a complexa interação entre os vários fatores que interagem com vista ao fenótipo e que podem ser de origem genética, epigenética ou ambiental, contribuem para o desenvolvimento de novas e mais eficazes abordagens terapêuticas.
- Nanomateriais manufaturados: avaliação de segurança através da caracterização dos seus efeitos genéticosPublication . Louro, Maria Henriqueta; Leite, Ema; Silva, Maria João[PT] Os nanomateriais manufaturados (NMs), isto é, fabricados deliberadamente para fins específicos, apresentam propriedades físico-químicas únicas como a dimensão, área superficial ou funcionalização, que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas muito vantajosas para aplicações industriais e biomédicas. Efetivamente, a tecnologia baseada nos NMs, ou nanotecnologia, foi identificada como uma key enabling technology, impulsionadora do crescimento económico dos países industrializados, devido ao seu potencial para melhorar a qualidade e desempenho de muitos tipos de produtos e de processos. Contudo, a expansão da utilização de NMs contrasta com a insuficiente avaliação de risco para a saúde humana e para o ambiente, sendo considerados como um risco emergente para a saúde pública. As incertezas sobre a segurança dos NMs para a saúde pública advêm sobretudo de estudos epidemiológicos em humanos expostos a nanomateriais produzidos como consequência dos processos e atividades humanas e da poluição. Uma das principais preocupações relativamente aos efeitos adversos dos NMs na saúde humana é o seu potencial efeito carcinogénico, que é sugerido por alguns estudos experimentais, como no caso dos nanomateriais de dióxido de titânio ou dos nanotubos de carbono. Para avaliar em curto termo as propriedades carcinogénicas de um composto, utilizam-se frequentemente ensaios de genotoxicidade em linhas celulares de mamífero ou ensaios em modelos animais, em que se analisa uma variedade de lesões genéticas potencialmente relacionados com o processo de carcinogénese. No entanto, a investigação sobre as propriedades genotóxicas dos NMs não foi, até hoje, conclusiva. O presente estudo tem por objectivo principal caracterizar os efeitos genotóxicos associados à exposição a nanomateriais manufaturados, de forma a contribuir para a avaliação da sua segurança. Constituíram objectivos específicos deste estudo: i) avaliar a genotoxicidade dos NMs em três tipos de células humanas expostas in vitro: linfócitos humanos primários, linha celular de epitélio brônquico humano (BEAS-2B) e linha celular de adenocarcinoma epitelial de pulmão humano (A549); ii) avaliar a sua genotoxicidade num modelo de ratinho transgénico; iii) investigar alguns mecanismos de acção que poderão contribuir para a genotoxicidade dos nanomateriais, como a contribuição de lesões oxidativas para a genotoxicidade induzida pelos NMs in vitro, e a investigação da sua bioacumulação e localização celular in vivo. Foram analisados os efeitos genotóxicos associados à exposição a duas classes de NMs, dióxido de titânio e nanotubos de carbono de parede múltipla, bem como a um NM de óxido de zinco, candidato a ser utlilizado como controlo positivo de dimensão nanométrica. Os NMs utilizados foram previamente caracterizados com detalhe relativamente às suas características físico-químicas e também relativamente à sua dispersão em meio aquoso e no meio de cultura. A metodologia incluiu ensaios de citotoxicidade e de genotoxicidade in vitro, designadamente, ensaios de quebras no DNA (ensaio do cometa) e nos cromossomas (ensaio do micronúcleo) em células humanas expostas a várias concentrações de NMs, por comparação com células não expostas. Também foram realizados ensaios in vivo de quebras no DNA, quebras cromossómicas e ainda um ensaio de mutações em vários órgãos de grupos de ratinhos transgénicos LacZ, expostos por via intravenosa a duas doses de dióxido de titânio. Foi investigada a existência de uma relação dose-resposta após exposição das células humanas ou dos animais a NMs. A contribuição de lesões oxidativas para a genotoxicidade após exposição das células aos NMs in vitro foi explorada através do ensaio do cometa modificado com enzima. Realizaram-se estudos histológicos e citológicos para deteção e localização celular dos NMs nos órgãos-alvo dos ratinhos expostos in vivo. Os resultados demonstraram efeitos genotóxicos em alguns dos NMs analisados em células humanas. No entanto, os efeitos genotóxicos, quando positivos, foram em níveis