Biology subjects

Sermet-Gaudelus, I.

Publications and source records attributed to Sermet-Gaudelus, I..

8 recordsLinked to original sources

bioRxiv2023

From CFTR to a CF signalling network: a systems biology approach to study Cystic fibrosis

BackgroundCystic Fibrosis (CF) is a monogenic disease caused by mutations in the gene coding the Cystic Fibrosis Transmembrane Regulator (CFTR) protein, but its overall physio-pathology cannot be solely explained by the loss of the CFTR chloride channel function. Indeed, CFTR belongs to a yet not fully deciphered network of proteins participating in various signalling pathways. MethodsWe propose a systems biology approach to study how the absence of the CFTR protein at the membrane leads to perturbation of these pathways, resulting in a panel of deleterious CF cellular phenotypes. ResultsBased on publicly available transcriptomic datasets, we built and analyzed a CF network that recapitulates signalling dysregulations. The CF network topology and its resulting phenotype was found to be consistent with CF pathology. ConclusionAnalysis of the network topology highlighted a few proteins that may initiate the propagation of dysregulations, those that trigger CF cellular phenotypes, and suggested several candidate therapeutic targets. Although our research is focused on CF, the global approach proposed in the present paper could also be followed to study other rare monogenic diseases.

systems biology↗

bioRxiv2023

TLN468 changes the pattern of tRNA used to readthrough premature termination codons in CFTR

Nonsense mutations account for 12% of cystic fibrosis (CF) cases. The presence of a premature termination codon (PTC) leads to gene inactivation, which can be countered by the use of drugs stimulating PTC readthrough, restoring production of the full-length protein. We recently identified a new readthrough inducer, TLN468, more efficient than gentamicin. We measured the readthrough induced by these two drugs with different cystic fibrosis transmembrane conductance regulator (CFTR) PTCs. We then determined the amino acids inserted at the S1196X, G542X, W846X and E1417X PTCs of CFTR during readthrough induced by gentamicin or TLN468. TLN468 significantly promoted the incorporation of one specific amino acid, whereas gentamicin did not greatly modify the proportions of the various amino acids incorporated relative to basal conditions. The function of the engineered missense CFTR channels corresponding to these four PTCs was assessed with and without potentiator. For the recoded CFTR, except for E1417Q and G542W, the PTC readthrough induced by TLN468 allowed the expression of CFTR variants that were correctly processed and had significant activity that was enhanced by CFTR modulators. These results suggest that it would be relevant to assess the therapeutic benefit of TLN468 PTC suppression in combination with CFTR modulators in preclinical assays.

molecular biology↗

bioRxiv2022

Representation and quantification Of Module Activity from omics data with rROMA

The efficiency of analyzing high-throughput data in systems biology has been demonstrated in numerous studies, where molecular data, such as transcriptomics and proteomics, offers great opportunities for understanding the complexity of biological processes. One important aspect of data analysis in systems biology is the shift from a reductionist approach that focuses on individual components to a more integrative perspective that considers the system as a whole, where the emphasis shifted from differential expression of individual genes to determining the activity of gene sets. Here, we present the rROMA software package for fast and accurate computation of the activity of gene sets with coordinated expression. The rROMA package incorporates significant improvements in the calculation algorithm, along with the implementation of several functions for statistical analysis and visualizing results. These additions greatly expand the packages capabilities and offer valuable tools for data analysis and interpretation. It is an open-source package available on github at: www.github.com/sysbio-curie/rROMA. Based on publicly available transcriptomic datasets, we applied rROMA to cystic fibrosis, highlighting biological mechanisms potentially involved in the establishment and progression of the disease and the associated genes. Results indicate that rROMA can detect disease-related active signaling pathways using transcriptomic and proteomic data. The results notably identified a significant mechanism relevant to cystic fibrosis, raised awareness of a possible bias related to cell culture, and uncovered an intriguing gene that warrants further investigation. Contact: loredana.martignetti@curie.fr

systems biology↗

bioRxiv2022

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha 1 antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild type (WT) A1AT with a 13-fold and 4-fold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the s heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

cell biology↗

bioRxiv2021

Elexacaftor/Tezacaftor/Ivacaftor alters branching morphogenesis of the mouse embryonic lung

