Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis.

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

Toward High Spatially Resolved Proteomics Using Expansion Microscopy.

Expansion microscopy (EM) is an emerging approach for morphological examination of biological specimens at nanoscale resolution using conventional optical microscopy. To achieve physical separation of cell structures, tissues are embedded in a swellable polymer and expanded several fold in an isotropic manner. This work shows the development and optimization of physical tissue expansion as a new method for spatially resolved large-scale proteomics. Herein we established a novel method to enlarge the tissue section to be compatible with manual microdissection on regions of interest and MS-based proteomic analysis. A major issue in expansion microscopy is the loss of protein information during the mechanical homogenization phase due to the use of proteinase K. For isotropic expansion, different homogenization agents were investigated, both to maximize protein identification and to minimize protein diffusion. Best results were obtained with SDS for homogenization. Using our modified protocol, we were able to enlarge a tissue section more than 3-fold and identified up to 655 proteins from 1 mm in size after expansion, equivalent to 330 µm in their real size corresponding thus to an average of 260 cells. This approach can be performed easily without any expensive sampling instrument. We demonstrated the compatibility of sample preparation for expansion microscopy and proteomic study in a spatial context.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

A Hidden Human Proteome Signature Characterizes the Epithelial Mesenchymal Transition Program.

BACKGROUND: Molecular changes associated with the initiation of the epithelial to mesenchymal transition (EMT) program involve alterations of large proteome-based networks. The role of protein products mapping to non-coding genomic regions is still unexplored. OBJECTIVE: The goal of this study was the identification of an alternative protein signature in breast cancer cellular models with a distinct expression of EMT markers. METHODS: We profiled MCF-7 and MDA-MB-231 cells using liquid-chromatography mass/spectrometry (LCMS/ MS) and interrogated the OpenProt database to identify novel predicted isoforms and novel predicted proteins from alternative open reading frames (AltProts). RESULTS: Our analysis revealed an AltProt and isoform protein signature capable of classifying the two breast cancer cell lines. Among the most highly expressed alternative proteins, we observed proteins potentially associated with inflammation, metabolism and EMT. CONCLUSION: Here, we present an AltProts signature associated with EMT. Further studies will be needed to define their role in cancer progression.

Carbonic Anhydrase XII Expression Is Modulated during Epithelial Mesenchymal Transition and Regulated through Protein Kinase C Signaling.

Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

Optimized Sample Preparation Workflow for Improved Identification of Ghost Proteins.

Large scale proteomic strategies rely on database interrogation. Thus, only referenced proteins can be identified. Recently, Alternative Proteins (AltProts) translated from nonannotated Alternative Open reading frame (AltORFs) were discovered using customized databases. Because of their small size which confers them peptide-like physicochemical properties, they are more difficult to detect using standard proteomics strategies. In this study, we tested different preparation workflows for improving the identification of AltProts in NCH82 human glioma cell line. The highest number of identified AltProts was achieved with RIPA buffer or boiling water extraction followed by acetic acid precipitation.

Distinct Protein Expression Networks are Activated in Microglia Cells after Stimulation with IFN-γ and IL-4.

Microglia cells are the primary immune population of the central nervous system with a role in the regulation of several physiological and pathological conditions. Upon appropriate stimulation, microglia cells can be polarized in a pro-inflammatory M1-like or anti-inflammatory M2-like status. Biological processes and pathways engaged in microglia polarization are starting to be elucidated. To help clarify this, we used a liquid chromatography-mass spectrometry (LC-MS/MS) label free approach to characterize the proteomic profile of human microglia cell line (CHME-5) stimulated with gamma-interferon (IFN-γ) and interleukin-4 (IL-4) to induce a M1 or M2 phenotype, respectively. Outside the classical M1/M2 polarization markers, the M1 status appears to center around the activation of a classical inflammatory response and through the activation of multiple signaling pathways. M2 polarization resulted in a different pattern of protein modulation related to RNA and cellular metabolic processes. Together, our findings provide information regarding the protein changes specific to M1 and M2 activation states, and potentially link the polarization of microglia cells to the acquisition of a specific proteomic profile.

Nuclei of HeLa cells interactomes unravel a network of ghost proteins involved in proteins translation.

