Anti-apoptotic BH3-only proteins inhibit Bak-dependent apoptosis

Bcl-2 family proteins regulate induction of intrinsic apoptosis through initiating mitochondrial outer membrane permeabilization (MOMP). Activation of the MOMP effectors Bax and Bak is controlled by interplay levels 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 two Bcl-2 family proteins, BNIP5 and Bcl-G, as inhibitors of Bak, but not Bax induced apoptosis. BNIP5 was able to block 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 domains of BNIP5 and Bcl-G act as a selective Bak activators, while not inhibiting anti-apoptotic proteins. This led to increased binding of activated Bak to Mcl-1, which prevented apoptosis engagement, identifying BNIP5 and Bcl-G as anti-apoptotic BH3-only proteins.