Browsing by Issue Date, starting with "2019-10-18"
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- A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membranePublication . Loureiro, ClaudiaMutations in the CFTR gene cause the recessive genetic disease Cystic Fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the NBD1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial-epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine binding (PTB) domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how epithelial chloride secretion is regulated.
- Establishing priorities for Food Composition Table update – Nutrients and foodPublication . Lopes, Andreia; Oliveira, Luísa; Fernandes, Paulo; Dias, Maria da GraçaAim: To define the priority foods/analites to be analysed/determined in order to contribute to high quality data for FCT update with new foods and nutrients.
- Natural Language Processing applied to Food Data: a smart food description mapping systemPublication . Tomé, SidneyIntroduction: For the past years we have been working with EFSA on report of food data for the domains of chemical contaminants and food additives; Over the years, INSA has been the central point for gathering data from multiple national entities for processing and redirecting these information to EFSA; A National Data Management Systems was built from the start in order to facilitate such tasK; A consortium between 3 partners (INSA, ASAE & HAPIH) was created and they share the common interest of further developing their own official control National Data Management Systems (NDMS); INSA has developed PT.ON.DATA NDMS based on SSD and SSD2 data models in cooperation with ASAE and other national competent authorities. HAPIH has developed a NDMS with a similar approach; Both ASAE and HAPIH are interested in implementing real-time sample data collection based on preparatory digital forms already existing in PT.ON.DATA. The three partners are committed to investigate and implement an automatic approach to FoodEx2 classification of food samples using the knowledge and databases existing in HAPIH and INSA. Consortium aim: The consortium envisages improving the quality of raw occurrence data for risk assessment by reducing error, incrementing completeness and timeliness both in data fields and food classification, and simultaneously reducing human time and work and therefore releasing time of scientists for data analysis and for performing risk assessment; Together, the improvements will be reflected on the strengthening of food safety risk assessment capacity of the countries involved and contributing to a better assess on risks associated with the food chain by EFSA. Project IDRisk (Improving Data quality for RISK assessment) - This project emerged from two distinct problems regarding data report: Technicians/Data experts, whether they are working on-field sampling data or cleaning and preparing data for report, an absurd amount of manual work is done: too much time is spent; The more manual work we have, the more prone to errors the data will become. Main objectives: Improve/Restructure the dynamic sampling forms module; Develop the application that will run on the mobile devices; Plan and implement an automatic NDMS FoodEx2 classification system for sampling descriptions.
- Occurrence of acesulfame K, saccharin and aspartame in table-top intense sweetenersPublication . Veiga, Cláudia; Serra, Celeste; Vasco, ElsaAim: Determination of acesulfame K, saccharin and aspartame contents in table-top intense sweeteners samples.
- Content of Vitamin A and E in representative samples of the Portuguese consumptionPublication . Ravasco, Francisco; Vasco, Elsa; Dias, Maria da GraçaVitamin A (all-trans-retinol and 13-cis-retinol) is naturally present in animal products. Vitamin E (dl-α-tocopherol) occurs naturally in foods of plant and animal origin. Vitamin A plays an important role in the mechanisms associated with vision and vitamin E as an antioxidant. To evaluate the ingestion of vitamins A and E by the Portuguese population, through a Total Diet Study (TDS), using the daily reference doses, 800 μg and 12 mg, respectively for vitamins A and E, for adult subjects, analytical determinations in representative samples were done. Objectives: To determine the vitamins A and E contents of 228 composite samples representative of the consumption of the Portuguese population.
- Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3Publication . Loureiro, Claudia; barros, Patricia; Matos, Paulo; Jordan, PeterIntroduction: Cellular chloride transport has a fundamental role in cell volume regulation and membrane potential, both in normal and tumour cells (1,2). Cellular chloride 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. Experimental: Co-transporter constructs were transfected into HEK293 cells and the activity of SYK kinase modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Co-transporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (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. Conclusions: 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 have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.
- Protein kinase WNK1 contributes to the regulation of GLUT1 expression in the plasma membranePublication . Henriques, Andreia; Matthiesen, Rune; Matos, Paulo; Jordan, PeterIntroduction: One mechanism by which tumour cells regulate the uptake of glucose is overexpression of glucose transporter proteins (GLUT). Besides their expression level, the number of GLUT present at the plasma membrane is regulated by signaling mechanisms (1). Previously we found that protein kinase WNK1 phosphorylates TBC1D4 (2), a GTPase activating protein for RAB-family proteins involved in membrane traffic regulation and regulates the surface expression of the constitutive glucose transporter GLUT1. Phosphorylation of either the TBC1D4 or its paralogue TBC1D1 is a key regulatory step in the kinase cascades leading to changes in glucose uptake (1). Experimental: Putative WNK1 phosphorylation sites in TBC1D1 and 4 were determined by MS following in vitro kinase assays with recombinant proteins. RNA interference, transfection of phosphorylation site mutants, and cell surface protein biotinylation assays were used to analyze the impact of the identified phosphorylation events on GLUT1 plasma membrane abundance. Results: We compared phosphorylation by AKT1, WNK1 and SGK1 and identified two novel WNK1-specific phosphorylation sites at TBC1D1-Ser565 and TBC1D4-Ser704. Transfection of the corresponding phosphomimetic or unphosphorylatable mutants revealed that phosphorylation of either RabGAP by WNK1 at these novel sites participates in the delivery of GLUT1 to the plasma membrane (PM). Consistently, downregulation of WNK1 by RNA interference decreased GLUT1 PM abundance by over 2-fold, which translates to a 60% decrease in Glucose uptake by these cells. Conclusions: Together, our data contribute to a better understanding of the pathways regulating glucose uptake via GLUT1, the upregulation of which is related to cancer progression.
- Portuguese Food Composition Table: from origin to presentPublication . Oliveira, LuísaAbout development history of the Portuguese Food Composition Table.
- Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stabilityPublication . Matos, Ana Margarida; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, PauloCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
- Estudo Português de Hipercolesterolemia FamiliarPublication . Medeiros, Ana MargaridaA hipercolesterolemia familiar é uma das dislipidemias genéticas mais comuns, caracterizada por valores elevados de LDL, xantomas tendinosos e desenvolvimento precoce de aterosclerose e doença coronária prematura. É uma patologia autossómica, monogénica, dominante, cujo diagnóstico precoce é essencial para aumentar a esperança média de vida dos doentes, bem como para identificação dos seus familiares em risco.