Ghost proteins are issued from alternative Open Reading Frames (ORFs) and are missing a genome annotation. Indeed, historical filters applied for the detection of putative translated ORFs led to a wrong classification of transcripts considered as non-coding although translated proteins can be detected by proteomics. This Ghost (also called Alternative) proteome was neglected, and one major issue is to identify the implication of the Ghost proteins in the biological processes. In this context, we aimed to identify the protein-protein interactions (PPIs) of the Ghost proteins. For that, we re-explored a cross-link MS study performed on nuclei of HeLa cells using cross-linking mass spectrometry (XL-MS) associated with the HaltOrf database. Among 1679 cross-link interactions identified, 292 are involving Ghost Proteins. Forty-Four of these Ghost proteins are found to interact with 7 Reference proteins related to ribonucleoproteins, ribosome subunits and zinc finger proteins network. We, thus, have focused our attention on the heterotrimer between the RE/poly(U)-binding/degradation factor 1 (AUF1), the Ribosomal protein 10 (RPL10) and AltATAD2. Using I-Tasser software we performed docking models from which we could suggest the attachment of AUF1 on the external part of RPL10 and the interaction of AltATAD2 on the RPL10 region interacting with 5S ribosomal RNA as a mechanism of regulation of the ribosome. Taken together, these results reveal the importance of Ghost Proteins within known protein interaction networks.

ALK4/5-dependent TGF-ß signaling contributes to the crosstalk between neurons and microglia following axonal lesion.

Neuronal activity is closely influenced by glia, especially microglia which are the resident immune cells in the central nervous system (CNS). Microglia in medicinal leech are the only cells able to migrate to the injury site within the 24 hours post-lesion. The microglia-neuron interactions constitute an important mechanism as there is neither astrocyte nor oligodendrocyte in the leech CNS. Given that axonal sprouting is impaired when microglia recruitment is inhibited, the crosstalk between microglia and neurons plays a crucial role in neuroprotection. The present results show that neurons and microglia both use ALK4/5 (a type of TGF-ß receptor) signaling in order to maintain mutual exchanges in an adult brain following an axonal injury. Indeed, a TGF-ß family member (nGDF) is immediately released by injured axons contributing to the early recruitment of ALK4/5+ microglia to the lesion site. Surprisingly, within the following hours, nGDF from microglia activates ALK4/5+ neurons to maintain a later microglia accumulation in lesion. Taken together, the results demonstrate that ALK4/5 signaling is essential throughout the response to the lesion in the leech CNS and gives a new insight in the understanding of this pathway. This latter is an important signal contributing to a correct sequential mobilization over time of microglia recruitment leading to axon regeneration.

The multiverse nature of epithelial to mesenchymal transition.

The epithelial mesenchymal transition (EMT) program is defined as a cellular transition from an epithelial to a mesenchymal state. This process occurs to provide the cell with new phenotypic assets and new skills to perform complex processes. EMT is regulated at multilayer levels, including transcriptional control of gene expression, regulation of RNA splicing, and translational/post-translational control. Although transcriptional regulation by EMT-inducing transcription factors (EMT-TFs), including Zeb, Snail and Slug members, is generally considered the master step in this process, emerging data indicate that all these regulatory networks may have a role in the control of EMT. There is a sort of parallelism between the biological and still unrevealed EMT complexity and the cosmological hypothesis that sustains the universe may exist as a multiverse. The presence of different EMT transition states together with the occurrence of multiple layers of regulation support the idea that EMT is just one on many out there. Is the activation of a single layer of regulation sufficient to initiate the whole EMT program? Can we postulate the activation of different EMT "dimensions"? If we think about these layers as multiple separate "universes", various scenarios can be revealed.

A specific lipid metabolic profile is associated with the epithelial mesenchymal transition program.

Several studies have identified a specific metabolic program that is associated with the process of epithelial-mesenchymal transition (EMT). Whereas much is known about the association between glucose metabolism and EMT, the contribution of lipid metabolism is not still completely understood. Here, we studied epithelial and mesenchymal breast cancer cells by proteomic and lipidomic approaches and identified significant differences that characterised these models concerning specific metabolic enzymes and metabolites including fatty acids and phospholipids. Higher levels of monounsaturated fatty acids together with increased expression of enzymes of de novo fatty acid synthesis is the distinct signature of epithelial with respect to mesenchymal cells that, on the contrary, show reduced lipogenesis, higher polyunsaturated fatty acids level and increased expression of genes involved in the triacylglycerol (TAG) synthesis and lipid droplets formation. In the mesenchymal model, the diacylglycerol acyltransferase (DGAT)-1 appears to be the major enzyme involved in TAG synthesis and inhibition of DGAT1, but not DGAT2, drastically reduces the incorporation of labeled palmitate into TAG. Moreover, knockdown of ß-catenin demonstrated that this metabolic phenotype in under the control of a network of transcriptional factors and that ß-catenin has a specific role in the regulation of lipid metabolism in mesenchymal cells.

