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.
Summary SINEUPs are antisense long non-coding RNAs that enhance translation of overlapping sense mRNAs through the activity of two domains: a SINE B2 sequence UP- regulating translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD). In this study, we demonstrate that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA is an Internal Ribosomal Entry Site (IRES) when acting in cis and that known viral and cellular IRES sequences can act as Effector Domain in synthetic SINEUPs. To identify natural IRES-containing, non-coding RNAs with SINEUP-like activity, we focused on circular RNAs showing that the non-coding circ5533 , transcribed from the c-myc locus , enhances endogenous protein expression of its target PX Domain Containing Serine/Threonine Kinase Like ( Pxk) by increasing mRNA association to polysomes. In summary, this study shows that natural and synthetic SINEUPs include linear and circular transcripts with an embedded IRES sequence as ED.
The benefits of ginseng have been mainly attributed to its triterpenoids, called ginsenosides. Recent genome sequencing of the Panax ginseng has paved the way for in-depth proteomic studies of this medicinal plant. The current study was conducted to deepen the proteomic information on the root proteome of Korean ginseng. Proteomic workflow was optimized by testing two different strategies, characterized by the phenol extraction procedure, the presence or the absence of SDS-PAGE fractionation step, and nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) analysis. The results highlighted an evident improvement of proteome extraction by the combination of phenol extraction with SDS-PAGE before the nLC-MS/MS analysis. In addition, a dramatic impact of the steaming process (the treatment to produce red ginseng from ginseng) on protein properties was observed. Overall, the analyses of Korean ginseng permitted the characterization of a total of 2412 proteins. A large number of identified proteins belonged to the functional categories of protein and carbon/energy metabolism (22.4% and 14.6%, respectively). The primary and secondary metabolisms are major metabolic pathways, which emerged from the proteomic analysis. In addition, a large number of proteins known to play an important role in response to (a)biotic stresses were also identified. The current proteomic study not only confirmed the previous transcriptomic and proteomic reports but also extended proteomic information, including the main metabolic pathways involved in Korean ginseng.
Abstract Increasing 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.
The waste stream of low-grade wool is an underutilized source of keratin-rich materials with appropriate methods for upcycling into high value-added products still being an open challenge. In the present work, keratins were precipitated from their water solution to produce hierarchical keratin particles via isoelectric precipitation. Matrix-assisted laser desorption/ionization coupled with time-of-flight tandem mass spectrometry analysis (MALDI-TOF/TOF MS/MS) showed the presence of the amino acid sequence leucine-aspartic acid-valine (LDV) in the extracted keratin. This well-known cell adhesion motif is recognized by the cell adhesion molecule α4β1 integrin. We showed that keratin particles had this tripeptide exposed on the surface and that it could be leveraged, via patterns obtained with microcontact printing, to support and facilitate dermal fibroblast cell adhesion and direct their growth orientation. The zeta potential, isoelectric point, morphological structures, chemical composition, and biocompatibility of keratin particles and the influence of the surfactant sodium dodecyl sulfate (SDS) were investigated. An appropriate ink for microcontact printing of the keratin particles was developed and micron-sized patterns were obtained. Cells adhered preferentially to the patterns, showing how this strategy could be used to functionalize biointerfaces.
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 LC-MS run. In the subsequent data analysis phase, the corresponding dataset is searched in a "triple quadrupole-like" 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 "any" 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,[5], [6], [7], 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).
A comparative journey into biomolecular corona features involving proteomics, lipidomics, high throughput in vitro screening, and molecular feature analysis to investigate the in vivo / in vitro bias for nanomaterials testing in biology.
The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis (MS) is gaining scientific interest. We applied a single transcription factor (SOX10)-based protocol to accelerate oligodendrocyte differentiation from human induced pluripotent stem cell (hiPSC)-derived neural precursor cells, generating self-organizing forebrain organoids. These organoids include neurons, astrocytes, oligodendroglia, and hiPSC-derived microglia to achieve immunocompetence. Over 8 weeks, organoids reproducibly generated mature CNS cell types, exhibiting single-cell transcriptional profiles similar to the adult human brain. Exposed to inflamed cerebrospinal fluid (CSF) from patients with MS, organoids properly mimic macroglia-microglia neurodegenerative phenotypes and intercellular communication seen in chronic active MS. Oligodendrocyte vulnerability emerged by day 6 post-MS-CSF exposure, with nearly 50% reduction. Temporally resolved organoid data support and expand on the role of soluble CSF mediators in sustaining downstream events leading to oligodendrocyte death and inflammatory neurodegeneration. Such findings support the implementation of this organoid model for drug screening to halt inflammatory neurodegeneration.
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.
