Inhibition of BAK-mediated apoptosis by the BH3-only protein BNIP5

BCL-2 family proteins regulate apoptosis by initiating mitochondrial outer membrane permeabilization (MOMP). Activation of the MOMP effectors BAX and BAK is controlled by the interplay of

anti-apoptotic BCL-2 proteins (e.g., MCL-1) and pro-apoptotic BH3-only proteins (e.g., BIM). Using a genome-wide CRISPR-dCas9 transactivation screen we identified BNIP5 as an inhibitor of

BAK-, but not BAX-induced apoptosis. BNIP5 blocked BAK activation in different cell types and in response to various cytotoxic therapies. The BH3 domain of BNIP5 was both necessary and

sufficient to block BAK activation. Mechanistically, the BH3 domain of BNIP5 acts as a selective BAK activator, but a poor de-repressor of complexes between BAK and pro-survival BCL-2 family

proteins. By promoting the binding of activated BAK to MCL-1 or BCL-xL, BNIP5 inhibits apoptosis when BAX is absent. Based on our observations, BNIP5 can act functionally as an

anti-apoptotic BH3-only protein.

All relevant datasets are available in the online version of the manuscript.

We thank Xiaofei Wang and Tanya Khan for the help with animal experiments. We thank Dr. Katherine Verbist for proofreading the manuscript. Figure S6 was created using Biorender.

These authors contributed equally: Sebastian Rühl, Zhenrui Li.

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

Sebastian Rühl, Zhenrui Li, Luigi Mari, Clifford S. Guy, Mao Yang & Douglas R. Green

Department of Biochemistry and Molecular Biology, UAMS College of Medicine, Little Rock, AR, 72205, USA

Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

SR and DRG conceived the study, SR and ZL performed most experiments, TM, SS GC, MY and LM performed experiments. SR and DRG wrote the initial draft of the manuscript. All authors commented

on the manuscript. DRG supervised the research.

The authors declare no competing interests. During the course of the work, D.R.G. consulted for Sonata Therapeutics, Ventus Therapeutics, and ASHA pharmaceuticals. S.R. is an employee of T3

Pharmaceuticals AG.

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