Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.Work supported by the Portuguese Fundação para a Ciência e Tecnologia (fellowship grant SFRH/BSAB/105741/2014—to CF, grant PTDC/SAU-ORG/119782/2010—to PJ, and centre grant UID/MULTI/04046/2013—to BioISI); Gilead Génese PGG/039/2014 and ERS Romain Pauwels 2012 (to CF); CFF SWIATE14P0 (to AS); NIH NIDDK P30 072506 Basic and Translational Studies of Cystic Fibrosis (P&F to AS)

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR

Publisher Copyright: © 2015 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.publishersversionpublishe

Cell type-specific regulation of CFTR trafficking—on the verge of progress

Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a complex process that starts with its biosynthesis and folding in the endoplasmic reticulum. Exit from the endoplasmic reticulum (ER) is coupled with the acquisition of a compact structure that can be processed and traffic through the secretory pathway. Once reaching its final destination—the plasma membrane, CFTR stability is regulated through interaction with multiple protein partners that are involved in its post-translation modification, connecting the channel to several signaling pathways. The complexity of the process is further boosted when analyzed in the context of the airway epithelium. Recent advances have characterized in detail the different cell types that compose the surface epithelium and shifted the paradigm on which cells express CFTR and on their individual and combined contribution to the total expression (and function) of this chloride/bicarbonate channel. Here we review CFTR trafficking and its relationship with the knowledge on the different cell types of the airway epithelia. We explore the crosstalk between these two areas and discuss what is still to be clarified and how this can be used to develop more targeted therapies for CF

Changes in metabolic and inflammatory markers after a combined exercise program in workers : a randomized controlled trial

Purpose: We investigated the effects of a 16-week combined exercise training on body composition, metabolic and inflammatory markers in sedentary middle-aged workers. We also assessed whether significant alterations in metabolic markers were associated with changes in health-related outcomes. Methods: This randomized controlled trial involved 46 participants randomly allocated into control and exercise groups. The exercise group performed 16-week combined aerobic and resistance training for 75 min/session, 3 times/week. Fasting blood samples were collected at baseline and after 16-week intervention to determine lipid profile, metabolic and inflammatory markers as primary outcomes. Results: A total of 36 participants completed the intervention (53.70 ± 6.92 years old) (n = 18 in each group). Waist circumference (interaction effect: F = 7.423, p = 0.002), fat mass (interaction effect: F = 5.070, p = 0.011), and muscle mass (interaction effect: F = 5.420, p = 0.007) were improved in the exercise group compared to the control group. Fasting glucose increased after the 16-week follow-up (time effect: F = 73.253, p < 0.001), without an intergroup difference. Insulin levels were greater in the control compared to exercise group (group effect: F = 6.509, p = 0.015). The control group tended to increase the HOMA-IR index (interaction effect: F = 3.493, p = 0.070) and to decrease the QUICKI index (interaction effect: F = 3.364, p = 0.075) to a greater extent compared to the exercise group. Exercise group reduced leptin (interaction effect: F = 11.175, p = 0.002) and adiponectin (interaction effect: F = 4.437, p = 0.043) concentrations in a greater magnitude than control group. IL-6 (time effect: F = 17.767, p < 0.001) and TNF-α (time effect: F = 9.781, p = 0.004) concentrations decreased after the intervention, without an intergroup difference. IL-17A levels increased in the control compared to exercise group (interaction effect: F = 5.010, p = 0.033). Effects on adiponectin, IL-6 and IL-17A levels seem to depend on baseline BMI, age, and sex. Percentage changes in leptin correlated positively with changes in HOMA-IR index in the exercise (r = 0.565, p = 0.015) and control (r = 0.670, p = 0.002) groups. Conclusions: A combined training program can be an effective strategy to improve body composition and inflammatory markers and prevent marked reductions in insulin sensitivity among middle-aged workers.info:eu-repo/semantics/acceptedVersio

Impact of different aquatic exercise programs on body composition, functional fitness and cognitive function of non-institutionalized elderly adults: a randomized controlled trial

