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Emission of ionizing radiation (IR) in the environment is a natural phenomenon which can be enhanced by human activities. Ecosystems are then chronically exposed to IR. But environmental risk assessment of chronic exposure suffers from a lack of knowledge. Extrapolation of data from acute to chronic exposure is not always relevant, and can lead to uncertainties as effects could be different between the two irradiation modes, especially regarding reproduction endpoint, which is an ecologically relevant parameter. In the present study, we decided to refine the understanding of the molecular mechanisms involved in response to acute and chronic γ-irradiation by a global proteome label free LC-MS/MS analysis. C. elegans were exposed to 3 common cumulated radiation doses for acute or chronic exposure condition and global modification of the proteome was studied. This analysis of protein expression has demonstrated the modulation of proteins involved in regulatory biological processes such as lipid transport, DNA replication, germ cell development, apoptosis, ion transport, cuticle development, and aging at lower doses than those for which individual effects on reproduction have been previously observed. Thus, these proteins could constitute early and sensitive markers of radio-induced reprotoxicity; more specifically HAT-1, RPS-19 in acute and VIT-3 for chronic conditions that are expressed in a dose-dependent manner. Finally, to focus on reproduction process, this analysis showed either repression or overexpression of 12 common proteins in organisms exposed to acute or chronic irradiation, respectively. These proteins include the vitellogenin cluster notably involved in lipid transport and oocyte maturation and proteins involved in cuticle development and molting i.e. COL-14, GLF-1, NOAH-1, NOAH-2, ACN-1. These results show that protein expression modulation is a sensitive and predictive marker of radio-induced reproductive effects, but also highlight limitation of data extrapolation from acute to chronic exposure for environmental risk assessment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cell polarity is a vital biological process involved in the building, maintenance and normal functioning of tissues in invertebrates and vertebrates. Unsurprisingly, molecular defects affecting polarity organization and functions have a strong impact on tissue homeostasis, embryonic development and adult life, and may directly or indirectly lead to diseases. Genetic studies have demonstrated the causative effect of several polarity genes in diseases; however, much remains to be clarified before a comprehensive view of the molecular organization and regulation of the protein networks associated with polarity proteins is obtained. This challenge can be approached head-on using proteomics to identify protein complexes involved in cell polarity and their modifications in a spatio-temporal manner. We review the fundamental basics of mass spectrometry techniques and provide an in-depth analysis of how mass spectrometry has been instrumental in understanding the complex and dynamic nature of some cell polarity networks at the tissue (apico-basal and planar cell polarities) and cellular (cell migration, ciliogenesis) levels, with the fine dissection of the interconnections between prototypic cell polarity proteins and signal transduction cascades in normal and pathological situations. This review primarily focuses on epithelial structures which are the fundamental building blocks for most metazoan tissues, used as the archetypal model to study cellular polarity. This field offers broad perspectives thanks to the ever-increasing sensitivity of mass spectrometry and its use in combination with recently developed molecular strategies able to probe in situ proteomic networks., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The O-linked-N-acetyl-d-glucosaminylation (O-GlcNAcylation) modulates numerous aspects of cellular processes. Akin to phosphorylation, O-GlcNAcylation is highly dynamic, reversible, and responds rapidly to extracellular demand. Despite the absolute necessity to determine post-translational sites to fully understand the role of O-GlcNAcylation, it remains a high challenge for the major reason that unmodified proteins are in excess comparing to the O-GlcNAcylated ones. Based on a click chemistry approach, O-GlcNAcylated proteins were labelled with azido-GalNAc and coupled to agarose beads. The proteome extracted from C2C12 myotubes was submitted to an intensive fractionation prior to azide-alkyne click chemistry. This combination of fractionation and click chemistry is a powerful methodology to map O-GlcNAc sites; indeed, 342 proteins were identified through the identification of 620 peptides containing one or more O-GlcNAc sites. We localized O-GlcNAc sites on proteins involved in signalling pathways or in protein modification, as well as structural proteins. Considering the recent role of O-GlcNAcylation in the modulation of sarcomere morphometry and interaction between key structural protein, we focused on proteins involved in the cytoarchitecture of skeletal muscle cells. In particular, several O-GlcNAc sites were located into protein-protein interaction domains, suggesting that O-GlcNAcylation could be strongly involved in the organization and reorganization of sarcomere and myofibrils., Significance: O-GlcNAcylation is an atypical glycosylation involved in the regulation of almost all if not all cellular processes, but its precise role