Proteomic expression profile of injured rat peripheral nerves revealed biological networks and processes associated with nerve regeneration.

Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins at the injury-site of rat sciatic nerve at 20 days after damage. By a label-free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signaling and remodeling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells at the injury site in the sciatic nerve. This further supports a specific local regulation of lipid metabolism in peripheral nerve after damage.

Spatially-Resolved Top-down Proteomics Bridged to MALDI MS Imaging Reveals the Molecular Physiome of Brain Regions.

Tissue spatially-resolved proteomics was performed on 3 brain regions, leading to the characterization of 123 reference proteins. Moreover, 8 alternative proteins from alternative open reading frames (AltORF) were identified. Some proteins display specific post-translational modification profiles or truncation linked to the brain regions and their functions. Systems biology analysis performed on the proteome identified in each region allowed to associate sub-networks with the functional physiology of each brain region. Back correlation of the proteins identified by spatially-resolved proteomics at a given tissue localization with the MALDI MS imaging data, was then performed. As an example, mapping of the distribution of the matrix metallopeptidase 3-cleaved C-terminal fragment of α-synuclein (aa 95-140) identified its specific distribution along the hippocampal dentate gyrus. Taken together, we established the molecular physiome of 3 rat brain regions through reference and hidden proteome characterization.

ß-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells.

ß-catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via ß-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after ß-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and ß-catenin knockout cells (shßcat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of ß-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in shßcat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in shßcat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in shßcat cells. ß-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, ß-catenin knockout cells showed increased incorporation of [1-14C]acetate and decreased utilization of [U-14C]glucose for fatty acid synthesis. Our data highlight a role of ß-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer.

BACKGROUND: Recently, it was demonstrated that proteins can be translated from alternative open reading frames (altORFs), increasing the size of the actual proteome. Top-down mass spectrometry-based proteomics allows the identification of intact proteins containing post-translational modifications (PTMs) as well as truncated forms translated from reference ORFs or altORFs. METHODS: Top-down tissue microproteomics was applied on benign, tumor and necrotic-fibrotic regions of serous ovarian cancer biopsies, identifying proteins exhibiting region-specific cellular localization and PTMs. The regions of interest (ROIs) were determined by MALDI mass spectrometry imaging and spatial segmentation. FINDINGS: Analysis with a customized protein sequence database containing reference and alternative proteins (altprots) identified 15 altprots, including alternative G protein nucleolar 1 (AltGNL1) found in the tumor, and translated from an altORF nested within the GNL1 canonical coding sequence. Co-expression of GNL1 and altGNL1 was validated by transfection in HEK293 and HeLa cells with an expression plasmid containing a GNL1-FLAG(V5) construct. Western blot and immunofluorescence experiments confirmed constitutive co-expression of altGNL1-V5 with GNL1-FLAG. CONCLUSIONS: Taken together, our approach provides means to evaluate protein changes in the case of serous ovarian cancer, allowing the detection of potential markers that have never been considered.

Progress and Potential of Imaging Mass Spectrometry Applied to Biomarker Discovery.

Mapping provides a direct means to assess the impact of protein biomarkers and puts into context their relevance in the type of cancer being examined. To this end, mass spectrometry imaging (MSI) was developed to provide the needed spatial information which is missing in traditional liquid-based mass spectrometric proteomics approaches. Aptly described as a "molecular histology" technique, MSI gives an additional dimension in characterizing tumor biopsies, allowing for mapping of hundreds of molecules in a single analysis. A decade of developments focused on improving and standardizing MSI so that the technique can be translated into the clinical setting. This review describes the progress made in addressing the technological development that allows to bridge local protein detection by MSI to its identification and to illustrate its potential in studying various aspects of cancer biomarker discovery.