Aquatic physical exercise programs have become progressively more popular among elderly people. Some of the major physical exercise program disadvantages on land are minimized due to the specific properties of the aquatic environment. The purpose of the present randomized controlled study is to verify the effects of different aquatic physical exercise programs on body composition, functional fitness and cognitive function in non-institutionalized elderly people. For this study, 102 elderly individuals were randomly allocated into four different groups: AerG (n = 25, 71.44 ± 4.84 years); IntG (n = 28, 72.64 ± 5.22 years); ComG (n = 29, 71.90 ± 5.67 years) and CG (n = 20, 73.60 ± 5.25 years). Individuals from the groups AerG, IntG and ComG participated in three different aquatic physical exercise programs for a period of 28 weeks. The CG participants kept to their usual routines. All participants were evaluated for body composition, functional fitness and cognitive function at two time moments, i.e., pre- (M1) and post-intervention (M2). Significant differences for body composition were found between M1 and M2 for FM (p < 0.001), LBM (p < 0.001) and WCir (p < 0.01) in the AerG, for BMI (p < 0.05), FM (p < 0.05), LBM (p < 0.001) and LCir-R (p < 0.05) in the IntG, and for WGT (p < 0.01), FM (p < 0.05), LBM (p < 0.01), LCir-R (p < 0.05) and LCir-L (p < 0.01) in the ComG groups. For functional fitness, differences were found between M1 and M2 for 2m-ST (p < 0.000), 30s-CS (p < 0.000), 30s-AC (p < 0.05), HG-T-R (p < 0.000) and HG-T-L (p < 0.000) in the AerG, for 2m-ST (p < 0.05), BS-R (p < 0.05), 30s-CS (p < 0.000), 30s-AC(p < 0.01), HG-T-R (p < 0.000) and HG-T-L (p < 0.000) in the IntG, and for 30s-CS (p < 0.000), HG-T-R (p < 0.000) and HG-T-L (p < 0.000) in the ComG groups. The present study evidenced the beneficial effects of physical exercise in an aquatic environment on body composition, functional fitness and cognitive function in non-institutionalized elderly adults. The ComG water-based exercise program showed more beneficial effects in the improvement of body composition and cognitive function variables, while the IntG and AerG programs were more effective in the improvement of functional fitness.info:eu-repo/semantics/publishedVersio

Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31231217/In 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.This work was supported by FCT, Portugal, through center grant UID/MULTI/04046/2019 to BioISI and the BioSys PhD program PD65-2012 (fellowships SFRH/BD/52488/2014, SFRH/ BD/106084/2015, and SFRH/BD/52490/2014 to CL, JS, and AM, respectively).info:eu-repo/semantics/publishedVersio

Combined Exercise Training, Body Composition, Glucose and Insulin Metabolism in Sedentary Workers

Sedentary behaviour (SB) and physical inactivity are two major risk factors for obesity, insulin resistance, type 2 diabetes, and cardiovascular disease development (Davies et al., 2018). Effective stress-reducing interventions, such as regular exercise, are essential to prevent the harmful impact of SB on metabolic outcomes (Pedersen, 2017). We aimed to analyse the effects of a combined training program on body composition, blood pressure (BP), and glucose and insulin metabolism markers in non-diabetic sedentary workers and to assess the associations between the changes promoted by the exercise intervention and the different variables studied. This is a single-blinded two-arm randomised controlled trial. Thirty-six participants (53.7±6.9 years old; BMI: 27.2±3.9 kg/m2) underwent 16 weeks of combined exercise training [CET (n=18), resistance (6-15 reps at 45-90% 1RM) and aerobic exercise (60-95% HRmax)], 3 times/week, 75 min/session, or a control group (n=18). Body composition, blood pressure (BP), and fasting blood samples were taken pre- and post-intervention. Body composition was determined by bioimpedance (Inbody 270, USA). Blood pressure was measured using an automated oscillometric cuff (NBP-24 NG, USA). Fasting glucose and insulin were analysed using standard enzymatic assays (Atellica CH Analyzer, USA) and ELISA (Crystal Chem, USA), respectively. Plasma HbA1c level was determined by the Ion-exchange HPLC method (Tosoh Bioscience, California). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as (glucose x insulin/22.5). The CET group significantly decreased BMI, waist circumference, fat mass, visceral adipose tissue, and increased muscle mass after the exercise intervention (p0.05). Bivariate correlations showed a significant positive correlation between the change (D) in systolic BP and D HOMA-IR index (r=0.475, p=0.046). Our results suggest that a combined training program could be an effective strategy to improve body composition and maintain normal glucose and insulin metabolism, preventing the development of metabolic disorders as well as the vascular dysfunction associated with the insulin resistance phenotype

