A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Yasuhiko Nakao,Chieh-Yu Liao,Akhilesh Pandey,Carrie Jo Heppelmann,K Sreekumaran Nair,Mayank Saraswat,Debanjali Dasgupta,Patrick M Vanderboom,Masanori Fukushima,Harmeet Malhi,Kazuhiko Nakao,Alina M Allen,Amy S Mauer,Anya Ferris

doi:10.3389/fcell.2021.735001

Abstract

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

Highlights

Extracellular vesicles (EVs) are emerging mediators of liver injury and inflammation in nonalcoholic steatohepatitis (NASH), a lipotoxic disorder characterized by the accumulation of the toxic saturated fatty acid, palmitate (Hirsova et al, 2016a; Kakazu et al, 2016)
Of identified proteins, the number of the total protein IDs that were found in the ExoCarta database (Keerthikumar et al, 2016), and the number of proteins from each method that were found in the ExoCarta top 100 EV proteins, respectively (Keerthikumar et al, 2016)
EVs are being widely appreciated for their role in cell-to-cell communication and as important mediators in multiple biological functions in NASH pathogenesis, which are mediated by unique EV cargoes

Summary

Introduction

Extracellular vesicles (EVs) are emerging mediators of liver injury and inflammation in nonalcoholic steatohepatitis (NASH), a lipotoxic disorder characterized by the accumulation of the toxic saturated fatty acid, palmitate (Hirsova et al, 2016a; Kakazu et al, 2016). We have previously demonstrated that palmitateinduced activation of the endoplasmic reticulum (ER) stress sensor inositol requiring enzyme 1 alpha (IRE1α) mediates the release of EVs via the de novo synthesis of ceramide (Fukushima et al, 2018). These lipotoxic EVs are enriched in ceramides and other sphingolipids (Kakazu et al, 2016; Dasgupta et al, 2020). Whether protein selection or trafficking into lipotoxic EVs is impacted by ceramide trafficking remains unknown

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