reduzidos, ainda que superiores aos valores dos controlos, e a sua reprodutibilidade era dependente do sistema experimental utilizado. Para outros NMs, a evidência de genotoxicidade revelou-se equívoca, conduzindo à necessidade de esclarecimento através de ensaios in vivo. Para esse fim, recorreu-se a uma análise integrada de múltiplos parâmetros num modelo animal, o ratinho transgénico baseado em plasmídeo contendo o gene LacZ exposto a um NM de dióxido de titânio, NM-102. Embora tenha sido demonstrada a exposição e a acumulação do NM no fígado, não se observaram efeitos genotóxicos nem no fígado, nem no baço nem no sangue dos ratinhos expostos a esse NM. Neste estudo concluiu-se que algumas formas de dióxido de titânio e nanotubos de carbono de parede múltipla produzem efeitos genotóxicos em células humanas, contribuindo para o conjunto de evidências sobre o efeito genotóxico desses NMs. As diferenças observadas relativamente à genotoxicidade entre NMs do mesmo tipo, mas distintos em algumas das suas características físico-quimicas, aparentemente não são negligenciáveis, pelo que os resultados obtidos para um NM não devem ser generalizados ao grupo correspondente. Para além disso, a genotoxicidade equívoca verificada para o NM-102 em células humanas expostas in vitro, não foi confirmada no modelo in vivo, pelo que o valor preditivo da utilização dos ensaios in vitro para a identificação de NMs com efeitos genotóxicos (e portanto potencialmente carcinogénicos) ainda tem de ser esclarecido antes de ser possível extrapolar as conclusões para a saúde humana. Por sua vez, como a informação aqui produzida pelas metodologias in vitro e in vivo não reflete os efeitos de exposição continua ou prolongada, que poderá conduzir a efeitos genotóxicos distintos, esta deverá ser complementada com outras linhas de evidência relativamente à segurança dos NMs. Perante a incerteza dos níveis de exposição real do organismo humano e do ambiente, a segurança da utilização dos NMs não pode ser garantida a longo prazo e, tendo em conta a elevada produção e utilização destes NMs, são prementes futuros estudos de monitorização ambiental e humana.
- Nutritional Status and Metabolic Syndrome in patients with PhenylketonuriaPublication . Rocha, Júlio César; Borges, Nuno; Guimarães, João Tiago; van Spronsen, Francjan[PT] A Fenilcetonúria (PKU; MIM 261600) é a doença hereditária do metabolismo dos aminoácidos mais frequente. Após o diagnóstico, atualmente realizado através do rastreio neonatal, é implementada uma dieta restrita em proteína natural e em fenilalanina (fen), simultaneamente suplementada com uma mistura de aminoácidos isenta de fen e com alimentos especiais hipoproteicos. Este tratamento nutricional tem mostrado um tremendo sucesso na prevenção do atraso mental. Contudo, a nutrição na PKU é desafiante na medida em que podem surgir desequilíbrios importantes. Enquanto não há consenso relativo à ingestão ideal de proteína, o balanço em termos do status em micronutrientes revela frequentemente resultados extremos, sublinhando a necessidade de cuidadosamente monitorizar o estado nutricional nestes doentes. A definição precisa do estado nutricional proteico na PKU constitui um desejo antigo de todos os clínicos. Enquanto o melhor marcador bioquímico ainda não está definido, a prealbumina plasmática parece ser um parâmetro útil para decidir a ingestão proteica adequada nos doentes com PKU. Uma ingestão proteica de acordo com as recomendações para os doentes com PKU parece resultar no atingimento de patamares de crescimento e de composição corporal comparável a indivíduos controlo. A dieta tipicamente usada no tratamento dos doentes com PKU é restrita em proteína natural mas é normalmente rica em glícidos. Não obstante as características obesogénicas desta dieta, o tratamento dietético na PKU não aumenta o risco de excesso de peso, de obesidade central ou de incremento na massa gorda. Cumulativamente, os doentes com PKU revelam tendência para uma menor prevalência de síndrome metabólica, embora tenha sido constatada a presença de dislipidemia aterogénica. Embora estes doentes com obesidade central não manifestem um estado próinflamatório aumentado comparativamente aos controlos, são necessários mais estudos que ajudem a clarificar o seu risco cardiovascular a longo prazo, considerando a presença de dislipidemia aterogénica, mesmo apresentando concentrações plasmáticas reduzidas de colesterol total e de glicose.
