Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax,and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-x_L according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.The Bcl-2 family of proteins controls the mitochondrial pathway of apoptosis, a process often dysregulated in cancer and other diseases.^{1, 2, 3} Apoptotic triggers including DNA damage and oncogene activation cause the synthesis or activation of one or more pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins,^{1, 2, 3, 4} a subfamily that includes Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk. These proteins then engage via their BH3 domain with other Bcl-2 family members. BH3-only proteins that can directly bind and activate the Bcl-2 effector proteins Bak or Bax are called ‘activators''.⁵ When Bak or Bax become activated and oligomerize in the mitochondrial outer membrane (MOM), the apoptotic ‘switch'' has flipped and the cell is committed to cell death. The prosurvival members (Bcl-2, Bcl-x_L, Mcl-1, Bcl-w, Bfl-1/A1 and Bcl-B) inhibit apoptosis by specifically binding both the BH3-only proteins and activated Bak and Bax.^{6, 7, 8, 9, 10, 11} Thus, the cell''s complement of prosurvival proteins, Bak, and Bax, determines the sensitivity of that cell to each BH3-only protein, and by extension to each type of pro-apoptotic stimulus.A thorough understanding of BH3-only proteins is crucial for the development of cancer therapeutics such as the new class of anti-cancer molecules called BH3 mimetics that are showing significant promise in clinical trials.^{12, 13} The binding of BH3-only proteins to prosurvival proteins has been well-characterized and revealed significant preferences for engaging different members.^{6, 8, 9} How BH3-only proteins bind and activate Bak and Bax remains less understood for several reasons. First, generating stable recombinant BH3-only proteins is difficult because, except for Bid, they are intrinsically disordered^{14, 15, 16} and because most contain hydrophobic C-terminal membrane anchors.¹⁷ Thus, most in vitro studies of BH3-only proteins have used synthetic peptides corresponding to the BH3 domains, C-terminally truncated recombinant proteins or in vitro translated (IVT) proteins. Second, BH3-only reagents bind poorly to recombinant Bak and Bax in the absence of membranes, although detergents and liposomes may substitute for the MOM.^{18, 19, 20} Third, activation of Bak and Bax on mitochondria can be complicated by the presence of other proteins such as prosurvival proteins. Indeed, genetically altering BH3-only protein levels in mice resulted in complex phenotypes due to multiple interactions between family members, precluding firm conclusions as to which BH3-only proteins are direct activators.^{18, 21, 22}Bid and Bim are direct activators according to a variety of approaches,^{5, 8, 9, 23, 24} and were recently proposed to be specific for Bak and Bax, respectively.²⁵ Early studies using Noxa BH3 peptides^{5, 8} and IVT Noxa⁹ concluded that Noxa was not an activator. However, in more recent studies a Noxa BH3 peptide²³ and purified recombinant NoxaΔC²⁰ were found to be activators of both Bak and Bax. Puma has also been described as both an activator^{26, 27} and not an activator.^{8, 28} Du et al.²³ analyzed the full panel of BH3 peptides and classified Bim as a strong activator, Bid, Noxa and Bmf as moderate activators, and Puma, Bik and Hrk as weak activators. The only BH3-only member that has never been described as an activator is Bad.While BH3 peptides and recombinant truncated BH3-only proteins have been useful for in vitro studies, new reagents that target mitochondria may better reflect the behavior of the parent proteins. As Bid is stable as a recombinant protein, we generated chimeras of Bid in which the BH3 domain of Bid was replaced with that of seven other BH3-only proteins. This is a similar approach to the Bim chimeras used for expression in cells¹⁸ and in mice.²⁹ More recently, truncated Bid (tBid) chimeras containing the BH3 domains of Bim, Bak and Bax as well as those of the prosurvival proteins, have been generated as IVT proteins.¹¹To compare the ability of BH3-only proteins to activate Bak and Bax in vitro, we incubated Bid chimeras and BH3 peptides with mitochondria containing either Bak or Bax. We found that the membrane-targeted Bid chimeras were much more potent activators than their related BH3 peptides, and that all BH3 domains except for Bad and Noxa were activators to some extent. We conclude that activation of Bak and Bax may be underestimated by studies using BH3 peptides, and that even BH3-only proteins such as Bik, Bmf and Hrk that are often considered unable to activate Bak or Bax, may act as activators under certain conditions.     

Inhibition of apoptotic Bax translocation to the mitochondria is a central function of parkin

Parkinson''s disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive Parkinson''s disease (PD).¹ Multiple functions have been ascribed to parkin, most notably the inhibition of apoptosis^{2, 3, 4, 5, 6, 7} and the induction of autophagic mitochondrial turnover (mitophagy).^{8, 9} However, the relative scale of these effects mediated by endogenous parkin and whether these processes can occur concomitantly or are mutually exclusive, is not known.Bax is a primary effector of cell death that translocates from the cytosol to the mitochondria upon stress, where it facilitates cytochrome c release and the subsequent caspase cascade.¹⁰ We previously identified Bax as a parkin substrate, and found that the anti-apoptotic effects of parkin can be directly linked to the parkin-dependent ubiquitination of Bax and inhibition of its mitochondrial translocation.³ Recent corroborative evidence showed that primary cultured neurons from parkin knock-out (KO) mice accumulate greater levels of activated Bax at the mitochondria than wild-type (WT) neurons after apoptotic stimulation,¹¹ while a separate report showed the parkin-dependent ubiquitination of Bax during mitophagy.¹²In addition to its anti-apoptotic function, parkin facilitates a depolarization-induced and autophagy-dependent turnover of mitochondria. This process is robustly observed in immortalized cell lines expressing human parkin, where exposure to the mitochondrial depolarizing agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) causes rapid recruitment of parkin from the cytosol to the mitochondrial outer membrane and a coordinated proteasome and autophagosome-mediated turnover of the entire organelle.^{8, 13, 14, 15} Examination of this process in primary neuronal cultures with endogenous parkin expression, however, has been challenging,^{16, 17, 18, 19} and a cooperative role for inhibition of mitochondria-dependent cell death has not been investigated in the context of mitophagy.In this study, we sought further insight into the biological functions of parkin across multiple cell types. Our data showed that whole-cell biochemical techniques were not sufficient to observe the participation of endogenous parkin in mitochondrial turnover but were able to confirm the parkin-dependent regulation of apoptosis. Further examination of the parkin-dependent regulation of apoptosis identified two specific lysines of Bax that are critical for recognition and inhibition of its translocation to the mitochondria by parkin. In addition, the BH3 domain of Bax was critical for its association with parkin. Importantly, we observed parkin-dependent mitophagy and inhibition of apoptotic Bax translocation in the same cell culture systems, suggesting that these two pathways coexist and likely cooperate within neurons. Taken together, our data indicate that the parkin-dependent regulation of Bax is critical for cell survival, irrespective of the nature of cell stress involved.     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.The removal of apoptotic cells, known as efferocytosis, is a series of arranged events from the recruitment of phagocytes to sites where apoptotic cells are generated to the digestion of apoptotic cells by phagocytes.^{1, 2, 3} One of the key steps during efferocytosis is the recognition of dying cells by phagocytes. Phagocytes can detect apoptotic cells by the direct or indirect association of multiple receptors on phagocytes with ligands on apoptotic cells.^{4, 5, 6, 7, 8, 9} Some receptors on the surface of phagocytic cells not only bind to apoptotic cells but also transduce apoptotic cell recognition signals into phagocytes in order to mediate the ingestion of apoptotic cells. For instance, brain-specific angiogenesis inhibitor 1 (BAI1) and stabilin-2, which are phosphatidylserine (PtdSer) receptors, recognize PtdSer on apoptotic cells and relay signals to the Elmo-Dock-Rac module and Gulp, respectively, via their cytoplasmic tails.^{8, 10, 11} By contrast, it has been suggested that other receptors, called tethering receptors, merely tether apoptotic cells to phagocytes without mediating downstream signal transduction, following which the internalization of apoptotic cells is mediated by the association of these receptors with co-receptors or other engulfment receptors located nearby.