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/

Pherotypes are driving genetic differentiation within Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. <it>Streptococcus pneumoniae </it>populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in <it>S. pneumoniae </it>is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.</p> <p>Results</p> <p>We studied the distribution of the two major pherotypes in the pneumococcal population and their association with serotype, antimicrobial resistance and genetic lineage. Using multilocus sequence data we evaluated pherotype influence on the dynamics of horizontal gene transfer. We show that pherotype is a clonal property of pneumococci. Standard population genetic analysis and multilocus infinite allele model simulations support the hypothesis that two genetically differentiated populations are defined by the major pherotypes.</p> <p>Conclusion</p> <p>Severe limitations to gene flow can therefore occur in bacterial species in the absence of geographical barriers and within highly recombinogenic populations. This departure from panmixia can have important consequences for our understanding of the response of pneumococci to human imposed selective pressures such as vaccination and antibiotic use.</p

Group A Streptococci clones associated with invasive infections and pharyngitis in Portugal present differences in emm types, superantigen gene content and antimicrobial resistance

© 2012 Friães et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedBackground: A few lineages of Group A streptococci (GAS) have been associated with a reemergence of severe invasive streptococcal disease in developed countries. However, the majority of the comparisons between invasive and non-invasive GAS isolates have been performed for collections of reduced genetic diversity or relied on limited typing information to distinguish clones. We characterized by several typing methods and compared a collection of 160 isolates recovered from normally sterile sites with 320 isolates associated with pharyngitis and recovered in the same time period in Portugal. Results: Although most of the isolates belonged to clones that were equally prevalent in invasive infections and pharyngitis, we identified markers of invasiveness, namely the emm types 1 and 64, and the presence of the speA and speJ genes. In contrast, emm4, emm75, and the ssa and speL/M genes were significantly associated with pharyngitis. There was a strong agreement between the emm type, the superantigen (SAg) genes and the clusters defined by pulsed-field gel electrophoresis (PFGE) profiling. Therefore, combinations of particular emm types and SAg genes frequently co-occurred in the same PFGE cluster, but there was no synergistic or antagonistic interaction between them in determining invasiveness. Only macrolide-susceptible PFGE clones were significantly associated with invasive infections or pharyngitis, while the clones of resistant isolates sharing all other molecular properties analyzed were equally prevalent in the two groups of isolates. Conclusions: This study confirmed the importance of the widely disseminated emm1-T1-ST28 clone in invasive infections but also identified other clones linked to either invasive infections (emm64-ST164) or pharyngitis (emm4-T4-ST39), which may be more limited in their temporal and geographical spread. Clonal properties like some emm types or SAg genes were associated with disease presentation, highlighting the importance of bacterial genetic factors to the outcome of GAS infections, although other, yet unidentified factors may also play an important role.This work was partially supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-ESA/72321/2006), Fundação Calouste Gulbenkian and unrestricted research grant from Glaxo SmithKline.info:eu-repo/semantics/publishedVersio

Tree type and forest management effects on the structure of strem wood following wilfires

Wildfires are an increasingly common disturbance influencing wood recruitment to streams, and thereby affecting their physical and biological condition. Mediterranean countries such as Portugal, where more than 25% of the land area has burned since 1990, are ideal areas to study impacts of wildfire effects on streams. We evaluated the physical structure of 2206 downed wood pieces (DWP) across 27 first- to third-order streams in central Portugal, all of which had experienced recent wildfires. The streams flowed through monospecific upland forests of Eucalyptus, Maritime pines, or Cork oaks and were fringed by a mixture of riparian tree species. DWP structure differed between tree types and between burned and unburned pieces. Post-fire timber-production forests (Maritime pines and Eucalyptus) contributed a higher quantity of thinner, longer and straighter DWP to streams than Cork oak stands. Pieces from Maritime pines had more rootwads and branches than DWP from the other tree types. Pieces from Cork oak and riparian species generally had a bent form, were shorter and had no rootwads. Burned DWP in streams were often from riparian trees. Relative to unburned DWP, the burned DWP occurred more frequently, were larger and straighter, had branches less often, and were more decayed. With more complex branches, rootwads, and a larger diameter, inputs from burned Maritime pine forests are more likely to change stream hydraulics and habitat complexity, relative to inputs from Eucalyptus forests with their simpler structure. This study shows that, less than a decade after wildfires, structure of downed wood in and near streams is strongly influenced by wildfire, but also still reflects intrinsic species characteristics and respective silviculture practices, even after the effects of fire have been accounted for. Under an anticipated shift in landscape cover with higher shrubland proportions and more mixing of Maritime pine and Eucalyptus forests, our results suggest that instream large wood will become scarcer and more structurally homogeneous

Analyzing the Implementation of Lean Methodologies and Practices in the Portuguese Industry: A Survey

The mass production paradigm on which much of the industry was based has changed. The market is increasingly demanding, requesting diversity and products that are more and more adapted to personal wishes and requirements. This implies producing a greater diversity of products in smaller quantities. Competitiveness is enormous, which forces most companies to be truly effective and efficient, taking care of product quality, delivery time, and final cost. Lean methodologies have been a valuable aid in this field. The diversity of Lean tools has been shown to have answers to the most diverse challenges, and companies are aware of this, increasingly adopting methodologies and processes that aim to progressively reduce waste and adapting their production paradigm to what the market requires. This work intends to provide a vision, as global as possible, of the pathway of Lean implementation in the Portuguese industry. For this purpose, a survey was carried out with a significant sample of Portuguese industrial companies from a wide range of activity sectors. The data collected through the survey were treated statistically, and then a SWOT analysis of the results was performed, which provided a collection of precious information on the evolution of industrial companies in Portugal.The author CP was partially supported by CMUP, which is financed by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project with reference UIDB/00144/2020. F.J.G.S. would like to thank INEGI Research Center due to its continuous support, namely to Jorge Seabra, as well as all remaining CETRIB/INEGI/LAETA teal.info:eu-repo/semantics/publishedVersio

Studies on the interaction of the carbohydrate binding module 3 from the Clostridium thermocellum CipA scaffolding protein with cellulose and paper fibres

The adsorption of a carbohydrate binding module (CBM3) from the Clostridium thermocellum scaffolding protein (CipA) to cellulose was analysed in this work. The effect of CBM-PEG on the drainability of E. globulus and P. sylvestris pulps and on the physical properties of the respective papersheets was also studied. The CBM binding to cellulose is often described as “irreversible”, but this classification does not fully characterize this interaction. Indeed, the results obtained demonstrate that, although the adsorption on cellulose is rather stable, CBM inter-fibre mobility may be observed. The results also showed that the CBM-PEG conjugate improves the drainability of E. globulus and P. sylvestris pulps without affecting the physical properties of the papersheets.This research was supported by Fundacao para a Ciencia e a Tecnologia under grant POCTI/BIO/45356/2002

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