- Post-transcriptional regulation of HFE gene expressionPublication . Martins, Rute; Faustino, Paula; Romão, LuísaIron is a key element for numerous metabolic processes in living cells. The challenge for most organisms is to acquire the adequate amounts of iron for these processes yet avoiding the toxicity associated with free iron. In fact, disruptions of iron homeostasis account for some of the most common human diseases. Amongst these, lays hereditary hemochromatosis, which is mainly associated with mutations in the HFE gene, leading to iron overload in specific organs. HFE protein acts in iron homeostasis by regulating the expression of hepcidin in the liver. Besides the major HFE transcript, which is expressed at low levels in a wide range of human tissues, several additional alternative HFE transcripts have been described. The work presented in this dissertation addresses the characterization of HFE alternative transcripts, the biological mechanisms involved in their genesis as well as their physiological significance and regulation. A variety of human tissues was analysed and shown to express several alternatively spliced HFE transcripts. Functional analysis of the corresponding proteins revealed that alternative splicing can either generate non-functional HFE protein variants or produce a soluble HFE variant that is secreted by cells associated with beta2-microglobulin. This soluble HFE may have a vital role in iron homeostasis by acting as an agonist or antagonist of the full length HFE. Furthermore, HFE transcripts were found to be physiologically regulated by the nonsense-mediated mRNA decay (NMD), since its levels are significantly increased when depleting human cells from a key NMD effector. Through the analysis of several human tissues, it is shown that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites. This tissue-specific polyadenylation pattern seems to respond to cellular iron status, acting coordinately with NMD to fine-tune HFE’s expression levels. The regulation of HFE gene expression is here shown to be post-transcriptionally influenced by alternative splicing, alternative polyadenylation and nonsense-mediated mRNA decay mechanisms. These findings may hint future directions in the active field of iron biology research and provide interesting cues that could be translated into new therapeutics for iron homeostasis disorders through the HFE-mediated regulation of hepcidin.
- 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 glucose uptake in mammalian cells by protein phosphorylation networksPublication . Henrique, Andreia; Jordan, Peter; Clarke, LukaGlucose uptake is a key mechanism to maintain cell, tissue and body homeostasis. Among others, glucose transporter proteins (GLUTs) are responsible for glucose transport into the cell. Besides their expression level, the GLUT number present at the plasma membrane (PM) is regulated by signaling mechanisms. Previously, protein kinase WNK1 was found to phosphorylate TBC1D4, a Rab-GAP involved in membrane traffic regulation, and to regulate the surface expression of the constitutive glucose transporter GLUT1. In this work the phosphorylation of either TBC1D4 or its paralogue TBC1D1 was studied as a key step in regulatory cascades modulating glucose uptake. First, we showed that downregulation of WNK1 through RNA interference translates in a 2-fold decrease in PM GLUT1 expression and a 60% decrease in glucose uptake. Then, we compared by mass spectrometry (MS) the in vitro phosphorylation of TBC1D1 and 4 by AKT1, WNK1 and SGK1 and 3. We identified two novel WNK1-specific phosphorylation sites at TBC1D1-Ser565 and TBC1D4-Ser704 and showed that transfection of their phosphomimetic or unphosphorylatable mutants affected cell surface abundance of GLUT1. To define new biological pathways regulated by WNK1, we determined the interactome of WNK1 by MS. Interestingly, the bioinformatic and gene ontology analysis pointed to a previously unrecognized function related to mRNA processing. Our studies identified a novel function of WNK1 in alternative splicing using RAC1B in colorectal HT29 cells as a model. In particular, WNK1 acts as a scaffolding protein through complex formation with GSK3β. This complex protects GSK3β from an inhibitory phosphorylation at Ser9. The active GSK3β allows the translocation of kinase SRPK1 and splicing factor SRSF1 to the nucleus, which is important for RAC1B generation. Considering that RAC1B is known to be essential for cell survival and malignant progression, the results establish a new link between WNK kinases and tumorigenesis. Altogether, this work reinforced a role for WNK1 in cell metabolism and uncovered a new function in regulation of alternative splicing, two events that can contribute to tumor development. The data may provide new targets for pharmacological modulation of RAC1B expression and cellular metabolism, with potential impact for the treatment of cancer and type 2 diabetes.
