Jerry E. Chipuk

The BCL-2 Family Reunion

B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.

Dissecting p53-dependent apoptosis

The complexity of the p53 protein, coupled with the vast cellular responses to p53, is simply astonishing. As new isoforms, functional domains and protein–protein interactions are described; each morsel of information forces us to think (and re-think) about how it ‘fits’ into the current p53 paradigm. One aspect of p53 signaling that is under refinement is the mechanism(s) leading to apoptosis. Here we discuss what is known about p53-induced apoptosis, what proteins and protein–protein interactions are responsible for regulating apoptosis, how can this cascade be genetically dissected, and what pharmacological tools are available to modulate p53-dependent apoptosis. While everything may not comfortably fit into our understanding of p53, all of these data will certainly broaden our viewpoint on the complexity and significance of the p53-induced apoptotic pathway. Here, our discussion is primarily focused on the works presented at the 12th International p53 Workshop, except where appropriate background is required.

Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario

Mitochondrial outer membrane permeabilization (MOMP) is considered the ‘point of no return’ as this event is responsible for engaging the apoptotic cascade in numerous cell death pathways. MOMP is directly governed by a subset of the BCL-2 family of proapoptotic proteins, which induce disruptions in the outer mitochondrial membrane (OMM) and subsequent release of death-promoting proteins like cytochrome c. The proposal here is centered on our hypothesis that MOMP is dictated by an interaction between the cytosol and the OMM, and although proteins of the OMM may be important in the process, the ‘decision’ to undergo apoptosis originates within the cytosol with no participation (in terms of yes, no and when) by mitochondria.

Do inducers of apoptosis trigger caspase-independent cell death?

Apoptotic cell death is mediated by molecular pathways that culminate in the activation of a family of cysteine proteases, known as the caspases, which orchestrate the dismantling and clearance of the dying cell. However, mounting evidence indicates that a cell that has been treated with an apoptotic inducer can also initiate a suicide programme that does not rely on caspase activation. Here, we present recent findings and discuss the physiological relevance of caspase-independent cell death.

Pharmacologic activation of p53 elicits Bax-dependent apoptosis in the absence of transcription

Recent efforts to develop pharmacologic agents that restore function to mutant forms of p53 hold significant promise in cancer therapy. Here, we examine the effects of such pharmacologic activation of p53 function using a small molecule, PRIMA-1, and a model system employing a p53 protein fused to a mutant steroid binding domain of the murine estrogen receptor (p53ERtam) that renders it responsive only in the presence of 4-hydroxytamoxifen. In either case, p53 activation triggered apoptosis that was not inhibited by the presence of macromolecular synthesis inhibitors. This p53-induced, transcription-independent apoptosis is Bax dependent, proceeds in the absence of a nucleus, and involves Bax translocation and cytochrome c release. Hence, pharmacologic p53 modulators can activate a transcription-independent apoptotic program.

Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins

Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or marked cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the effect of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires “covert” levels of direct activators of BAX and BAK at the OMM.

p53 and Metabolism: Inside the TIGAR

The p53 tumor suppressor pathway coordinates DNA repair, cell-cycle arrest, apoptosis, and senescence to preserve genomic stability and prevent tumor formation. The discovery of three new target genes for p53 reveals unexpected functions for this tumor suppressor in the regulation of glucose metabolism and autophagy.