remains sometimes obscure because of the ignorance of the O-GlcNAc site localization; thus, it remains indispensable to precisely map the O-GlcNAcylated sites to fully understand the role of O-GlcNAcylation on a given protein. For this purpose, we combined extensive fractionation of skeletal muscle cells proteome with click chemistry to map O-GlcNAc sites without an a priori consideration. A total of 620 peptides containing one or more O-GlcNAc sites were identified; interestingly, several of them belong to low expressed proteins, in particular proteins involved in signalling pathways. We also focused on structural proteins in view of recent data supporting the role of O-GlcNAcylation in the modulation of sarcomere cytoarchitecture; importantly, some of the O-GlcNAc sites were mapped into protein-protein interaction domains, reinforcing the involvement of O-GlcNAcylation in the organization and reorganization of sarcomere, and in larger extent, of myofibrils., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Unlabelled: Colorectal cancer (CRC) remains a major cause of cancer related-death in developed countries. The mortality risk is correlated with the stage of CRC determined at the primary diagnosis and early diagnosis is associated with enhanced survival rate. Currently, only faecal occult blood tests are used to screen for CRC. Consequently, there is an incentive to identify specific markers of CRC. We used quantitative proteomic analysis of serum samples to characterize protein profiles in adenoma, CRC and healthy control samples. We identified 89 distinct proteins modulated between normal, colorectal adenoma and carcinoma patients. This list emphasizes proteins involved in enzyme regulator activities and in particular the serpin family. In serum samples, protein profiles of three members of the serpin family (SERPINA1, SERPINA3 and SERPINC1) were confirmed by ELISA assays. We obtained sensitivity/specificity values of 95%/95% for both SERPINA1 and SERPINC1, and 95%/55% for SERPINA3. This study supports the idea that serum proteins can discriminate adenoma and CRC patients from unaffected patients and reveals a panel of regulated proteins that might be useful for selecting patients for colonoscopy. By evaluating SERPINA1, SERPINA3 and SERPINC1, we highlight the potential role of the serpin family during the development and progression of CRC., Significance: Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world. However, very few CRC biomarkers have satisfactory sensitivity and specificity in clinical practice. To the best of our knowledge our study is the first to profile sera proteomes between adenoma, CRC and healthy patients. We report a comprehensive list of proteins that may be used as early diagnostic biomarkers of CRC. It is noteworthy that 17% of these modulated proteins have been previously described as candidate biomarkers in CRC. Enzyme regulator activity was found to be the main molecular function among these proteins and, in particular, there was an enrichment of members of the serpin family. The subsequent verification on a new cohort by ELISA demonstrates that these serpins could be useful to discriminate healthy from colorectal carcinoma patients with a high sensitivity and specificity. The combination of these biomarkers should increase predictive powers of CRC diagnosis. The remaining candidates form a reserve for further evaluation of additional biomarkers for CRC diagnosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity® analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein footprinting is a new methodology that is based on probing, typically with the use of MS, of reactivity of different amino acid residues to a modifying reagent. Data thus obtained allow one to make inferences about protein conformations and their intermolecular interactions. Most of the protein footprinting studies so far have been performed on individual proteins in vitro. We explore whether a similar approach is possible with the proteins inside of living cells, employing dimethylsulfate (DMS), a reagent widely used for the in vivo footprinting of nucleic acids. DMS can induce methylation of the lysine, histidine and glutamate residues on proteins. Using models of the histone H2B/H2AZ heterodimer assembled in vitro and from chromatin treated in vivo, we show that the methylation by deuterated DMS allows one to distinguish the accessibility of a particular residue in and out of the protein's environmental/structural context. The detection of changes in protein conformations or their interactions in vivo can provide a new approach to the identification of proteins involved in various intracellular pathways and help in the search for perspective drug targets and biomarkers of diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Wnt/beta-catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying beta-catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2-D DIGE combined with MS, in mice with liver-specific deletion of Apc resulting in acute activation of beta-catenin signaling (Apc(KOliv) mice). We identified 94 protein spots showing differential expression between mutant Apc(KOliv) and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that beta-catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the "Warburg effect". Imaging with (18)F-fluoro-2-deoxy-D-glucose-positron emission tomography suggests that the specific metabolic reprogramming induced by beta-catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro-2-deoxy-D-glucose-positron emission tomography imaging.