Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Cystic 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.This work was supported by a center grant UID/MULTI/04046/2019 to BioISI and project PTDC/BIA-CEL/28408/2017 and IF2012 to PM, both from FCT, Portugal. AMM was recipient of fellowship SFRH/BD/52490/2014 from BioSYS PhD programme PD65-2012, and PB of fellowship SFRH/BPD/94322/2013.N/

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

Laughlin states change under large geometry deformations and imaginary time Hamiltonian dynamics

We study the change of the Laughlin states under large deformations of the geometry of the sphere and the plane, associated with Mabuchi geodesics on the space of metrics with Hamiltonian $S^1$-symmetry. For geodesics associated with the square of the symmetry generator, as the geodesic time goes to infinity, the geometry of the sphere becomes that of a thin cigar collapsing to a line and the Laughlin states become concentrated on a discrete set of $S^1$--orbits, corresponding to Bohr-Sommerfeld orbits of geometric quantization. The lifting of the Mabuchi geodesics to the bundle of quantum states, to which the Laughlin states belong, is achieved via generalized coherent state transforms, which correspond to the KZ parallel transport of Chern-Simons theory

Uso de CLAE no controle de qualidade em produtos comerciais de Nim: reprodutibilidade da ação inseticida

The Neem tree, Azadirachta indica, provides many useful compounds that are used as pesticides. However, the efficiency in field of products like neem oil can be committed because they have not been observed reproductive content of secondary metabolic like azadirachtin. Based on reverse-phase high-performance liquid chromatography (HPLC) a new method was developed to permit the rapid quantitative analysis of azadirachtin from seeds, extracts and oil of Neem. In the present study it was evaluated the azadirachtin quantitative variation among various Neem's extracts and seeds showing the importance of quality control for reproduction of the insecticide efficiency, using S. frugiperda as target insect

Association between dietary patterns and renal function in a cross-sectional study using baseline data from the ELSA-Brasil cohort

Previous analyses of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) identified four main dietary patterns (DP). The aim of this study was to explore the association between the previously defined DP and renal function (RF). A crosssectional study using the ELSA-Brasil baseline data was carried out. DP (‘‘traditional’’, ‘‘fruits and vegetables’’, ‘‘bakery’’, and ‘‘low sugar/low fat), metabolic syndrome (MS) using the Joint Interim Statement criteria, microalbuminuria (MA), and glomerular filtration rate (eGFR) through the CKD-EPI equation were evaluated. Abnormal RF was defined as eGFRo60 mL min–1 (1.73 m2 ) –1 and MAX3.0 mg/dL. Factors associated with RF were determined and mediation analysis was performed to investigate the association between DP, MS, and RF. A total of 15,105 participants were recruited, with a mean age of 52±9 years; 8,134 participants (54%) were females. The mediation analysis identified indirect associations between ‘‘bakery’’ and ‘‘fruits and vegetables’’, and both were associated with decreased eGFR and albuminuria in both genders, compared with ‘‘traditional’’ and ‘‘low sugar/low fat’’ patterns in the general population. There was a direct association of the ‘‘bakery’’ pattern with MA in men (OR: 1.17, 95%CI: 1.92–1.48). The ‘‘fruits and vegetables’’ pattern also showed a direct association with reduced eGFR in women (OR: 1.65, 95%CI: 1.28–2.12), although there was no significance after adjustment. The ‘‘fruits and vegetables’’ and ‘‘bakery’’ DPs were associated with renal dysfunction. The only independent, direct association was between ‘‘bakery’’ DP and MA in men, raising concerns about DP and renal damage in men

The BLue Amazon Brain (BLAB): A Modular Architecture of Services about the Brazilian Maritime Territory

We describe the first steps in the development of an artificial agent focused on the Brazilian maritime territory, a large region within the South Atlantic also known as the Blue Amazon. The "BLue Amazon Brain" (BLAB) integrates a number of services aimed at disseminating information about this region and its importance, functioning as a tool for environmental awareness. The main service provided by BLAB is a conversational facility that deals with complex questions about the Blue Amazon, called BLAB-Chat; its central component is a controller that manages several task-oriented natural language processing modules (e.g., question answering and summarizer systems). These modules have access to an internal data lake as well as to third-party databases. A news reporter (BLAB-Reporter) and a purposely-developed wiki (BLAB-Wiki) are also part of the BLAB service architecture. In this paper, we describe our current version of BLAB's architecture (interface, backend, web services, NLP modules, and resources) and comment on the challenges we have faced so far, such as the lack of training data and the scattered state of domain information. Solving these issues presents a considerable challenge in the development of artificial intelligence for technical domains

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

Acknowledgements This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01- 0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/136814/2018 to S.M.G., SFRH/BD/141127/2018 to C.D.O., PD/BD/137680/2018 to D.A., IF/00474/2014 to N.S.O., IF/01390/2014 to E.T., IF/00959/2014 to S.C., IF/00021/2014 to R.S., PTDC/SAU-SER/29635/2017 and CEECIND/04601/2017 to C.C., and CEECIND/03628/2017 to A.C.), the Institut Mérieux (Mérieux Research Grant 2017 to C.C.), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to A.C.). M.G.N. was supported by a Spinoza grant of the Netherlands Organization for Scientific Research. A.A.B. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center/Transregio TR124 FungiNet (project A1). G.D.B. was funded by the Wellcome Trust (102705), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD