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Identification of Novel Scaffold Proteins for Improved Endogenous Engineering of Extracellular Vesicles

ABSTRACT: Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. In this study 244 candidate scaffold proteins were studied, resulting in the identification of 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 were detected as the lead candidates for cargo loading, outperforming the well-known CD63 scaffold. EVs from human embryonic kidney epithelial (HEK-293T) cells, either wild type or overexpressing TSPAN2, TSPAN3 or CD63, were analyzed with label-free top-down proteomics to understand the effect of EV-engineering on protein signatures. The proteomics findings demonstrated that TSPAN2/TSPAN3-based engineering gives rise to EV subpopulations distinct from CD63. The discovery of these novel scaffolds provides a new platform for EV-based therapies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Helena Sork

LAB HEAD: Samir EL Andaloussi

PROVIDER: PXD043840 | Pride | 2023-08-18

REPOSITORIES: Pride

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			Action	DRS
	230118_03_Orbi3_MS_SER_G60_T82_TYTI_Sork_1ug_CD63_2.raw	Raw
	230118_05_Orbi3_MS_SER_G60_T82_TYTI_Sork_1ug_WT_2.raw	Raw
	230118_07_Orbi3_MS_SER_G60_T82_TYTI_Sork_1ug_TSPAN3_2.raw	Raw
	230118_09_Orbi3_MS_SER_G60_T82_TYTI_Sork_1ug_TSPAN2_2.raw	Raw
	230118_11_Orbi3_MS_SER_G60_T82_TYTI_Sork_1ug_WT_3.raw	Raw

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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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Identification of Novel Scaffold Proteins for Improved Endogenous Engineering of Extracellular Vesicles

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