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Exosomal proteome from mouse CSF

ABSTRACT: Exosomes were isolated from the mouse CSF using total exosome isolation kit (Life Technologies) from 0h and 6h LPS treated mice. These exosomes were separated on the RP-HPLC in to 20 fractions and analysed on the Qexactive.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Sriram Balusu

LAB HEAD: Kris Gevaert

PROVIDER: PXD001346 | Pride | 2016-09-27

REPOSITORIES: Pride

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Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles.

Balusu Sriram S Van Wonterghem Elien E De Rycke Riet R Raemdonck Koen K Stremersch Stephan S Gevaert Kris K Brkic Marjana M Demeestere Delphine D Vanhooren Valerie V Hendrix An A Libert Claude C Vandenbroucke Roosmarijn E RE

EMBO molecular medicine 20161004 10

Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood-brain communication. Systemic inflammation induced an increase in EVs and associated pro-inflammatory miRNAs, including miR-146a and miR-155, in the CSF Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS-stimulated primary CPE cells and choroid pl ...[more]

PMID: 27596437

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Choroid plexus proteome with and without LPS

Project description:Choroid plexus was isolated and pooled from the 5 mice after the perfusion with bromo phenol blue from 0 h and 6 h LPS treated mice. Proteins were isolated and trypsin digested and labelled with the NHS-porp (0h –LPS and 6h +LPS). Pre-fractionated using RP-HPLC and analysed on the Qexactive.

2016-09-27 | PXD001347 | Pride

Novel surface functionalized superparamagnetic nanoparticles with engineered cellular uptake as a means for intracellular omics

Project description:Superparamagnetic nanoparticles (SPMNPs) are appealing for use in organelle isolation strategies. Yet, this potential remains largely unexplored because thus far research has focused on either physicochemical design or on their application in micron-sized beads. For their use in life sciences, the biocompatibility of SPMNPs goes beyond their chemical composition and shape, and features like their size and more importantly their surface properties are becoming more important to exploit extra- and intracellular interactions. Here we introduce thermal decomposition to manufacture iron oxide based SPMNPs (Ø10nm) and demonstrate how different surface functionalizations can lead to different types of cellular interactions. Cationic aminolipid-coated SPMNPs reside surprisingly strong at the outer cell surface. In contrast, anionic dimercaptosuccinic acid-coated SPMNPs are efficiently internalized and accumulate in a time-dependent manner in endosomal and lysosomal populations. These features allowed us to establish a standardized magnetic isolation procedure to selectively isolate plasma membranes and intracellular late endosomes/lysosomes with high yields and purities as consolidated by biochemical and ultrastructural analyses. Subsequent quantitative and qualitative proteome analysis underpins the overall high enrichment for hydrophobic (membrane) proteins as well as plasma membrane and lysosomal constituents in the respective purified fractions. This nano based technology provides therefore a breakthrough in the field of subcellular ‘omics’ as it allows the identification of subtle alterations in the biomolecular composition of different SPMNP-isolated compartments that would be otherwise not detected in total cell or tissue analysis.

2017-02-06 | PXD001225 | Pride

Thylakoid membranes from Arabidopsis LC-MSMS

Project description:The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps, with a single conserved -sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants, which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labelling, of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered

2017-06-21 | PXD006734 | Pride

Targeting the lineage-specific long non-coding RNA LINC01212 as an effective anti-melanoma therapeutic strategy

Project description:Focal amplifications of 3p13-3p14 occur in about 10% of melanoma and are associated with poor prognosis. The melanoma-specific oncogene MITF resides at the epicenter of this amplicon1 . However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA gene LINC01212 is consistently cogained with MITF. In addition to being amplified, LINC01212 is a target of the lineage-specific transcription factor SOX10 and, consequently, it is expressed in more than 90% of human melanomas, but not in normal adult tissues. Whereas exogenous LINC01212 functions in trans to increase melanoma clonogenic potential, LINC01212 knock-down drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state, BRAF, NRAS or TP53 status and increases their sensitivity to MAPK-targeting therapeutics both in vitro and in Patient-Derived Xenograft (PDX) models. Mechanistically, LINC01212 interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that targeting the lineage addiction oncogene LINC01212, especially in combination with BRAFV600Einhibitors, disrupts vital mitochondrial functions in a cancer-cell specific manner and is therefore expected to deliver highly effective and tissuerestricted anti-melanoma therapeutic responses.