Combined MALDI Mass Spectrometry Imaging and Parafilm-Assisted Microdissection-Based LC-MS/MS Workflows in the Study of the Brain.

Proteins and other biomolecules such as lipids are significant players in the central nervous system and are implicated in various neurological disorders. Their identification, quantification, and distribution are thus important not only in understanding the disease but also in developing treatments. A combined workflow allowing the localized microextraction of discrete regions identified by a matrix-assisted laser desorption/ionization mass spectrometry (MSI) imaging experiment for proteomics analysis by liquid chromatography/tandem mass spectrometry (LC MS/MS) is described in this chapter. MSI was initially used to map lipid distributions allowing for the identification of regions of interest (ROIs) that are then subjected to microextraction in a consecutive tissue section. Mounting of consecutive tissue on parafilm allows microdissection of the ROIs, where proteins can then be recovered for processing and LC MS/MS analysis. The PAM method provides a fast and cheap means to perform further downstream analysis after an MSI experiment.

Droplet-Based Liquid Extraction for Spatially-Resolved Microproteomics Analysis of Tissue Sections.

Obtaining information on protein content while keeping their localization on tissue or organ is of importance in different domains to understand pathophysiological processes. There is increasing interest in studying the microenvironment and heterogeneity of tumors, which currently is difficult with existing proteomics techniques. The advent of new techniques, like MALDI Mass Spectrometry Imaging, made a significant progress in the last decade but is characterized by a number of inherent drawbacks. One of these is the limited identification of proteins. New alternative approaches such as spatially-resolved liquid microextraction have recently been proposed to overcome this limitation. In this chapter, we describe strategies using liquid microjunction to perform extraction of previously digested peptides or of intact proteins from tissue section in a localized manner.

Integrated mass spectrometry imaging and omics workflows on the same tissue section using grid-aided, parafilm-assisted microdissection.

BACKGROUND: In spite of the number of applications describing the use of MALDI MSI, one of its major drawbacks is the limited capability of identifying multiple compound classes directly on the same tissue section. METHODS: We demonstrate the use of grid-aided, parafilm-assisted microdissection to perform MALDI MS imaging and shotgun proteomics and metabolomics in a combined workflow and using only a single tissue section. The grid is generated by microspotting acid dye 25 using a piezoelectric microspotter, and this grid was used as a guide to locate regions of interest and as an aid during manual microdissection. Subjecting the dissected pieces to the modified Folch method allows to separate the metabolites from proteins. The proteins can then be subjected to digestion under controlled conditions to improve protein identification yields. RESULTS: The proof of concept experiment on rat brain generated 162 and 140 metabolite assignments from three ROIs (cerebellum, hippocampus and midbrain/hypothalamus) in positive and negative modes, respectively, and 890, 1303 and 1059 unique proteins. Integrated metabolite and protein overrepresentation analysis identified pathways associated with the biological functions of each ROI, most of which were not identified when looking at the protein and metabolite lists individually. CONCLUSIONS: This combined MALDI MS imaging and multi-omics approach further extends the amount of information that can be generated from single tissue sections. GENERAL SIGNIFICANCE: To the best of our knowledge, this is the first report combining both imaging and multi-omics analyses in the same workflow and on the same tissue section.

A Proteomic Analysis of Human Uterine Myoma.

Uterine leiomyoma is a benign smooth muscle tumor characterized by a high incidence in women of reproductive age. The aetiology of this tumor is still unknown but established risk factors include high levels of female hormones, family history, African ancestry, early age of menarche and obesity. Here, to identify proteomic features associated with this tumor type, we performed a liquid chromatography-mass spectrometry (LC-MS/MS) analysis of uterine myomas. The identified proteins were subjected to a gene ontology analysis to generate biological functions, molecular processes, and protein networks that were relevant to the uploaded dataset. Pathway-based analysis was an effective approach to investigate the molecular mechanisms underlying the disease and to create biological hypotheses about regulation of our proteins including the identification of upstream regulators and main protein nodes. Moreover, proteomic and in silico data were combined with immunohistochemistry and western blotting to identify a group of proteins representative of some selected pathways, with a dysregulated expression in myoma, pseudocapsule, and normal myometrium samples. Based on these results, we confirmed the over-expression of extracellular matrix components, and estrogen and progesterone receptors in uterine myomas, and proposed biological networks, canonical pathways and functions that may be relevant to the pathophysiology of this tumor.