Dataset Information

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles.

ABSTRACT: The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.

SUBMITTER: Sanwald JL

PROVIDER: S-EPMC7349886 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles.

Sanwald Julia L JL Poschmann Gereon G Stühler Kai K Behrends Christian C Hoffmann Silke S Willbold Dieter D

Cells 20200616 6

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using s ...[more]

PMID: 32560054

Similar Datasets

Project description:Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed-Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (?-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1?, IL-6, and TNF-?), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-?/NF-?B-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote ?-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.

Dataset Information

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles.

Publications

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets