16 research outputs found
NEW PHARMACOLOGICAL TARGETS FOR CYSTIC FIBROSIS TREATMENT FROM OMICS PROFILING OF F508del-CFTR EXPRESSING CELLS.
Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding an anion channel expressed on the epithelial cells of a variety of tissues. The deletion of phenylalanine in position 508 (F508del) is the most frequent CF-causing mutation, causing impaired trafficking and activity of the mutant channel. Today, two classes of drugs are available to treat CF: potentiators (molecules that increase the mutant CFTR function on the cell surface) and correctors (molecules that improve the processing and the delivery of mutant CFTR to the cell surface). It has been demonstrated that the combination of molecules exploiting different mechanisms of action is needed to achieve a therapeutically relevant rescue of CFTR. The aim of this project is to apply mass spectrometry (MS)-based omics techniques to understand what is associated to CFTR rescue and thus finding new potential targets for CF pharmacological treatment. Several strategies were applied for the functional rescue of CFTR at the plasma membrane (both pharmacological and genetic rescue). Proteomic and lipidomic profilings of F508del-CFTR expressing cells were performed after the application of these rescue strategies. The proteomic experiments were performed following the SWATH label-free quantification workflow, after the optimization of the panhuman ion library for CF research. Together with these experiments, Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) workflow was also applied to detect proteins that change their subcellular localization after the treatment with the most used corrector available for CF therapy (VX-809). Several candidate targets were found to be involved in F508del-CFTR rescue. Compounds aiming at the inhibition of these putative targets were tested on both immortalized and primary cells. Further validation studies are needed to confirm these results
Recommended from our members
Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.
Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR
CFTR Rescue by Lumacaftor (VX-809) Induces an Extensive Reorganization of Mitochondria in the Cystic Fibrosis Bronchial Epithelium
Background: Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome. Methods: We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR. Results: The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol. Conclusions: We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM
Proteomics and Metabolomics for Cystic Fibrosis Research
The aim of this review article is to introduce the reader to the state-of-the-art of the contribution that proteomics and metabolomics sciences are currently providing for cystic fibrosis (CF) research: from the understanding of cystic fibrosis transmembrane conductance regulator (CFTR) biology to biomarker discovery for CF diagnosis. Our work particularly focuses on CFTR post-translational modifications and their role in cellular trafficking as well as on studies that allowed the identification of CFTR molecular interactors. We also show how metabolomics is currently helping biomarker discovery in CF. The most recent advances in these fields are covered by this review, as well as some considerations on possible future scenarios for new applications
A new SWATH ion library for mouse adult hippocampal neural stem cells
Over the last years, the SWATH data-independent acquisition protocol (Sequential Window acquisition of All THeoretical mass spectra) has become a cornerstone for the worldwide proteomics community (Collins et al., 2017) [1]. In this approach, a high-resolution quadrupole-ToF mass spectrometer acquires thousands of MS/MS data by selecting not just a single precursor at a time, but by allowing a broad m/z range to be fragmented. This acquisition window is then sequentially moved from the lowest to the highest mass selection range. This technique enables the acquisition of thousands of high-resolution MS/MS spectra per minute in a standard LCâMS run. In the subsequent data analysis phase, the corresponding dataset is searched in a âtriple quadrupole-likeâ mode, thus not considering the whole MS/MS scan spectrum, but by searching for several precursor to fragment transitions that identify and quantify the corresponding peptide. This search is made possible with the use of an ion library, previously acquired in a classical data dependent, full-spectrum mode (Fabre et al., 2017; Wu et al., 2017) [2,3]. The SWATH protocol, combining the protein identification power of high-resolution MS/MS spectra with the robustness and accuracy in analyte quantification of triple-quad targeted workflows, has become very popular in proteomics research. The major drawback lies in the ion library itself, which is normally demanding and time-consuming to build. Conversely, through the realignment of chromatographic retention times, an ion library of a given proteome can relatively easily be tailored upon âanyâ proteomics experiment done on the same proteome. We are thus hereby sharing with the worldwide proteomics community our newly acquired ion library of mouse adult hippocampal neural stem cells. Given the growing effort in neuroscience research involving proteomics experiments (Pons-Espinal et al., 2017; Sarnyai and Guest, 2017; Sethi et al., 2015; Bramini et al., 2016) [4,5â7], we believe that this data might be of great help for the neuroscience community. All the here reported data (RAW files, results and ion library) can be freely downloaded from the SWATHATLAS (Deutsch et al., 2008) [8] website (http://www.peptideatlas.org/PASS/PASS01110) Keywords: Proteomics, SWATH, Neural stem cells, Neuroscienc
