Proteomic analysis of necroptotic extracellular vesicles

Inbar Shlomovitz,Liat Edry-Botzer,Hadar Cohen,Ziv Erlich,Sefi Zargarian,Tomer Cooks,Yifat Ofir-Birin,Tal Manko,Neta Regev-Rudzki,Gali Arad,Motti Gerlic

doi:10.1038/s41419-021-04317-z

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.

Highlights

Necroptosis is a well-studied form of regulated necrosis, defined as receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-/ mixed lineage kinase domain-like (MLKL)-dependent, caspaseindependent cell death [1–4].The ligation of tumor necrosis factor-α (TNF-α) results in caspase8 cleavage and activation and, apoptosis [5–7]
We reported that PS-exposing necroptotic cells release extracellular vesicles (EVs) early during necroptosis, which contain Phosphorylated MLKL (pMLKL) [16]
tripartite motif containing 27 (TRIM27), which we found to be significantly upregulated in the necroptotic EVs, positively regulates TNF-α-induced apoptosis by inducing ubiquitinspecific-processing protease 7 (USP7)-mediated RIPK1 deubiquitination [33]

Summary

INTRODUCTION

Necroptosis is a well-studied form of regulated necrosis, defined as receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-/ mixed lineage kinase domain-like (MLKL)-dependent, caspaseindependent cell death [1–4]. Necroptotic EVs are characterized by a unique shared 1172 common proteins with the Vesiclepedia database, proteome signature with 352 significantly upregulated proteins, as i.e., 35% of their total protein content (Fig. 1F) This includes 65 revealed by MS and supported by validation with immunoblotting. Of the 75 most frequently identified proteins in both databases (Supplementary Fig. S2 and Table S1) Among these are EV Components of ESCRTIII machinery and inflammatory biogenesis factors, e.g., Clathrin heavy chain 1 (CLTC) and the signaling are enriched in the necroptotic EVs. ESCRT accessory proteins programmed cell death 6-interacting To explore the distinctive signature of TBQ EV-enriched proteins we protein (PDCD6IP, known as Alix), as well as tetraspanins, performed gene ontology (GO) and Kyoto encyclopedia of genes such as CD63 and Flotillin-1 (FLOT1), and intracellular EV and genomes (KEGG) pathways enrichment analysis (Fig. 3A and trafficking proteins from the annexin and Ras-related protein Supplementary Table S3). The enriched processes represent the research, and presenting additional unique content to be studied. distinct signature of the necroptotic EVs in comparison to other EV

Shlomovitz et al 3

Findings

MATERIALS AND METHODS