TY - JOUR
T1 - Label-free proteomic analysis of exosomes derived from inducible hepatitis B virus-replicating HepAD38 cell line
AU - Jia, Xiaofang
AU - Chen, Jieliang
AU - Megger, Dominik A.
AU - Zhang, Xiaonan
AU - Kozlowski, Maya
AU - Zhang, Lijun
AU - Fang, Zhong
AU - Li, Jin
AU - Chu, Qiaofang
AU - Wu, Min
AU - Li, Yaming
AU - Sitek, Barbara
AU - Yuan, Zhenghong
N1 - Funding Information:
This work was supported by The National Natural Science Foundation of China (81461130019) to Zhenghong Yuan; the German Research Foundation (SFB/Transregio TRR60 to Zhenghong Yuan (A1) and to Barbara Sitek (Z3)); the National Natural Science Foundation of China (91542207) to Zhenghong Yuan; the Natural Science Foundation of Shanghai (13ZR1460100) to Xiaofang Jia; The National High-tech R&D Program (863 Program) (2014AA021403) and the National Natural Science Foundation of China (81271834) to Lijun Zhang.
Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2017/4
Y1 - 2017/4
N2 - Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometersized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox-)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox-)- exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox-)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox-)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox-)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.
AB - Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometersized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox-)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox-)- exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox-)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox-)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox-)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.
UR - http://www.scopus.com/inward/record.url?scp=85017139597&partnerID=8YFLogxK
U2 - 10.1074/mcp.M116.063503
DO - 10.1074/mcp.M116.063503
M3 - Article
C2 - 28242843
AN - SCOPUS:85017139597
SN - 1535-9484
VL - 16
SP - 144
EP - 160
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 4
ER -
