Proteomic profiling of subsets of NK cell-derived extracellular vesicles by LC-MS/MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Tuula Nyman
LAB HEAD: Tuula Nyman
PROVIDER: PXD034603 | Pride | 2022-09-09
REPOSITORIES: Pride
Dataset's files
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| EH3301.raw | Raw | |||
| EH3303.raw | Raw | |||
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| EH3310.raw | Raw |
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Project description:In this study we have compared the proteomic profile of subsets of extracellular vesicles (EVs) prepared from primary human NK cells cultured for 48hrs in serum-free conditions supplemented with 10 ng/ml human recombinant IL-12, IL-15, and IL-18. The aim was to isolate and define an EV subset with cytolytic activity against tumor cells. For this purpose, bulk EVs were separated according to density (density gradient ultracentrifugation; DG-UC) to yield 3 distinct subsets.
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Project description:In this study we have compared the proteomic profile of extracellular vesicles (EVs) prepared from primary, human NK cells or the human NK cell lines NK-92 and KHYG-1 cultured for 48hrs in serum-free conditions. EVs were harvested from cells either under resting conditions (culture in IL-15) or upon activation (combination of IL-12, IL-15, and IL-18). In addition, primary NK cells were activated in the presence of anti-CD16-coated beads, and EVs harvested after 48hrs. The aim was to compare their ability to target and kill a variety of tumor cell line-derived spheroids
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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.
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