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Deciphering the ciliary extracellular vesicle (EV) proteome

ABSTRACT: Cilia are conserved EV shedding organelles that release ciliary EVs for transmitting 13 signals. However, the content of ciliary EVs produced by living animals has not been explored. 14 Here we used evolutionarily conserved PKD-2::GFP EV cargo of Caenorhabditis elegans as a 15 marker of ciliary EVs to identify animal EV proteome. Top candidates were experimentally 16 confirmed using fluorescent reporter tracking and live animal imaging. Our findings indicate that 17 cilia produce multiple EVs with distinct protein content and that EVs from different neuronal 18 types carry different EV cargo. We discover that ciliary EVs carry nucleic acid binding proteins, 19 implying the role of cilia in epigenetic regulation and transfer of genetic information in trans. 20 Our dataset also contains non-ciliary EV cargo candidates from other tissues, such as cuticle, 21 somatic gonad, and germline, that can be explored using our MyEVome Web App.

INSTRUMENT(S): Orbitrap Eclipse, Q Exactive HF

ORGANISM(S): Escherichia Coli (ncbitaxon:562) Caenorhabditis Elegans (ncbitaxon:6239)

SUBMITTER: Maureen M Barr

PROVIDER: MSV000087943 | MassIVE | Wed Aug 04 10:50:00 BST 2021

REPOSITORIES: MassIVE

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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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Deciphering the ciliary extracellular vesicle (EV) proteome

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