CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis

Author:

Zhang Jingtian1,Niu Han1,Zhao Zhizhuang Joe12,Fu Xueqi1,Wang Yuxiang1,Zhang Xu1,Zhang Fuqiang3,Zeng Linlin1ORCID

Affiliation:

1. Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun, 130012, China

2. Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

3. Scientific Research Centre of China-Japan Union Hospital, Jilin University, Changchun, 130033, China

Abstract

TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax—members of the Bcl-2 family—are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK−/− cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak−/− cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.

Funder

National Natural Science Foundation of China

Publisher

Hindawi Limited

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine

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