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.

Comparative Proteomic Analysis of Proteins Involved in Bioenergetics Pathways Associated with Human Sperm Motility.

Sperm motility is the most important parameter involved in the fertilization process and it is strictly required for 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 the molecular mechanism underlying sperm motility. The aim of this study was to identify proteins involved in human sperm motility deficiency by using label-free mass-spectrometry liquid chromatography (LC-MS/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.

Sources of biases in the in vitro testing of nanomaterials: the role of the biomolecular corona.

The biological fate of nanomaterials (NMs) is driven by specific interactions through which biomolecules, naturally adhering onto their surface, engage with cell membrane receptors and intracellular organelles. The molecular composition of this layer, called the biomolecular corona (BMC), depends on both the physical-chemical features of the NMs and the biological media in which the NMs are dispersed and cells grow. In this work, we demonstrate that the widespread use of 10% fetal bovine serum in an in vitro assay cannot recapitulate the complexity of in vivo systemic administration, with NMs being transported by the blood. For this purpose, we undertook a comparative journey involving proteomics, lipidomics, high throughput multiparametric in vitro screening, and single molecular feature analysis to investigate the molecular details behind this in vivo/in vitro bias. Our work indirectly highlights the need to introduce novel, more physiological-like media closer in composition to human plasma to produce realistic in vitro screening data for NMs. We also aim to set the basis to reduce this in vitro-in vivo mismatch, which currently limits the formulation of NMs for clinical settings.

Proteome Alterations and Nucleosome Activation in Rat Myoblasts Treated with Cerium Oxide Nanoparticles.

Oxidative stress is a widespread causative agent of disease. Together with its general relevance for biomedicine, such a dynamic is recognizably detrimental to space exploration. Among other solutions, cerium oxide nanoparticles (or nanoceria, NC) display a long-lasting, self-renewable antioxidant activity. In a previous experiment, we evaluated oxidative imbalance in rat myoblasts in space, aboard the International Space Station, and unveiled possible protective effects from NC through RNA sequencing. Here, we focus on the myoblast response to NC on land by means of proteomics, defining a list of proteins that putatively react to NC and confirming nucleosomes/histones as likely mediators of its molecular action. The proteomics data set we present here and its counterpart from the space study share four factors. These are coherently either up- (Hist1h4b) or down-regulated (Gnl3, Mtdh, Trip12) upon NC exposure.

A glia-enriched stem cell 3D model of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

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.

Glia-enriched stem-cell 3D model of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

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 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 MS patients, organoids properly mimic macroglia-microglia neuro-degenerative 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 implementing this organoid model for drug screening to halt inflammatory neurodegeneration.

Wool Keratin Nanoparticle-Based Micropatterns for Cellular Guidance Applications.

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 α4ß1 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.

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.

Quantification of Changes in Protein Expression Using SWATH Proteomics.

Sequential Window Acquisition of all THeoretical fragment ion spectra (SWATH) is a data independent acquisition mode used to accurately quantify thousands of proteins in a biological sample in a single run. It exploits fast scanning hybrid mass spectrometers to combine accuracy, reproducibility and sensitivity. This method requires the use of ion libraries, a sort of databases of spectral and chromatographic information about the proteins to be quantified. In this chapter, a typical workflow of SWATH experiment is described, from the sample preparation to the analysis of proteomics data.

Proteomics analysis of FUS mutant human motoneurons reveals altered regulation of cytoskeleton and other ALS-linked proteins via 3'UTR binding.

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.

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.

GADD34 is a modulator of autophagy during starvation.

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

SWATH label-free proteomics for cystic fibrosis research.

BACKGROUND: Label-free proteomics is a powerful tool for biological investigation. The SWATH protocol, relying on the Pan Human ion library, currently represents the state-of-the-art methodology for this kind of analysis. We recently discovered that this tool is not perfectly suitable for proteomics research in the CF field, as it lacks assays for several proteins crucial for the CF biology, including CFTR. METHODS: We extensively investigated the proteome of a very popular model for in vitro research on CF, CFBE41o-, and we used the corresponding data to improve the power of SWATH proteomics for CF investigation. We then used this improved tool to explore in depth the proteome of primary bronchial epithelial (BE) cells deriving from four CF individuals compared with that of four corresponding non-CF controls. By means of advanced bioinformatics tools, we outlined the presence of a number of protein networks being significantly altered by CF. RESULTS: Our analysis on patients' BE cells identified 154 proteins dysregulated by the CF pathology (94 upregulated and 60 downregulated). Some known CFTR interactors are present among them, but our analysis also revealed the alteration of other proteins not previously known to be related with CF. CONCLUSIONS: The present work outlines the power of SWATH label free proteomics applied to CF research.

MiR-135a-5p Is Critical for Exercise-Induced Adult Neurogenesis.

Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice, and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1 and inositol polyphosphate-4-phosphatase type I are among the modulated proteins, suggesting that IP3 signaling may act downstream miR-135. miR-135 is the first noncoding RNA essential modulator of the brain's response to physical exercise. Prospectively, the miR-135-IP3 axis might represent a novel target of therapeutic intervention to prevent pathological brain aging.

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.

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 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).

Pharmacophore Identification and Scaffold Exploration to Discover Novel, Potent, and Chemically Stable Inhibitors of Acid Ceramidase in Melanoma Cells.

Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abnormally high in melanoma cells. AC inhibition may thus be key to treating malignant melanoma. Here, we have used a systematic scaffold exploration to design a general pharmacophore for AC inhibition. This pharmacophore comprises a 6 + 5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are highly potent AC inhibitors. Their chemical and metabolic stabilities are comparable or superior to those of previously reported AC inhibitors. Moreover, they are potent against endogenous AC in intact melanoma cells. These novel inhibitors merit further characterization and can serve as a promising starting point for the discovery of new antimelanoma therapeutics.