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ABSTRACT 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS MITOCHONDRIAL E3 UBIQUITIN LIGASE MARCHF5 CONTROLS BAK APOPTOTIC ACTIVITY

INDEPENDENTLY OF BH3-ONLY PROTEINS Article 28 September 2022 MECHANISMS OF BCL-2 FAMILY PROTEINS IN MITOCHONDRIAL APOPTOSIS Article 12 July 2023 TOM20-MEDIATED TRANSFER OF BCL2 FROM ER TO

MAM AND MITOCHONDRIA UPON INDUCTION OF APOPTOSIS Article Open access 15 February 2021 DATA AVAILABILITY All relevant datasets are available in the online version of the manuscript.

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Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION Author notes * Sebastian Rühl Present address: T3 Pharmaceuticals, Allschwil, Switzerland * These authors contributed equally:

Sebastian Rühl, Zhenrui Li. AUTHORS AND AFFILIATIONS * 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 Shagun Srivastava & Tudor Moldoveanu *

Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA Tudor Moldoveanu Authors * Sebastian Rühl View author publications You can also search for

this author inPubMed Google Scholar * Zhenrui Li View author publications You can also search for this author inPubMed Google Scholar * Shagun Srivastava View author publications You can

also search for this author inPubMed Google Scholar * Luigi Mari View author publications You can also search for this author inPubMed Google Scholar * Clifford S. Guy View author

publications You can also search for this author inPubMed Google Scholar * Mao Yang View author publications You can also search for this author inPubMed Google Scholar * Tudor Moldoveanu

View author publications You can also search for this author inPubMed Google Scholar * Douglas R. Green View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to Douglas R. Green. ETHICS DECLARATIONS COMPETING INTERESTS 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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Rühl, S., Li, Z., Srivastava, S. _et al._ Inhibition of BAK-mediated apoptosis by the BH3-only protein BNIP5. _Cell Death Differ_ 32, 320–336 (2025).

https://doi.org/10.1038/s41418-024-01386-3 Download citation * Received: 18 July 2023 * Revised: 15 September 2024 * Accepted: 18 September 2024 * Published: 15 October 2024 * Issue Date:

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