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Syntaxin-17-Dependent Mitochondrial Dynamics Is Essential for Protection against Oxidative-Stress-Induced Apoptosis

by Binran Wang 1,2,†, Xiaoyue Xiao 1,2,†, Fanwei Huang 1,2,† and Rong Liu 1,3,4,*
1
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
2
Department of Pathogen Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan 430030, China
3
National Center for International Research on Animal Gut Nutrition, Nanjing 210095, China
4
Jiangsu Collaborative Innovation Center of Meat Production and Processing, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Antioxidants 2019, 8(11), 522; https://doi.org/10.3390/antiox8110522
Received: 25 September 2019 / Revised: 24 October 2019 / Accepted: 24 October 2019 / Published: 30 October 2019
In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome–lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome–lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome–lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)–GFP–LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER–Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome–lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome–lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy. View Full-Text
Keywords: lysosome; autophagy; membrane fusion; mitochondrial fission; ROS; oxidative stress lysosome; autophagy; membrane fusion; mitochondrial fission; ROS; oxidative stress
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MDPI and ACS Style

Wang, B.; Xiao, X.; Huang, F.; Liu, R. Syntaxin-17-Dependent Mitochondrial Dynamics Is Essential for Protection against Oxidative-Stress-Induced Apoptosis. Antioxidants 2019, 8, 522.

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