The response to fasting and refeeding reveals functional regulation of lipoprotein lipase proteoforms.

Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.

Shotgun proteomics to characterize wastewater proteins.

Classically, the characterization of wastewater components has been restricted to the measurement of indirect parameters (chemical and biological oxygen demand, total nitrogen) and small molecules of interest in epidemiology or for environmental control. Despite the fact that metaproteomics has provided important knowledge about the microbial communities in these waters, practically nothing is known about other non-microbial proteins transported in the wastewater. The method described here has allowed us to perform a large-scale characterization of the wastewater proteome. Wastewater protein profiles have shown to be very different in different collection sites probably reflecting their human population and industrial activities. We believe that wastewater proteomics is opening the doors to the discovery of new environmental and health biomarkers and the development of new, more effective monitoring devices for issues like monitorization of population health, pest control, or control of industry discharges. The method developed is relatively simple and combines procedures for the separation of the soluble and particulate fractions of wastewater and their concentration, and conventional shotgun proteomics using high-resolution mass spectrometry for protein identification. •Unprecedented method for wastewater proteome characterization.•Proteins as new potential biomarkers for sewage chemical-information mining, wastewater epidemiology and environmental monitoring.•Wastewater protein profiles reflect human and industrial activities.

Sewage Protein Information Mining: Discovery of Large Biomolecules as Biomarkers of Population and Industrial Activities.

Wastewater-based epidemiology has been revealed as a powerful approach for surveying the health and lifestyle of a population. In this context, proteins have been proposed as potential biomarkers that complement the information provided by currently available methods. However, little is known about the range of molecular species and dynamics of proteins in wastewater and the information hidden in these protein profiles is still to be uncovered. In this study, we investigated the protein composition of wastewater from 10 municipalities in Catalonia with diverse populations and industrial activities at three different times of the year. The soluble fraction of this material was analyzed using liquid chromatography high-resolution tandem mass spectrometry using a shotgun proteomics approach. The complete proteomic profile, distribution among different organisms, and semiquantitative analysis of the main constituents are described. Excreta (urine and feces) from humans, and blood and other residues from livestock were identified as the two main protein sources. Our findings provide new insights into the characterization of wastewater proteomics that allow for the proposal of specific bioindicators for wastewater-based environmental monitoring. This includes human and animal population monitoring, most notably for rodent pest control (immunoglobulins (Igs) and amylases) and livestock processing industry monitoring (albumins).

Diabetes mellitus and aortic stenosis head to head: toward personalized medicine in patients with both pathologies.

Diabetes mellitus (DM) and calcific aortic stenosis (CAS) are common morbidities in the elderly, which are both chronic, progressive and often concomitant diseases. Several studies revealed that DM increases the risk of developing severe CAS, yet clear information about the relationship between both these diseases and the influence of DM on the progression of CAS is currently lacking. To evaluate the effect of DM on aortic valves and on the process of calcification, and to achieve better patient management in daily clinical practice, we analysed calcified and noncalcified valve tissue from patients with severe CAS, with or without DM. A proteomic strategy using isobaric tags was adopted and the plasma concentrations of nine proteins were studied using 3 orthogonal methods and in a separate cell model. The differentially expressed proteins identified are implicated in biological processes like endopeptidase activity, lipid metabolism, coagulation, and fibrinolysis. The results obtained provide evidence that DM provokes changes in the proteome of aortic valves, affecting valve calcification. This finding may help enhance our understanding of the pathogenesis of CAS and how DM affects the evolution of this condition, an important step in identifying targets to personalize the treatment of these patients.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.

Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements.

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.

Single-cell RNA-seq-based proteogenomics identifies glioblastoma-specific transposable elements encoding HLA-I-presented peptides.

We analyze transposable elements (TEs) in glioblastoma (GBM) patients using a proteogenomic pipeline that combines single-cell transcriptomics, bulk RNA sequencing (RNA-seq) samples from tumors and healthy-tissue cohorts, and immunopeptidomic samples. We thus identify 370 human leukocyte antigen (HLA)-I-bound peptides encoded by TEs differentially expressed in GBM. Some of the peptides are encoded by repeat sequences from intact open reading frames (ORFs) present in up to several hundred TEs from recent long interspersed nuclear element (LINE)-1, long terminal repeat (LTR), and SVA subfamilies. Other HLA-I-bound peptides are encoded by single copies of TEs from old subfamilies that are expressed recurrently in GBM tumors and not expressed, or very infrequently and at low levels, in healthy tissues (including brain). These peptide-coding, GBM-specific, highly recurrent TEs represent potential tumor-specific targets for cancer immunotherapies.

Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA. METHODS: HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry. RESULTS: We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity. CONCLUSIONS: We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.

Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Inflammatory capacity of exosomes released in the early stages of acute pancreatitis predicts the severity of the disease.

As acute pancreatitis progresses to the severe form, a life-threatening systemic inflammation is triggered. Although the mechanisms involved in this process are not yet well understood, it has been proposed that circulating exosomes may be involved in the progression of inflammation from the pancreas to distant organs. Here, the inflammatory capacity and protein profile of plasma exosomes obtained during the first 24 h of hospitalization of patients diagnosed with acute pancreatitis were characterized and compared with the final severity of the disease. We found that the final severity of the disease strongly correlates with the inflammatory capacity of exosomes in the early stages of acute pancreatitis. Exosomes isolated from patients with mild pancreatitis had no effect on macrophages, while exosomes isolated from patients with severe pancreatitis triggered NFκB activation, TNFα and IL1ß expression, and free radical generation. To delve deeper into the mechanism involved, we performed a proteomic analysis of the different exosomes that allowed us to identify different groups of proteins whose concentration was also correlated with the clinical classification of pancreatitis. In particular, an increase in the amount of S100A8 and S100A9 carried by exosomes of severe pancreatitis suggests that the mechanism of action of exosomes is mediated by the effect of these proteins on NADPH oxidase. This enzyme is activated by S100A8/S100A9, thus generating free radicals and promoting an inflammatory response. Along these lines, we observed that inhibition of this enzyme abolished all the pro-inflammatory effects of exosomes from severe pancreatitis. All this suggests that the systemic effects, and therefore the final severity of acute pancreatitis, are determined by the content of circulating exosomes generated in the early hours of the process. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Epstein-Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report.

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa.

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H2O2 efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca2+-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca2+- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach.

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

Manganese-induced neurotoxicity in cerebellar granule neurons due to perturbation of cell network pathways with potential implications for neurodegenerative disorders.

Manganese (Mn) is essential for living organisms, playing an important role in nervous system function. Nevertheless, chronic and/or acute exposure to this metal, especially during early life stages, can lead to neurotoxicity and dementia by unclear mechanisms. Thus, based on previous works of our group with yeast and zebrafish, we hypothesized that the mechanisms mediating manganese-induced neurotoxicity can be associated with the alteration of protein metabolism. These mechanisms may also depend on the chemical speciation of manganese. Therefore, the current study aimed at investigating the mechanisms mediating the toxic effects of manganese in primary cultures of cerebellar granule neurons (CGNs). By exposing cultured CGNs to different chemical species of manganese ([[2-[(dithiocarboxy)amino]ethyl]carbamodithioato]](2-)-kS,kS']manganese, named maneb (MB), and [[1,2-ethanediylbis[carbamodithioato]](2-)]manganese mixture with [[1,2-ethanediylbis[carbamodithioato]](2-)]zinc, named mancozeb (MZ), and manganese chloride (MnCl2)), and using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, we observed that both MB and MZ induced similar cytotoxicity (LC50∼ 7-9 µM), which was higher than that of MnCl2 (LC50∼ 27 µM). Subsequently, we applied systems biology approaches, including metallomics, proteomics, gene expression and bioinformatics, and revealed that independent of chemical speciation, for non-cytotoxic concentrations (0.3-3 µM), Mn-induced neurotoxicity in CGNs is associated with metal dyshomeostasis and impaired protein metabolism. In this way, we verified that MB induced more post-translational alterations than MnCl2, which can be a plausible explanation for cytotoxic differences between both chemical species. The metabolism of proteins is one of the most energy consuming cellular processes and its impairment appears to be a key event of some cellular stress processes reported separately in other studies such as cell cycle arrest, energy impairment, cell signaling, excitotoxicity, immune response, potential protein accumulation and apoptosis. Interestingly, we verified that Mn-induced neurotoxicity shares pathways associated with the development of Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, and Parkinson's disease. This has been observed in baker's yeast and zebrafish suggesting that the mode of action of Mn may be evolutionarily conserved.

Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome.

Defects in mitochondrial function lead to severe neuromuscular orphan pathologies known as mitochondrial disease. Among them, Leigh Syndrome is the most common pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death. Mitochondrial diseases are characterized by a marked anatomical and cellular specificity. However, the molecular determinants for this susceptibility are currently unknown, hindering the efforts to find an effective treatment. Due to the complex crosstalk between mitochondria and their supporting cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us to assess the proteome and acetylome of a subset of susceptible neurons in a well characterized model recapitulating the human disease. Our results show a marked reduction of complex I N-module subunit abundance and an increase in the levels of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins such as PKCδ, and the complement subcomponent C1Q were also increased in Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to decreased PDH activity. We provide novel insight on the pathways involved in mitochondrial disease, which could underlie potential therapeutic approaches for these pathologies.

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade.

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.

Platelet membrane lipid rafts protein composition varies following GPVI and CLEC-2 receptors activation.

Lipid rafts are membrane microdomains that have been proposed to play an important role in several platelet-signalling cascades, including those mediated by the receptors Glycoprotein VI (GPVI), and C-type lectin domain family 1 member B (CLEC-2), both involved in thrombus formation. We have performed a LC-MS/MS proteomic analysis of lipid rafts isolated from platelets activated through GPVI and CLEC-2 as well as from resting platelets. Our aim was to determine the magnitude of changes in lipid rafts protein composition and to elucidate the relevance of these alterations in platelet function. A number of relevant signalling proteins were found enriched in lipid rafts following platelet activation (such as the tyrosine protein kinases Fyn, Lyn and Yes; the G proteins G(i) and G(z); and cAMP protein kinase). Interestingly, our results indicate that the relative enrichment of lipid rafts in these signalling proteins may not be a consequence of protein translocation to these domains upon platelet stimulation, but the result of a massive loss in cytoskeletal proteins after platelet activation. Thus, this study may help to better understand the effects of platelet activation in the reorganization of lipid rafts and set the basis for further proteomic studies of these membrane microdomains in platelets. SIGNIFICANCE: We performed the first proteomic comparative analysis of lipid rafts- protein composition in platelets activated through GPVI and CLEC-2 receptors and in resting state. We identified a number of signalling proteins essential for platelet activation relatively enriched in platelets activated through both receptors, and we show that lipid rafts reorganization upon platelet activation leads to a loss in cytoskeletal proteins, highly associated to these domains in resting platelets.

TCellXTalk facilitates the detection of co-modified peptides for the study of protein post-translational modification cross-talk in T cells.

MOTIVATION: Protein function is regulated by post-translational modifications (PTMs) that may act individually or interact with others in a phenomenon termed PTM cross-talk. Multiple databases have been dedicated to PTMs, including recent initiatives oriented towards the in silico prediction of PTM interactions. The study of PTM cross-talk ultimately requires experimental evidence about whether certain PTMs coexist in a single protein molecule. However, available resources do not assist researchers in the experimental detection of co-modified peptides. RESULTS: Herein, we present TCellXTalk, a comprehensive database of phosphorylation, ubiquitination and acetylation sites in human T cells that supports the experimental detection of co-modified peptides using targeted or directed mass spectrometry. We demonstrate the efficacy of TCellXTalk and the strategy presented here in a proof of concept experiment that enabled the identification and quantification of 15 co-modified (phosphorylated and ubiquitinated) peptides from CD3 proteins of the T-cell receptor complex. To our knowledge, these are the first co-modified peptide sequences described in this widely studied cell type. Furthermore, quantitative data showed distinct dynamics for co-modified peptides upon T cell activation, demonstrating differential regulation of co-occurring PTMs in this biological context. Overall, TCellXTalk facilitates the experimental detection of co-modified peptides in human T cells and puts forward a novel and generic strategy for the study of PTM cross-talk. AVAILABILITY AND IMPLEMENTATION: TCellXTalk is available at https://www.tcellxtalk.org. Source Code is available at https://bitbucket.org/lp-csic-uab/tcellxtalk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients.

Plasma-derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs-derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean-matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D-DIGE)-based study, and the other one for a label free LC-MS/MS-based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty-two and 23 differentially regulated features are detected from 2D-DIGE and label free LC-MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D-western-blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma-derived-EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.