Proteomic analysis of necroptotic extracellular vesicles

ABSTRACT: Necroptosis is a regulated but inflammatory form of cell death. We and others have previously reported that necroptotic cells release extracellular vesicles (EVs). We found that necroptotic EVs are loaded with proteins, including the key necroptosis executor factor, phosphorylated mixed lineage kinase domain-like (pMLKL). However, the exact necroptotic EVs proteins composition and impact have not been delineated yet. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated by ultracentrifugation and analyzed by mass spectrometry. A total of 3337 proteins were identified, sharing high similarity with exosome proteome databases. Unsupervised hierarchical clustering of identified proteins distinguished between necroptotic and control EVs. A total of 353 proteins was significantly upregulated in the necroptotic EVs. Among these are MLKL and caspase-8, as validated by immunoblot. Components of ESCRTIII machinery and inflammatory signaling were found to be enriched in the necroptotic EVs, as well as yet unreported components of vesicles formation and transport and necroptosis signaling pathways. Moreover, we show that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we reveal that necroptotic EVs contain tumor neoantigen and are enriched with proteins annotated as “antigen processing and presentation” biological process. In summary, our study reveals a new layer of regulation during the early stage of necroptosis by the secretion of specific EVs that influence their microenvironment and may instigate innate and adaptive immune responses. Future investigation will shed light on new players in the necroptosis signaling and its related EVs, and will uncover the functional tasks accomplished by the cargo of these necroptotic EVs.

INSTRUMENT(S): Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Tamar Geiger  

LAB HEAD: Tamar Geiger

PROVIDER: PXD018258 | Pride | 2021-11-11

REPOSITORIES: Pride