Proteomics of motor enriched fraction (ATP releasate) from Dictyostelium discoideum
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Dictyostelium Discoideum (slime Mold)
SUBMITTER: 
Apurwa Meghna
LAB HEAD: Roop Mallik
PROVIDER: PXD051777 | Pride | 2024-05-16
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DICTY-peptides.xlsx | Xlsx | |||
| DICTY-proteins.xlsx | Xlsx | |||
| DICTY.msf | Msf | |||
| DICTY.mzML | Mzml | |||
| DICTY.mzid.gz | Mzid |
Items per page: 5 1 - 5 of 23 |
Similar Datasets
Project description:Physiological stimuli such as thrombin or pathological stimuli such as lysophosphatidic acid (LPA) activate platelets. The activated platelets bind to monocyte through P-selectin-PSGL-1 interactions, but also release the contents of their granules, which were defined as platelet releasate. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS has been viewed as a potential regulator of protein function. In previous study, we employed a model of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that been influenced by reversible cysteine oxidation. To gain better understanding of the redox regulation of monocyte in atherosclerosis, we now extend the modified biotin switch to quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite in the modified biotin-switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites further highlighted biological processes of monocyte transendothelial migration including integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes on human primary monocyte upon platelet releasate treatments. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Moreover, the production of ROS and the activation of LFA-1 in human primary monocyte induced by platelet releasate were independent of P-selectin-PSGL-1 interaction. The modified biotin switch assay proved to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD003365 | Pride
Project description:Physiological stimuli such as thrombin or pathological stimuli such as lysophosphatidic acid (LPA) activate platelets. The activated platelets bind to monocyte through P-selectin-PSGL-1 interactions, but also release the contents of their granules, which were defined as platelet releasate. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS has been viewed as a potential regulator of protein function. In previous study, we employed a model of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that been influenced by reversible cysteine oxidation. To gain better understanding of the redox regulation of monocyte in atherosclerosis, we now extend the modified biotin switch to quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite in the modified biotin-switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites further highlighted biological processes of monocyte transendothelial migration including integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes on human primary monocyte upon platelet releasate treatments. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Moreover, the production of ROS and the activation of LFA-1 in human primary monocyte induced by platelet releasate were independent of P-selectin-PSGL-1 interaction. The modified biotin switch assay proved to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD003363 | Pride
Project description:Upon activation, platelets release a multitude of soluble and vesicular signals, collectively termed the ‘platelet releasate’ (PR). This PR plays an important role in haemostasis, wound healing and the inflammatory response. We and others have used qualitative/quantitative proteomic approaches to characterise the PR; however, with reports of marked inter-individual variability, confident and reliable insights have been hindered. Here we aimed to provide a reproducible and quantifiable proteomic analysis and establish a 'core' human PR, as well as to assess its variability in healthy human pregnancy where increased platelet activation is observed. 896 proteins from thrombin-induced PRs were quantified across 32healthy donors, with 277 proteins forming a ‘core’ PR with low variability. All 277 core PR proteins were identified in pregnancy. Furthermore 69 proteins were found differentially released from platelets in healthy human pregnancy. In summary, the PR comprises a ‘core’ set of proteins between healthy individuals. This dynamic proteome significantly changes in physiologic and pathologic conditions. Thus, the PR is a barcode for the health status of an individual at a given time.
2018-06-25 | PXD009310 | Pride
Project description:Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions, but also release the contents of their granules, commonly called “platelet releasate”. It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
2016-10-10 | PXD004862 | Pride
Project description:Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the ‘platelet releasate’ (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. We undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1U/ml thrombin-induced PR from 13 acute coronary syndrome (ACS-STEMI) versus 14 stable angina pectoris patients using a tandem mass spectrometry approach. We identified differentially released platet proteins including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5) and fibronectin (FN1). Strikingly, all 9 differential proteins were associated with the GO cellular component term ‘extracellular vesicle’ and reduced levels of EVs were detected in plasma of ACS-STEMI patients. Network analysis revealed 3 PR proteins either reduced (F5; FN1) or absent (CLEC3B) in ACS-STEMI patients, which are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated signature highlights the possible basis of platelet dysfunction in ACS-STEMI and may prove useful for non-invasive risk assessment of the progression of coronary artery disease.