2016-01-13 | PXD002565 | Pride

Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes

Project description:Type 1 and type 2 diabetes (T1D and T2D) share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, while beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alter the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role for beta cell fragility in genetic predisposition to diabetes.

2016-02-24 | PXD000859 | Pride

Cell-intrinsic regulation of interneuron migration controls cortical neurogenesis

Project description:Interneurons navigate along multiple tangential paths to settle into appropriate cortical layer. They undergo saltatory migration, which is paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. However, it remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes deglutamylation, is a pivotal regulator of pausing of cortical interneurons. Moreover, we show that pausing during migration controls the flow of interneurons invading the cortex by generating heterogeinity in movement at the population level. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also secondarily impairs the generation of age-matched upper layer projection neurons.

2018-02-14 | PXD006397 | Pride

The dynamics of protein-bound methionine oxidation events in Arabidopsis thaliana during oxidative stress

Project description:Reactive oxygen species such as hydrogen peroxide can modify proteins via direct oxidation of their sulfur-containing amino acids, cysteine and methionine. Methionine oxidation, studied here, is a reversible posttranslational modification, which is increasingly suggested as a mechanism by which proteins perceive oxidative stress and function in redox signalling. Identification of proteins with oxidized methionines is a first prerequisite towards understanding the functional effect of methionine oxidation on proteins and the biological processes in which they are involved. Here, we describe a proteome-wide study of in vivo protein-bound methionine oxidation in plants upon oxidative stress, using Arabidopsis thaliana catalase 2 knock-out plants as a model system. We identified approximately 500 sites of oxidation in about 400 proteins, and quantified the differences in oxidation between wild-type and catalase 2 knock-out plants. Further, by sampling over time, we mapped the dynamics of methionine oxidation and gained new insights into this complex and dynamic landscape of the part of the plant proteome that is sculpted by oxidative stress.

2015-03-24 | PXD001286 | Pride

development of the cell wall in alfalfa

Project description:In this study, the cell wall proteomes of 3 different regions of alfalfa stems were compared. These three regions correspond to three phases in the stem development. The sequential extraction of cell wall proteins with CaCl2, EGTA and LiCl-complemented buffers was combined with a gel-based proteome approach and multivariate analysis. Although the highest similarities were observed between the apical and intermediate stem regions, the three proteome patterns are characteristic for each region. In the basal stem region, proteins that bind carbohydrates and have proteolytic activity, as well as enzymes involved in glycan remobilization, accumulate. Beta-amylase and ferritin likewise accumulate more in the basal stem segment. Therefore remobilization of nutrients appears to be an important process in the oldest stem segment. The intermediate and the apical region are sites of cell wall polymer remodelling, as suggested by the high abundance of proteins involved in the remodelling of the cell wall such as xyloglucan endoglucosylase, polygalacturonase non-catalytic subunit or beta-galactosidase. The most striking change between the different stem parts is however the strong accumulation of a DUF642-conserved domain containing protein in the apical region of the stem which suggests a particular role of this protein during the early development of stem tissues.

2019-11-12 | PXD010666 | Pride

A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

Project description:Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

2023-09-22 | PXD044681 | Pride

Anisakis simplex L3 proteome stability under heat sterilization condition

Project description:Protein extracts from Anisakis simplex L3 larvae were autoclaved at 121C for 60 min. Subsequently, prepared samples were analyzed in LC-MS/MS to identify autoclave-resistant proteins (also allergens).

2021-09-09 | PXD018059 | Pride

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