IntroductionCFTR modulators triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) has proven to clinically benefit homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of the potential impact of these modulators on the development of the foetus are mandatory. MaterialsWe used the early mouse embryonic lung organ culture model to analyse ex vivo the lung branching process and the relative expression of Fgf10, Fgfr2IIIb, Shh, and Hhip development regulator genes in different conditions: standard culture medium, treatment with ETI or with Forskolin {+/-} Inh172. Development of lung branching and distal bud caliber were evaluated in lung explants from heterozygous F508del Cftrtm1Eur/+ and control Cftrtm1Eur+/+ (WT) mouse embryos at E12.5 during pseudo-glandular stage. ResultsExposure to ETI of the Cftrtm1Eur/+ and WT lung explants induced a significant decrease in lung branching after 48h culture and the percentage of terminal bud dilations was significantly increased. These results were recapitulated by cAMP-dependent CFTR continuous activation by Forskolin and reversed by addition of Inh172. ETI induced a significant decrease in Fgf10, Fgfr2IIIb, Shh and Hhip expression in lung explants of both E12.5 Cftrtm1Eur/+ and WT embryos treated with ETI for 72h. ConclusionOur results provide evidence that the triple association Elexacaftor/Tezacaftor/Ivacaftor alters lung branching morphogenesis of WT and heterozygous F508del mouse embryos during the pseudo-glandular stage. Those results argue for a close monitoring of pregnancies in patients treated with these drugs. Plain LanguageO_ST_ABSIntroductionC_ST_ABSThe triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) improves homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of this treatment on the development of the foetus are lacking. We incubated lungs of murine foetus not carrying CFTR mutation or F508del heterozygous. We show that ETI induces significant defect of lung development and the formation of cysts. These results are at least partly due to CFTR activation. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

pharmacology and toxicology↗

bioRxiv2021

CFTR corrector efficacy is associated with occupancy of distinct binding sites

CFTR misfolding due to cystic fibrosis causing mutations can be corrected with small molecules designated as correctors. VX-809, an investigational corrector compound, is believed to bind CFTR directly to either the first membrane-spanning domain (MSD1) and/or the first nucleotide-binding domain (NBD1). Blind docking onto the 3D structures of these domains, followed by molecular dynamics (MD) simulations, revealed the presence of two potential VX-809 binding sites which, when mutated, abrogated corrector rescue. Mutations altering protein maturation are also shown to be not equally sensitive to the occupancy of the two sites by VX-809, with the most frequent mutation F508del requiring integrity of both sites and allosteric coupling with the F508del region while L206W only requires the integrity of the MSD1 site. A network of charged amino acids in the lasso Lh2 helix and the intracellular loops ICL1 and ICL4 is involved in the allostery between MSD1 and NBD1. Corrector VX-445, which is used in combination in clinics with VX-661, a structurally close analog of VX-809, to fully correct F508del, is also shown to occupy two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. In conclusion, VX-809 and VX-445 appear to bind different CFTR domains to alleviate specific folding defects. These results provide new insights into therapeutics understanding and may help the development of efficient corrector combinations.

molecular biology↗

bioRxiv2021

Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10kb C-to-T splicing mutation

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2'-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43{+/-}8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.

genetics↗

bioRxiv2018

Persistent Staphylococcus aureus infections in children with chronic lung disease: a multi-omics analysis of bacterial adaptation to define novel therapeutic approaches

BackgroundChronic lung infection of cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Strikingly the molecular mechanisms underlying S. aureus persistency are not understood.\n\nMethodsWe selected pairs of sequential S. aureus isolates from 3 patients with CF and from one patient with non-CF chronic lung disease. We used a combination of genomic, proteomic and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence.\n\nResultsFor the first time, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CFBE-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated.\n\nFurthermore, we identified the underlying genetic modifications inducing altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets.\n\nConclusionsOur results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments.\n\nSummaryS. aureus persists for years in the lungs of patients with cystic fibrosis despite antibiotic therapies. We demonstrate that S. aureus adaptation leads to increased intracellular persistence suggesting a key role for intracellular niche during S. aureus chronic lung infection.

microbiology↗