Comparative ProteomicAnalysis of Proteins Involved in Bioenergetics Pathways Associated with Human Sperm Motility
none8Sperm motility is the most important parameter involved in the fertilization process and it is strictly required for the reproductive success. Although sperm movements are essential for the physiologic fertilization process, the data, deriving from studies focused on the research of altered cell pathways involved in asthenozoospermia, offer only limited information about molecular mechanism underlying sperm motility. The aim of this study was to identify proteins involved in human sperm motility deficiency by using LCâMS/MS. For this purpose, we selected sperm samples with three different classes of progressive motility: low, medium (asthenozoospermic samples) and high (normozoospermic samples). We found that several differential expressed proteins in asthenozoospermic samples were related to energetic metabolism, suggesting an interesting link between bioenergetics pathways and the regulation of sperm motility, necessary for the flagellum movement. Therefore, our results provide strong evidence that mass spectrometry-based proteomics represents an integrated approach to detect novel biochemical markers of sperm motility and quality with diagnostic relevance for male infertility and unravel the molecular etiology of idiopathic cases.mixedMoscatelli, Natalina; Lunetti, Paola; Braccia, Clarissa; Armirotti, Andrea; Pisanello, Ferruccio; De Vittorio, Massimo; Zara, Vincenzo; Ferramosca, Alessandra.Moscatelli, Natalina; Lunetti, Paola; Braccia, Clarissa; Armirotti, Andrea; Pisanello, Ferruccio; De Vittorio, Massimo; Zara, Vincenzo; Ferramosca, Alessandra
Comparative Proteomic Analysis of Proteins Involved in Bioenergetics Pathways Associated with Human Sperm Motility
Distinctive lipid signatures of bronchial epithelial cells associated with cystic fibrosis drugs, including Trikafta
In recent years, a number of drugs have been approved for the treatment of cystic fibrosis (CF). Among them, newly released Trikafta, a combination of 3 drugs (VX-661/VX-445/VX-770), holds great promise to radically improve the quality of life for a large portion of patients with CF carrying 1 copy of F508del, the most frequent CF transmembrane conductance regulator (CFTR) mutation. Currently available disease-modifying CF drugs work by rescuing the function of the mutated CFTR anion channel. Recent research has shown that membrane lipids, and the cell lipidome in general, play a significant role in the mechanism of CFTR-defective trafficking and, on the other hand, its rescue. In this paper, by using untargeted lipidomics on CFBE41o- cells, we identified distinctive changes in the bronchial epithelial cell lipidome associated with treatment with Trikafta and other CF drugs. Particularly interesting was the reduction of levels of ceramide, a known molecular player in the induction of apoptosis, which appeared to be associated with a decrease in the susceptibility of cells to undergo apoptosis. This evidence could account for additional beneficial roles of the triple combination of drugs on CF phenotypes
Proteomics analysis of FUS mutant human motoneurons reveals altered regulation of cytoskeleton and other ALS-linked proteins via 3âČUTR binding
International audienceIncreasing evidence suggests that in Amyotrophic Lateral Sclerosis (ALS) mutated RNA binding proteins acquire aberrant functions, leading to altered RNA metabolism with significant impact on encoded protein levels. Here, by taking advantage of a human induced pluripotent stem cell-based model, we aimed to gain insights on the impact of ALS mutant FUS on the motoneuron proteome. Label-free proteomics analysis by mass-spectrometry revealed upregulation of proteins involved in catabolic processes and oxidation-reduction, and downregulation of cytoskeletal proteins and factors directing neuron projection. Mechanistically, proteome alteration does not correlate with transcriptome changes. Rather, we observed a strong correlation with selective binding of mutant FUS to target mRNAs in their 3'UTR. Novel validated targets, selectively bound by mutant FUS, include genes previously involved in familial or sporadic ALS, such as VCP, and regulators of membrane trafficking and cytoskeleton remodeling, such as ASAP1. These findings unveil a novel mechanism by which mutant FUS might intersect other pathogenic pathways in ALS patients' motoneurons