J. Marie Hardwick

Caspase-3-dependent Cleavage of Bcl-2 Promotes Release of Cytochrome c

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Bax-type Apoptotic Proteins Porate Pure Lipid Bilayers through a Mechanism Sensitive to Intrinsic Monolayer Curvature

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and ΔN76Bcl-xL (the Bax-like cleavage fragment of Bcl-xL) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or ΔN76Bcl-xL. Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.

Aven, a Novel Inhibitor of Caspase Activation, Binds Bcl-xL and Apaf-1

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.

Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins

Intradermal vaccination by gene gun efficiently delivers DNA vaccines into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigen-specific T cells and DNA vaccine potency. Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL-2, XIAP, dominant negative caspase-9, or dominant negative caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs. More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. Among these antiapoptotic factors, BCL-xL demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects. Thus, coadministration of DNA vaccines with DNA encoding antiapoptotic proteins represents an innovative approach to enhance DNA vaccine potency.

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.

Multiple Functions of BCL-2 Family Proteins

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.

Bcl-xL increases mitochondrial fission, fusion, and biomass in neurons

Mitochondrial fission and fusion are linked to synaptic activity in healthy neurons and are implicated in the regulation of apoptotic cell death in many cell types. We developed fluorescence microscopy and computational strategies to directly measure mitochondrial fission and fusion frequencies and their effects on mitochondrial morphology in cultured neurons. We found that the rate of fission exceeds the rate of fusion in healthy neuronal processes, and, therefore, the fission/fusion ratio alone is insufficient to explain mitochondrial morphology at steady state. This imbalance between fission and fusion is compensated by growth of mitochondrial organelles. Bcl-xL increases the rates of both fusion and fission, but more important for explaining the longer organelle morphology induced by Bcl-xL is its ability to increase mitochondrial biomass. Deficits in these Bcl-xL–dependent mechanisms may be critical in neuronal dysfunction during the earliest phases of neurodegeneration, long before commitment to cell death.

Upgrading the BCL-2 Network

The BCL-2 oncogene promotes cancer progression by prohibiting cell death. BCL-2-related regulators were once thought to constitute a simple, two-class system of anti-death and pro-death factors. New data delineate a multistep hierarchy of BCL-2-family protein interactions that are driven by signal-dependent pathways.

c-IAP1 Is Cleaved by Caspases to Produce a Proapoptotic C-terminal Fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor α treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.