Dataset Information

Intact extracellular vesicle cross-linking on vesicles derived from MDA-MB-231 and LM2 cells

ABSTRACT: The development of any pharmaceutical requires characterization of the compound, usually be several techniques. Therefore, characterizing the protein content and biochemical state of EVs is of great importance for their useif they are to be used as biopharmaceuticals. Structural characterization of proteins in EVs remains a great challenge even for state-of-the-art technologies such as super-resolution and electron microscopy, partly due to the biophysical and biochemical heterogeneity of EVs. Here we overcome some of these challenges by using cross-linking mass spectrometry (XL-MS) to structurally characterize protein complexes and the interactome of intact EVs, at an unprecedented resolution of up to 30Å. By applying XL-MS to intact EVs derived from two metastatic breast cancer cell lines (MDA-MB-231 and LM2), we validate the presence of active forms of protein complexes that are known to play a role metastasis, revealing for example previously uncharacterized regions of Moesin. Moreover, a cross-linking data-driven molecular docking supports novel conformations of ANXA2 in LM2 EVs.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Julia Bauzá Martinez

LAB HEAD: Wei Wu

PROVIDER: PXD029591 | Pride | 2022-11-08

REPOSITORIES: Pride

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Publications

Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL).

Bauzá-Martinez Julia J Armony Gad G Pronker Matti F MF Wu Wei W

Journal of extracellular vesicles 20220701 8

Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spe ...[more]

PMID: 35918900

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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.

Dataset Information

Intact extracellular vesicle cross-linking on vesicles derived from MDA-MB-231 and LM2 cells

Publications

Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL).

Similar Datasets

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