Reversible imine crosslinking in waterborne self-healing polymer coatings

Waterborne polymer coatings have the potential to address the environmental concerns associated with solvent based systems. To improve their performance without using volatile organic compounds, we propose a new approach based on reconfigurable covalent crosslinking that provides mechanical resistance and self-healing properties. The new waterborne polymer coatings are based on mixtures of aldehyde- and amine-functionalized polymer nanoparticles (PNPs) that take advantage of the reversibility of imine bonds in the presence of water. Different degrees of functional monomer incorporation (10 % to 40 %) allowed us to balance crosslinking and interdiffusion during film formation, to obtain mechanically robust and solvent resistant films. A clear structure-properties relation was assessed by following the formation of water resulting from amine-aldehyde condensation crosslinking, measured by differential scanning calorimetry. The resulting polymer coatings further show self-healing properties at room temperature, triggered with residual amounts of water and featuring high recovery rates of the mechanical properties. Our mechanically robust waterborne polymer coatings based in imine reversible crosslinking, featuring self-healing in mild conditions, offer excellent prospects for application in smart coating materials.publishe

Inequality and crisis: conspicuous consumption as the missing link in the portuguese case

Portuguese household debt increased above GDP between 2000 and 2007. This article uses conspicuous consumption to explain credit demand dynamics. The author develops an institutionalist framework and consider how rapid high inequalities and increasing top income share favored conspicuous consumption and climbing household debt.info:eu-repo/semantics/publishedVersio

Multifunctional platform based on electroactive polymers and silica nanoparticles for tissue engineering applications

Poly(vinylidene fluoride) nanocomposites processed with different morphologies, such as porous and non-porous films and fibres, have been prepared with silica nanoparticles (SiNPs) of varying diameter (17, 100, 160 and 300 nm), which in turn have encapsulated perylenediimide (PDI), a fluorescent molecule. The structural, morphological, optical, thermal, and mechanical properties of the nanocomposites, with SiNP filler concentration up to 16 wt %, were evaluated. Furthermore, cytotoxicity and cell proliferation studies were performed. All SiNPs are negatively charged independently of the pH and more stable from pH 5 upwards. The introduction of SiNPs within the polymer matrix increases the contact angle independently of the nanoparticle diameter. Moreover, the smallest ones (17 nm) also improve the PVDF Youngs modulus. The filler diameter, physico-chemical, thermal and mechanical properties of the polymer matrix were not significantly affected. Finally, the SiNPs inclusion does not induce cytotoxicity in murine myoblasts (C2C12) after 72 h of contact and proliferation studies reveal that the prepared composites represent a suitable platform for tissue engineering applications, as they allow us to combine the biocompatibility and piezoelectricity of the polymer with the possible functionalization and drug encapsulation and release of the SiNP.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UID/FIS/04650/2013 and UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and project POCI-01-0145-FEDER-028237 funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI); and also under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. The authors also thank the FCT for the SFRH/BD/111478/2015 (S.R.), SFRH/BPD/96707/2013 (T.R.), SFRH/BPD/90870/2012 (C.R.) and SFRH/BPD/121526/2016 (D.C) grants. The authors acknowledge funding from the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) and from the Basque Government Industry and Education Departments under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively.info:eu-repo/semantics/publishedVersio