Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Apoptotic priming is defined by the dynamic exchange of Bcl-2 proteins between mitochondria and cytosol.

Apoptosis is regulated by interactions between the BH3-only and multi-domain Bcl-2 family proteins. These interactions are integrated on the outer mitochondrial membrane (OMM) where they set the threshold for apoptosis, known as mitochondrial priming. However, how mitochondrial priming is controlled at the level of single cells remains unclear. Retrotranslocation of Bcl-XL has been proposed as one mechanism, removing pro-apoptotic Bcl-2 proteins from the OMM, thus reducing priming. Contrary to this view, we now show that Bcl-XL retrotranslocation is inhibited by binding to its BH3-only partners, resulting in accumulation of these protein complexes on mitochondria. We find that Bcl-XL retrotranslocation dynamics are tightly coupled to mitochondrial priming. Quantifying these dynamics indicates the heterogeneity in priming between cells within a population and predicts how they subsequently respond to a pro-apoptotic signal.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

Apoptosis regulation at the mitochondria membrane level.

Mitochondrial outer membrane permeabilization (MOMP) is a key checkpoint in apoptosis that activates the caspase cascade and irreversibly causes the majority of cells to die. The proteins of the Bcl-2 family are master regulators of apoptosis that form a complex interaction network within the mitochondrial membrane that determines the induction of MOMP. This culminates in the activation of the effector members Bax and Bak, which permeabilize the mitochondrial outer membrane to mediate MOMP. Although the key role of Bax and Bak has been established, many questions remain unresolved regarding molecular mechanisms that control the apoptotic pore. In this review, we discuss the recent progress in our understanding of the regulation of Bax/Bak activity within the mitochondrial membrane.

BioID-based proteomic analysis of the Bid interactome identifies novel proteins involved in cell-cycle-dependent apoptotic priming.

Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how it is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to antimitotic drugs. Here, we report an unbiased proximity biotinylation (BioID) screen to identify regulators of apoptotic priming in mitosis, using Bid as bait. The screen primarily identified proteins outside of the canonical Bid interactome. Specifically, we found that voltage-dependent anion-selective channel protein 2 (VDAC2) was required for Bid phosphorylation-dependent changes in apoptotic priming during mitosis. These results highlight the importance of the wider Bcl-2 family interactome in regulating the temporal control of apoptotic priming.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

E2F1 interacts with BCL-xL and regulates its subcellular localization dynamics to trigger cell death.

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity.