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Human - Cells and Extracellular vesicles of Hela and MNT-1 WT vs CD63 KO Exosomes

ABSTRACT: CD63 is a tetraspanin, which organize on cholesterol enriched microdomains, especially on late endosomes. Previous work of our team has shown that CD63 could regulate the sorting of cargoes into ILVs through an ESCRT-independent manner in melanocytes. However, CD63 has also been involved in other trafficking events in other cell type and so far, we have no clear idea of the general process that CD63 would control and would unify these disparate reports. In order to elucidate to general role of CD63 we compared two cell lines, Hela and MNT-1 that we Knock-out for CD63 by CRISPR in both cell lines and investigated the EV generation and the expression level of proteins involved in this process in cell lysates and the EV pellet obtained by SEC.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lymph Node, Hela Cell

SUBMITTER: Vanessa Masson

LAB HEAD: Damarys Loew

PROVIDER: PXD037149 | Pride | 2024-04-19

REPOSITORIES: Pride

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Project description:The high incidence of pre-eclampsia, affecting 5-10% of pregnancies, makes it a major health problem. Early detection of pre-eclampsia, before the appearance of the first clinical symptoms, is therefore an urgent need, since it would allow the early intervention. Identification of plasma/serum biomarkers would pave the way to develop new strategies for the diagnosis of pre-eclampsia. Liquid biopsy emerges as a promising source of protein biomarkers that circumvent some of the inherent challenges of proteome wide analysis of plasma and serum. Besides, purified exosomes have the added interest of being communication vehicles between cells and tissues both in physiological and pathological processes. Methods We compared the protein abundance in purified exosomes from three different cohorts of control and preeclamptic serum samples, obtained around the sixth month and at the end of pregnancy. To this end, a shotgun label-free proteomics analysis was conducted and the relevant differential proteins were then validated by targeted MRM. Results An exosome purification method was developed that yielded highly enriched preparations, as indicated by the detection of a large number of exosomal markers. Likewise, the presence of specific pregnancy protein markers suggested that a very significant percentage of purified exosomes come from tissues related to pregnancy. Shotgun quantitative proteomic analysis allowed us to identify 10, 114 and 98 differentially regulated proteins in the three studied cohorts, with a high degree of concordance among them. Functional analysis suggests that these proteins participate in biological processes closely related to pre-eclampsia, such as angiogenesis, inflammation or cell migration. Differential abundance of 66 proteins were validated by MRM. Finally, we show the impact of the pre-eclampsia associated exosomes in the proteome of a cellular model, compared to normal exosomes. Conclusions We identified and validated differential proteins in liquid biopsy of patients that open new possibilities for the early diagnostic of pre-eclampsia. Additionally, the functional impact of the different proteome composition of purified pre-eclamptic exosomes in target cells provide new information to further understand changes on embryo-maternal interactions and therefore the pathogenesis of this disease.

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Human - Cells and Extracellular vesicles of Hela and MNT-1 WT vs CD63 KO Exosomes

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