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Proteomics characterization of extracellular vesicles derived from fermented food industry by-product: looking for new potential delivery vectors

ABSTRACT: In the last decade, the interest in extracellular vesicles (EVs) as safe and biocompatible nano-transporters has risen dramatically, as these nanoparticles can be used to increase the delivery efficiency rate and reduce secondary effects. However, the difficulty for its obtention in large quantities represents a major drawback for its utilization. Also, EVs are currently mainly derived from immortalized cell lines, which may arise safety problems when administered to humans. Hence, the shift from cell lines to safer EVs sources would suppose a step ahead for its use as delivery vectors in the biotechnology and biomedical research fields. Here, we provide proteomics characterization of EVs isolated by molecular weight cut-off filtration (MWCO) from four different fermented food industry by-products (FFIBPs), including: i) yogurt whey (named as milk in the raw data files), symbiotic culture of bacteria and yeast (SCOBY) from kombucha fermented tea (named as Com in the raw data files), wine pomace (named as Wine in the raw data files) and brewer spend yeast (named as beer in the raw data files).This proteomics characterization will provide key information about composition of by-product derived EVs (BP-EV) uncovering a safe and sustainable EVs source with potential as functional EVs delivery vectors.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Aida Serra

LAB HEAD: Aida Serra, PhD.

PROVIDER: PXD025780 | Pride | 2022-04-27

REPOSITORIES: Pride

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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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Proteomics characterization of extracellular vesicles derived from fermented food industry by-product: looking for new potential delivery vectors

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