2020-06-05 | PXD009356 | Pride
Project description:Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets’ tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1 (POFUT1), or a novel POFUT, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
2024-02-15 | PXD045535 | Pride
Project description:Redox imbalance in cells, due to the accumulation of reactive oxygen species (ROS), has been implicated in the pathogenesis of several diseases, including atherosclerosis. However, the molecular details of redox regulation in atherosclerosis remain to be established.1 During the progression of atherosclerotic plaque, NADPH oxidase (NOX) that are expressed in vascular cells, particularly in phagocytic cells like monocytes, mainly contribute to the production of ROS.2 ROS with exogenous or endogenous sources is able to modify DNA, lipids to proteins. Cysteine residue in protein, despite being one of the least abundant amino acid residues, is a major target of ROS-mediated cell-signaling modulation3 The ROS-mediated regulation is accomplished through a variety of reversible and irreversible oxidative modifications on cysteines such as sulfenic acid, S-nitrosylation, S-glutathionylation, sulfonic acid. Among the reversible cysteine oxidative modifications, sulfenic acid has emerged as an important post-translational modification in proteins.4 Sulfenic acid, as the principle product of the reaction between a thiol and hydrogen peroxide, is a pivotal reactive intermediate under physiological and oxidative stress conditions. Several studies have identified the catalytic or regulatory sulfenic acid formation in proteins such as NADH peroxidase, peroxiredoxins, and protein tyrosine phosphatase.4 Pinpointing the precise location of sulfenic acid in proteins enables us to track down the reactive site where oxidation is initiated. However, the detection of sulfenic acid is challenging as sulfenic acids is highly reactive and in most situations they are rapidly converted back to the reduced state or further oxidized to a more stable state. Nonetheless, certain protein microenvironments, such as the presence of polar uncharged residues, particularly threonine, and the absence of the charged residues close to the sulfenic acid sites, promote sulfenic acid formation and stabilization.5 To conduct proteomic study of this significant oxidative modification, various analytical methods have been developed to enrich proteins or peptides bearing sulfenic acid.6 Launched by Jaffrey in 2001, the biotin switch assay has been extensively adapted to identify various cysteine modifications in complex biological samples.7 We previously combined the biotin switch assay with stable isotope labeling by amino acid in cell culture (SILAC) to quantify total reversible cysteine oxidation and applied it on an atherosclerotic model with human platelets and monocytic THP-1 cells. In the development of atherosclerosis, activated platele facilitates the recruitment of monocytes to the sites of inflamed vascular endothelium not only via P-selectin-PSGL-1 mediated direct binding, but also via releasing the contents of their granules, which are defined as platelet releasate (PR). We demonstrated that thrombin- or LPA- induced PR leads to ROS production in THP-1 cells which in turn alters fundamental biological processes like glycolysis.8 To further digging into the redox regulation mechanism in the atherosclerotic model, we extend the modified biotin switch assay to quantify sulfenic acid. The Eaton group has modified the biotin switch assay using m-arsenite to selectively reduce sulfenic acid.9 Here we combined SILAC and m-arsenite in the biotin switch assay and quantified more than 100 sulfenic acid sites in the model system. Bioinformatics analysis of these proteins with sulfenic acid modification underlined the involvement of integrin β2 in leukocytes transendothelium migration. LFA-1 (αLβ2) and Mac-1 (αMβ2) are two integrin β2 complexes that are most relevant to the migration of monocyte on the endothelial cells.10 We validated the activation of LFA-1 and Mac-1 in primary monocytes upon platelet releasate treatment. Moreover, the activation of LFA-1 and Mac-1 were shown to be independent of P-selectin-PSGL1 interaction on monocytes. In a word, analysis of sulfenic acid reveals a new angle of the crosstalk between platelet releasate and monocytes in the early stage of atherosclerosis.
2016-10-10 | PXD003354 | Pride
Project description:Microvascular Endothelial Cell RNA-Seq: Treatment with Platelet Releasate from COVID-19 & Control Donors
| PRJNA736408 | ENA
Project description:Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential. Microarray hybridizations (Agilent: Whole Human Genome Oligo Microarray; 41k unique probe) were carried out with 5 µg Cy3 labeled cRNA and 5 µg Cy5 labeled cRNA, both prepared from each sample.
2010-08-28 | E-GEOD-23851 | biostudies-arrayexpress
Project description:Multiple Sclerosis is an inflammatory neurodegenerative disease characterised by blood-brain barrier dysfunction and leukocyte infiltration into the central nervous system. Platelets are best known for their contributions to haemostasis, however upon activation platelets release an abundance of soluble and vesicular-associated proteins, termed the platelet releasate (PR). This milieu contains numerous inflammatory and vasoactive proteins which function to attract leukocytes and alter endothelial permeability. Here, we aimed to characterise the PR of Relapsing-Remitting multiple sclerosis (RRMS) patients, previously characterized with respect to thrombin generation dynamics compared to healthy controls. We carried out LFQ proteomic profiling of the PR from 15 RRMS and 19 aged-matched healthy controls. The PR proteome identified 9 proteins increased and 16 proteins decreased in RRMS patients.
2025-01-09 | PXD050479 | Pride