Proteomic analysis of necroptotic extracellular vesicles

Abstract

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.

Necroptosis is associated with Rab27‐independent expulsion of extracellular vesicles containing RIPK3 and MLKL

“…Both EM and NTA showed that SEVs-isolated by two different methods-have similar size distributions regardless of their RIPK3 genotypes or whether they were undergoing necroptosis. NEEs appear to share about half the proteins (1023) identified in similar NEEs in macrophages (Shlomovitz et al, 2021) although RIPK3 was uniquely identified in our study (Table S5).…”

“…Close examinations of our proteomic data revealed the presence of several Rab-family members in SEVs including Rab21, Rab6a, Rab6b, Rab18 and others Rab11a and Rab11b. Rab11 isoforms-Rab11a, Rab11b, and Rab25-have previously been found in SEVs including those secreted by necroptotic cells (Shlomovitz et al, 2021). To address whether RIPK3 and Rab11 could be mechanistically involved in SEV release during necroptosis, we immunoprecipitated extract from WCL and NEEs with an anti-RIPK3 antibody and subjected the resulting complexes to mass-spectrometry analysis.…”

The roles of extracellular vesicles in the immune system

“…There is a large body of evidence that EVs generated by either non-inflammatory or inflammatory types of cell death have discordant effects upon uptake by phagocytic cells. Recently, it was shown that uptake of necroptotic cell-derived EVs by macrophages results in the secretion of pro-inflammatory cytokines (tumour necrosis factor (TNF) and CCL2) 26 . Inflammasome activation during pyroptosis induces a marked release of exosomes, and a connection has been established between inflammasome-mediated cleavage of Rab-interacting lysosomal protein (RILP) and the selective loading of microRNAs (miRNAs) into EVs 27 .…”

Ferroptosis, necroptosis, and pyroptosis in the occurrence and development of ovarian cancer

“…Oligomerized MLKL has the ability to bind directly to lipids, allowing polymerized MLKL to form membrane permeable pores, disrupt cell membrane integrity, and undergo necroptosis ( 71 , 85 ). However, it inhibits the formation of complex iib to inhibit necroptosis, so the role of RIPK1 in cells can be determined by targeting the drug to determine whether the cells survive or undergo necroptosis ( 74 , 86 , 87 ).…”