Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.     

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.     

MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells

The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 microM), PD98059 (40 microM), or PD184352 (10 microM) with 10-100 microM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (DeltaPsi(m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of DeltaPsi(m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways

Two main intracellular apoptosis cascades, the receptor and the mitochondria pathway, have been identified. The mitochondrial pathway is controlled by the Bcl-2 proteins. This protein family contains members with either pro- or anti-apoptotic activity. When activated the pro-apoptotic multidomain proteins permeabilized the outer mitochondrial membrane, resulting in the release of proteins from the intermembrane space. Several proteins, including cytochrome c, Smac/DIABLO, HtrA2/Omi, endonuclease G and AIF, normally sequestered in the mitochondria induce or promote apoptosis once released into the cytosol. Although, apoptosis is an essential physiological process in multicellular organisms it is also involved in a wide range of pathological conditions.     

Bcl-2-family proteins and the role of mitochondria in apoptosis

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.     

Multiple partners can kiss-and-run: Bax transfers between multiple membranes and permeabilizes those primed by tBid

During apoptosis Bid and Bax are sufficient for mitochondrial outer membrane permeabilization, releasing pro-apoptotic proteins such as cytochrome c and Smac/Diablo into the cytoplasm. In most cells, both Bid and Bax are cytoplasmic but bind to mitochondrial outer membranes to exert pro-apoptotic functions. Binding to membranes is regulated by cleavage of Bid to truncated Bid (tBid), by conformation changes in tBid and Bax, and by interactions with other proteins. At least at the peripherally bound stage, binding is reversible. Therefore, regulation of apoptosis is closely linked with the interactions of tBid and Bax with mitochondria. Here we use fluorescence techniques and cell-free systems containing mitochondria or liposomes that faithfully mimic tBid/Bax-dependent membrane permeabilization to study the dynamic interactions of the proteins with membranes. We confirm that the binding of both proteins to the membrane is reversible by quantifying the binding affinity of proteins for the membrane. For Bax, both peripherally bound (inactive) and oligomerized (active) proteins migrate between membranes but much slower than and independent of tBid. When re-localized to a new membrane, Bax inserts into and permeabilizes it only if primed by an activator. In the case of tBid, the process of transfer is synergetic with Bax in the sense that tBid ‘runs'' faster if it has been ‘kissed'' by Bax. Furthermore, Mtch2 accelerates the re-localization of tBid at the mitochondria. In contrast, binding to Bcl-XL dramatically impedes tBid re-localization by lowering the off-rate threefold. Our results suggest that the transfer of activated tBid and Bax to different mitochondria is governed by dynamic equilibria and potentially contributes more than previously anticipated to the dissemination of the permeabilization signal within the cell.     

Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity on bax-/-mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.     

The cardiolipin-binding domain of Bid affects mitochondrial respiration and enhances cytochrome c release

Bid is cleaved by caspase 8 during apoptosis and the truncated Bid (tBid) translocates to mitochondria by targeting cardiolipin. Amino acids 103-162 of Bid were reported as the cardiolipin-binding domain (CBD). The EGFP-CBD fusion protein targets to mitochondria and induces apoptosis. Using [(3)H]cardiolipin, we proved that recombinant CBD binds cardiolipin similar to tBid and tBid(G94E), a mutant with a defective BH3 domain. CBD could induce cytochrome c release from isolated mitochondria, but much less potent than tBid. Free cardiolipin inhibited the CBD-induced cytochrome c release, suggesting that it may be mediated by interfering with mitochondrial cardiolipin, especially with the interaction between cytochrome c and cardiolipin. This is consistent with the findings that CBD induced cytochrome c release in Bax-deficient cells, and that CBD suppressed mitochondrial respiration through directly interfering with cardiolipin, a critical lipid involved in oxidative phosphorylation. These results indicate the functional importance of CBD in tBid-induced apoptosis.     

Modulation of mitochondrial apoptosis by PI3K inhibitors

Most anticancer therapies exert their action by triggering programmed cell death (apoptosis) in cancer cells. The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome c or Smac from the mitochondrial intermembrane space into the cytosol. Mitochondrial outer membrane permeabilization is tightly controlled, for example by pro- and anti-apoptotic proteins of the Bcl-2 family. Recent evidence indicates that inhibition of the PI3K/Akt/mTOR pathway by small-molecule PI3K inhibitors primes cancer cells to mitochondrial apoptosis by tipping the balance towards pro-apoptotic Bcl-2 proteins, resulting in increased mitochondrial outer membrane permeabilization. Thus, mitochondrial apoptotic events play an important role in PI3K inhibitor-mediated sensitization for apoptosis.     

Ubiquitin-mediated degradation of the proapoptotic active form of bid. A functional consequence on apoptosis induction

Under basal conditions, the proapoptotic protein Bid is a long-lived protein. Pro-apoptotic stimuli such as tumor necrosis factor-alpha (TNFalpha) or Fas induce its caspase-8-mediated cleavage into two fragments. The COOH-terminal cleavage fragment of Bid (tBid) becomes localized to mitochondrial membranes and triggers the release of cytochrome c. Here we show that tBid is ubiquitinated and subsequently degraded by the 26 S proteasome. Degradation of tBid is significantly inhibited by the proteasome inhibitors MG-132 and lactacystin. In contrast, caspase-specific or lysosomal inhibitors do not affect tBid stability. Furthermore, mutation of the putative ubiquitin acceptor sites within tBid results in a stabilized protein as assessed by pulse-chase analysis. To address whether tBid degradation might be regulated by interaction with other Bcl-2-like proteins, cotransfection studies were performed. However, neither the presence of proapoptotic Bax nor antiapoptotic Bcl-2 or Bcl-XL affected tBid degradation. Finally, we determined the functional role of tBid degradation. Overexpression of stabilized tBid proteins significantly enhanced cytochrome c release and subsequent apoptosis induction approximately 2-fold compared with wild type tBid. Similarly, tBid-induced apoptosis was considerably amplified by inhibition of tBid degradation using the proteasome-specific inhibitor MG-132. Thus, proteasomal degradation of tBid limits the extent of apoptosis in living cells.     

Mitochondrial intermembrane space proteins in apoptosis process

Mitochondria, despite their function in cellular energy metabolism, play an important role in the apoptotic signaling pathways. These organelles in response to the death signal undergo changes resulting in the release of proteins which are essential to conduct apoptosis via mitochondrial pathway. This article is focused on the properties and functions of apoptogenic proteins released from the mitochondrial intermembrane space, i.e., caspases, cytochrome c, Smac/DIABLO, serine protease Omi/HtrA2, AIF and endonuclease G.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.     

Cardiolipin provides specificity for targeting of tBid to mitochondria

Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.     

Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.     

Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space.     

Mitochondria as the target of the pro-apoptotic protein Bax

During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of cytochrome c and other apoptogenic proteins such as Smac/DIABLO, AIF, EndoG, Omi/HtraA2 and DDP/TIMM8a. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. Factors involved in Bax conformational change and the function(s) of the distinct domains controlling the addressing and the insertion of Bax into mitochondria are described in this review. We also discuss our current knowledge on Bax oligomerization and on the molecular mechanisms underlying the different models accounting for OMM permeabilization during apoptosis.     

A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.