Intermolecular interactions in the TMEM16A dimer controlling channel activity

TMEM16A and TMEM16B are plasma membrane proteins with Ca2+ -dependent Cl- channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the "activating domain" to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl- transport

Ex vivo model predicted in vivo efficacy of CFTR modulator therapy in a child with rare genotype

Background: New drugs that target the basic defect in cystic fibrosis (CF) patients may now be used in a large number of patients carrying responsive mutations. Nevertheless, further research is needed to extend the benefit of these treatments to patients with rare mutations that are still uncharacterized in vitro and that are not included in clinical trials. For this purpose, ex vivo models are necessary to preliminary assessing the effect of CFTR modulators in these cases. Method: We report the clinical effectiveness of lumacaftor/ivacaftor therapy prescribed to a CF child with a rare genetic profile (p.Phe508del/p.Gly970Asp) after testing the drug on nasal epithelial cells. We observed a significant drop of the sweat chloride value, as of the lung clearance index. A longer follow-up period is needed to define the effects of therapy on pancreatic status, although an increase of the fecal elastase values was found. Conclusion: Drug response obtained on nasal epithelial cells correlates with changes in vivo therapeutic endpoints and can be a predictor of clinical efficacy of novel drugs especially in pediatric patients

Evaluation of Fused Pyrrolothiazole Systems as Correctors of Mutant CFTR Protein

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named "correctors". So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors

Novel tricyclic pyrrolo-quinolines as pharmacological correctors of the mutant CFTR chloride channel

F508del, the most frequent mutation in cystic fibrosis (CF), impairs the stability and folding of the CFTR chloride channel, thus resulting in intracellular retention and CFTR degradation. The F508del defect can be targeted with pharmacological correctors, such as VX-809 and VX-445, that stabilize CFTR and improve its trafficking to plasma membrane. Using a functional test to evaluate a panel of chemical compounds, we have identified tricyclic pyrrolo-quinolines as novel F508del correctors with high efficacy on primary airway epithelial cells from CF patients. The most effective compound, PP028, showed synergy when combined with VX-809 and VX-661 but not with VX-445. By testing the ability of correctors to stabilize CFTR fragments of different length, we found that VX-809 is effective on the amino-terminal portion of the protein that includes the first membrane-spanning domain (amino acids 1-387). Instead, PP028 and VX-445 only show a stabilizing effect when the second membrane-spanning domain is included (amino acids 1-1181). Our results indicate that tricyclic pyrrolo-quinolines are a novel class of CFTR correctors that, similarly to VX-445, interact with CFTR at a site different from that of VX-809. Tricyclic pirrolo-quinolines may represent novel CFTR correctors suitable for combinatorial pharmacological treatments to treat the basic defect in CF

Normal calcium-activated anion secretion in a mouse selectively lacking TMEM16A in intestinal epithelium

Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr-null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

ATP release during cell swelling activates a Ca2+-dependent Cl - Current by autocrine mechanism in mouse hippocampal microglia

Microglia cells, resident immune cells of the brain, survey brain parenchyma by dynamically extending and retracting their processes. Cl- channels, activated in the cellular response to stretch/swelling, take part in several functions deeply connected with microglia physiology, including cell shape changes, proliferation, differentiation and migration. However, the molecular identity and functional properties of these Cl- channels are largely unknown. We investigated the properties of swelling-activated currents in microglial from acute hippocampal slices of Cx3cr1+/GFP mice by whole-cell patch-clamp and imaging techniques. The exposure of cells to a mild hypotonic medium, caused an outward rectifying current, developing in 5-10 minutes and reverting upon stimulus washout. This current, required for microglia ability to extend processes towards a damage signal, was carried mainly by Cl- ions and dependent on intracellular Ca2+. Moreover, it involved swelling-induced ATP release. We identified a purine-dependent mechanism, likely constituting an amplification pathway of current activation: under hypotonic conditions, ATP release triggered the Ca2+-dependent activation of anionic channels by autocrine purine receptors stimulation. Our study on native microglia describes for the first time the functional properties of stretch/swelling-activated currents, representing a key element in microglia ability to monitor the brain parenchyma

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins

Perspectives on utilization of edible coatings and nano-laminate coatings for extension of postharvest storage of fruits and vegetables

It is known that in developing countries, a large quantity of fruit and vegetable losses results at postharvest and processing stages due to poor or scarce storage technology and mishandling during harvest. The use of new and innovative technologies for reducing postharvest losses is a requirement that has not been fully covered. The use of edible coatings (mainly based on biopolymers) as a postharvest technique for agricultural commodities has offered biodegradable alternatives in order to solve problems (e.g., microbiological growth) during produce storage. However, biopolymer-based coatings can present some disadvantages such as: poor mechanical properties (e.g., lipids) or poor water vapor barrier properties (e.g., polysaccharides), thus requiring the development of new alternatives to solve these drawbacks. Recently, nanotechnology has emerged as a promising tool in the food processing industry, providing new insights about postharvest technologies on produce storage. Nanotechnological approaches can contribute through the design of functional packing materials with lower amounts of bioactive ingredients, better gas and mechanical properties and with reduced impact on the sensorial qualities of the fruits and vegetables. This work reviews some of the main factors involved in postharvest losses and new technologies for extension of postharvest storage of fruits and vegetables, focused on perspective uses of edible coatings and nano-laminate coatings.María L. Flores-López thanks Mexican Science and Technology Council (CONACYT, Mexico) for PhD fellowship support (CONACYT Grant Number: 215499/310847). Miguel A. Cerqueira (SFRH/BPD/72753/2010) is recipient of a fellowship from the Fundação para a Ciência e Tecnologia (FCT, POPH-QREN and FSE Portugal). The authors also thank the FCT Strategic Project of UID/ BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project ‘‘BioInd Biotechnology and Bioengineering for improved Industrial and AgroFood processes,’’ REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP, CE Brazil (CI10080-00055.01.00/13)

The Peter Pan paradigm

Genetic and environmental agents that disrupt organogenesis are numerous and well described. Less well established, however, is the role of delay in the developmental processes that yield functionally immature tissues at birth. Evidence is mounting that organs do not continue to develop postnatally in the context of these organogenesis insults, condemning the patient to utilize under-developed tissues for adult processes. These poorly differentiated organs may appear histologically normal at birth but with age may deteriorate revealing progressive or adult-onset pathology. The genetic and molecular underpinning of the proposed paradigm reveals the need for a comprehensive systems biology approach to evaluate the role of maternal-fetal environment on organogenesis