- Search for new modulators of Phe508del-CFTR retention at the plasma membranePublication . Matos, Ana Margarida; Matos, Paulo; Pepperkok, RainerCystic fibrosis (CF) is a complex inherited disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Around 2000 disease causing mutations are known for this gene, which encodes a Chloride (Cl−) channel expressed at the plasma membrane (PM) of epithelial cells. The most frequent CFTR mutation, the deletion of phenylalanine 508 (Phe508del), causes the protein to misfold and be prematurely degraded. Low temperature or pharmacological “correctors” can partly rescue Phe508del-CFTR processing defect and enhance the channel traffic to the cell surface. Nevertheless, the rescued channels show partial channel function and a highly decreased PM half-life, due to accelerated endocytosis and fast turnover. Given this accelerated endocytic rate, new strategies aiming to retain rescued Phe508del-CFTR at the cell surface could be relevant as to enhance the efficacy of currently available pharmacological correctors. For that reason, the major objective of this dissertation is to identify novel cellular pathways or key interactors for the modulation of CFTR surface retention. Previous results from the host laboratory had showed that stimulation of endogenous RAC1 by Hepatocyte Growth Factor (HGF) signaling potentiated the retention of rescued Phe508del-CFTR at the PM by promoting an interaction between the actin-binding adaptor ezrin (EZR) and the Na+/H+ exchange regulatory factor-1 (NHERF1), enhancing CFTR anchoring to the actin cytoskeleton. In chapter 2 we showed that the mechanism behind this stabilization lies on a conformational change in NHERF1, triggered by EZR activation upon RAC1 signaling, which is then able to bind and stabilize misfolded CFTR at the PM. However, HGF/RAC1 signaling pathway is known to have proliferative and pleiotropic biological functions, which limit its application for therapeutic intervention. Therefore, in chapter 3, we investigated the effect of HGF treatment in epithelium-like cellular models, in combination with the most common administrated drugs. Contrary to what would be commonly assumed, we found that prolonged co-administration of HGF actually prevented previously unrecognized epithelial dedifferentiation effects of prolonged exposure to the FDA-approved Phe508del-CFTR corrector VX-809. It also significantly increased the Phe508del-CFTR functional rescue by the FDA- and EMA-approved VX-809/VX-770 drug combination, preventing the destabilization of the PM rescued channels by prolonged exposure to the VX-770 potentiator drug. These results suggest that HGF co-administration could indeed be beneficial for CF patients and should be further clinically explored. Lastly, since we showed that the type of protein interactions that wt- and rescued Phe508del-CFTR establish at the cell surface can be major determinants of their different PM stabilities, in chapter 4 we identified, for the first time, the core components of the macromolecular complexes assembled around wt- and rescued Phe508del-CFTR proteins at the PM. By identifying exclusive PM interactions between rescued Phe508del-CFTR, NHERF1 and EZR, we were able to recognize Calpain 1 as a key contributor for the decreased surface stability of pharmacologically rescued Phe508del-CFTR, probably acting through the disruption of the EZR-actin cytoskeleton binding.
- Signal transduction pathways involving the hypertension-related WNK1 and WNK4 protein kinasesPublication . Mendes, Ana IsabelThe genes WNK1 and WNK4 belong to the subfamily of WNK protein kinases and their mutation causes pseudohypoaldosteronism type II, a rare familial form of hypertension with hyperkalemia and hypercalciuria. The molecular mechanisms underlying this condition involve the regulation of renal electrolyte homeostasis and the modulation of diverse ion channels and transporters via WNK kinases. Additionally, WNKs have also been reported to participate in signal transduction pathways related to cell survival and proliferation. The objective of the present thesis was to identify novel WNK1 and WNK4 interacting proteins and the underlying signal transduction pathways. First, it was found that WNK1 forms a protein complex with the Rab-GAP TBC1D4 and phosphorylates it in vitro. It was shown that the expression levels of WNK1 regulate surface expression of the constitutive glucose transporter GLUT1 in HEK293 cells. WNK1 is shown to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. Moreover, these effects were kinase activity-dependent. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. Second, WNK4 was found to promote the cell surface expression of the CFTR chloride channel in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with the tyrosine kinase Syk, which we found to phosphorylate tyrosine 512 (Tyr512) in the first nucleotide-binding domain of CFTR. The presence of WNK4 prevents this in vitro phosphorylation in a kinase-independent manner. In BHK21 cells stably expressing CFTR, Syk reduces, while WNK4 promotes, the cell surface expression of CFTR. Mutation of Tyr512 revealed that its phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk play an antagonistic role in this process. Finally, ten WNK4 variants were detected in a cohort of Portuguese patients and control individuals, which subsequently were tested for association to hypertension and/or osteoporosis. Despite none of the variants yield any significant association to hypertension, a rare missense alteration (rs56116165) in a highly conserved arginine residue showed a nominal association to osteoporosis. This finding advocates that this polymorphism is a rare allelic variant, in a candidate gene with a biological function in renal calcium homeostasis, that may contribute to a genetic predisposition to osteoporosis.