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The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any

Proper functioning of pancreatic β-cells is a crucial for glucose homeostasis control, and therefore a main problem regarding type 2 diabetes onset and evolution. The ability of β-cells to proliferate upon certain stimuli, such as elevated glucose concentration, is an essential property to overpass a major problem of the pathology: the decrease of β-cell mass leading to a lack of insulin production. However, high glucose concentrations are also an inducer of β-cell dysfunction, when proliferation become unable to overcome insulin demand. The control of β-cell proliferation could represent an interesting target for the development of therapeutic molecules for type 2 diabetes treatment. To get new insights on β-cell replication, we investigated the modulation of nuclear proteins of INS-1E cells submitted to medium and high glucose concentrations for 24 h. Indeed, the nucleus should contain proteins responsible of proliferation-related events. The SILAC approach allowed us identifying 24 nuclear proteins whose expressions were modified by chronic high glucose. A wide Downstream Effects Analysis assigned the majority of the differentially expressed proteins to functions such as proliferation and cell cycle. Interestingly, our study linked for the first time the increase of expression of the 6 MCM components to glucose-induced stimulation in β-cells. Biological significance The current study represents a progress in the understanding of glucose-induced proliferation mechanisms in β-cells. We applied the SILAC strategy to INS-1E cells cultivated with medium or high glucose concentrations for 24 h, and we targeted nuclear proteins which have a central role in proliferation-related mechanisms. It allows quantifying 24 nuclear proteins, which are regulated by high glucose exposure. The vast majority of them are shown to be related to proliferation and cell cycle. We describe here for the first time than the 6 proteins of the MCM complex are involved in glucose-mediated proliferation in β-cells.

UNLABELLED In the present study we performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Eight hundred and twenty seven proteins were identified most of them being associated to granules and to the granule's secretory machinery. Functional pathway analysis revealed 30 pathways including the major histocompatibility complex class 1 (MHC I) presenting antigen pathway. This pathway was of particular interest for its potential interrelation between platelets and the immune system. Key proteins belonging to this metabolic route such as ß 2 microglobulin 26S protease regulatory subunit 10B from the proteasome and proteins 1 and 2 of the transporter associated with antigen processing were shown to co localize with von Willebrand factor in resting platelets and to be located on the plasma membrane when platelets were activated. Key proteins of the MHC1 antigen presenting pathway are located in platelet alpha granules. These results suggest a possible functional role of platelet granules in platelet related immune modulation. BIOLOGICAL SIGNIFICANCE In this study we described the largest dataset related to platelet granule proteins. We performed a functional pathway analysis that evidenced several expected granule related pathways. We also highlighted the "Antigen processing and presentation" pathway that has drawn our attention. Using immunofluorescence technique we confirmed the presence of several key proteins for antigen presentation in platelet granules. This study suggests a putative functional role of MHC1 and platelet granules in the immune modulation.

Type 2 diabetes is a worldwide disease reaching epidemic dimensions. The rapid progression of the disease urgently calls for both a broader and deeper understanding of its pathophysiology. In line with this statement, the Human Diabetes Proteome Project (HDPP) was officially launched at the 11th HUPO meeting in Boston, 2012. A special session was dedicated to this new initiative, gathering experts in the main topics related to diabetes and its associated complications. Key issues were debated with a focus on how deranged circulating glucose and free fatty acids induce dysfunction. It has been decided that HDPP will therefore focus on studying the early stages of diabetes that lead to abnormal glucose and lipid levels. The initiative will initially focused on islets of Langerhans, insulin-producing cell lines, and blood human samples from diabetes-related cohorts. In subsequent stages HDPP will investigate target tissues in which glucose and lipids could promote protein dysfunctions. Omics-rooted systems approaches enhanced by bioinformatics will be deployed to unravel effects of lipids and glucose triggering diabetes initiation and progression. A first milestone has been defined for the 12th HUPO meeting in Yokohama, 2013: the 1000 diabetes-associated protein (the 1000-HDPP) database, i.e. a freely available internet resource ( www.HDPP.info ) of more than 1000 proteins with links to their corresponding proteotypic peptides, affinity reagents and protein-specific biological/biomedical information.

Insulin secretory granules (ISGs) are pivotal organelles of pancreatic ß-cells and represent a key participant to glucose homeostasis. Indeed, insulin is packed and processed within these vesicles before its release by exocytosis. It is therefore crucial to acquire qualitative and quantitative data on the ISG proteome, in order to increase our knowledge on ISG biogenesis, maturation and exocytosis. Despites efforts made in the past years, the coverage of the ISG proteome is still incomplete and comprises many potential protein contaminants most likely coming from suboptimal sample preparations. We developed here a 3-step gradient purification procedure combined to Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to further characterize the ISG protein content. Our results allowed to build three complementary proteomes containing 1/ proteins which are enriched in mature ISGs, 2/ proteins sharing multiple localizations including ISGs, and finally 3/ proteins sorted out from immature ISGs and/or co-purifying contaminants. As a proof of concept, the ProSAAS, a neuronal protein found in ISGs was further characterized and its granular localization proved. ProSAAS might represent a novel potential target allowing to better understand the defaults in insulin processing and secretion observed during type 2 diabetes progression. This article is part of a special issue entitled: Translational Proteomics.

Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.

Chronic hyperglycaemia is one of the main characteristics of a diabetic state. This is also the first cause of diabetic complications. However, it is now generally accepted that glucotoxicity also participates in the worsening of type 2 diabetes, by affecting the secretion of beta-cells. So far, different mechanisms have been proposed to explain the adverse effects of chronic hyperglycaemia. One of them suggests that the modulation of expression of several key proteins during a hyperglycaemia state, may explain the toxic effect of glucotoxicity. Therefore, proteomic analysis of biological samples represents an interesting method to study the effect of chronic hyperglycaemia on protein expression. The discovery of new proteins for which the expression could be modulated by chronic hyperglycaemia may probably help to better understand the mechanisms underlying glucotoxicity. In this review, we will first present an introduction of the different mechanisms known to be involved in the control of glucose homeostasis and in the development of glucotoxicity. In a second part, some proteomic data linked with the effect of glucotoxicity in pancreas, pancreatic islets and beta-cells will be presented and discussed.

Introduction: Early changes in protein expression and signaling mechanisms (MAPKs cascade or the cAMP pathway) in acute pancreatitis (AP) play important roles in the further development of the disease. Therefore, proteomic analyses of pancreatic subcellular fractions might be very useful for deciphering AP at the molecular level. Aims:WereportedhereaTandemMassTag(TMT)proteomicsanalysisof the soluble fraction of the pancreas during the early phase of experimental AP and under MAPKs or type IV phosphodiesterase (PDEIV) inhibition. Materials & methods: The early phase of AP was induced in rats by two subcutaneous injections of 20 mg of cerulein (Cer)/kg body weight at hourly intervals. PDEIV and the MAPKs cascade were inhibited by rolipram and SP600125 or PD98059, respectively, before the induction of AP. TMT 6 analysis weredesignedtocharacterizeeachexperimentalconditioninacross-sectional study. Changes in protein expression were validated by immunoblotting. Results: Out of the around 540 identified proteins, 298 were differentially expressed during the early phase of AP (Isobar, p < 0.05). Also, we identified 27 (SP600125), 39 (PD98059) or 38 (rolipram) proteins differentially regulated by the distinct pretreatments compared to the Cer class. These proteins are mainly involved in protein and membrane trafficking, apoptosis, mitochondrial function, calcium signaling and cytoskeleton impairment. We are currently characterizing novel candidates that might be relevant to AP pathogenesis. Conclusion: These data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP and might unmask new potential biomarkers of the disease.