^{12, 13, 14, 15, 16} However, it is unclear whether co-receptors for tethering receptors exist in tethering receptor-mediated phagocytosis of apoptotic cells, and, if they do, whether they are indispensable for this process.One intriguing characteristic of tethering receptors is that they have cytoplasmic tails lacking any signaling motifs or are anchored via glycophosphatidylinositol (GPI) to the outer leaflet of the plasma membrane. For example, Tim-4, a PtdSer receptor with a short cytoplasmic tail that promotes the engulfment of apoptotic cells by the binding of its IgV domain to PtdSer on apoptotic cells, lacks signaling motifs in its cytoplasmic tail. It has been known that neither the cytoplasmic tail nor the transmembrane region of Tim-4 is essential for Tim-4-mediated engulfment of apoptotic cells. Accordingly, it functions as a tethering receptor to secure apoptotic cells on phagocytes.^{9, 14} CD14 is located at the exofacial leaflet of the plasma membrane through its GPI anchor, which rules out the possibility that it mediates direct signal transduction into phagocytes after binding to apoptotic cells. Consequently, it is also considered to be a tethering receptor.¹⁵Phospholipids such as PtdSer and phosphatidylcholine (PtdCho) are unequally distributed between the inner and outer leaflets of the plasma membrane in the normal state. For instance, uncharged phospholipids such as PtdCho and sphingomyelin are primarily located in the outer leaflet, whereas positively or negatively charged phospholipids (such as phosphatidylethanolamine or PtdSer, respectively) are restricted to the inner leaflet facing the cytosol.^{17, 18, 19} However, this asymmetric distribution of phospholipids in the plasma membrane is disrupted during apoptosis. In the plasma membrane of apoptotic cells, PtdSer is exposed to the outer leaflet of the plasma membrane by the activity of scramblases and flippases.^{18, 20, 21} Thus, exposed PtdSer is a hallmark of apoptotic cells and is the best characterized ligand on apoptotic cells for efferocytosis. PtdSer on the surface of apoptotic cells can be recognized by various PtdSer-sensing membrane proteins on phagocytes, collectively called PtdSer receptors, including tethering receptors.Besides PtdSer receptors, many PtdSer-binding proteins have been identified. These proteins are involved in various biological processes such as blood coagulation, synaptic vesicle fusion, membrane scaffolding, and signal transduction.²² One of the best known proteins is annexin A5, which has been extensively studied as a PtdSer-binding protein. Annexin A5 belongs to the family of annexins, which are characterized by their Ca²⁺-dependent ability to bind to negatively charged phospholipids and share structural properties. Annexins are considered to be cytosolic proteins because they lack a 5′ leader sequence; however, some annexins, including annexin A5, are also found on the cell surface and in the circulation. This and related properties imply that annexins participate in diverse biological events from membrane dynamics to cell differentiation and migration.^{23, 24, 25} However, the physiological significance of this family is poorly understood. Among annexins, annexin A5 binds to PtdSer with high affinity. Because of this property, annexin A5 has been harnessed as a molecular probe to distinguish apoptotic cells from live cells both in vivo and in vitro for decades.^{25, 26}In this study, annexin A5 was expressed on the cell surface through a GPI anchor to delineate whether a tethering receptor without its co-receptor can promote efferocytosis. GPI-anchored annexin A5 (Anxa5-GPI) should not interact with any plasma membrane or extracellular protein, at least those involved in the engulfment of apoptotic cells. Thus, it is possible to exclude the effects of co-receptors on Anxa5-GPI-mediated phagocytosis of apoptotic cells. The expression of Anxa5-GPI in phagocytes promoted not only the binding but also the internalization of apoptotic cells. By contrast, phagocytosis of carboxylate beads and Escherichia coli was not affected by the expression of Anxa5-GPI in phagocytes. Anxa5-GPI-induced efferocytosis was not only partially dependent on a specific engulfment pathway but also relied on the generally known cytoskeletal engulfment machinery. Our observations suggest that co-receptors are dispensable for tethering receptor-mediated efferocytosis. In addition, tethering receptors could enhance efferocytosis through diverse engulfment machinery located nearby.     

Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0'' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.The intrinsic pathway to apoptosis is regulated by interactions between members of three factions of the Bcl-2 protein family: the BH3-only proteins such as Bim and Bid, which initiate the process, the essential effectors Bax and Bak, and the prosurvival members, which oppose the action of both other factions.¹ The interactions between prosurvival Bcl-2 family members and BH3 peptides have been well characterised as the earliest studies with Bcl-x_L and a BakBH3 peptide.² Such complexes are readily formed in solution by incubating the C-terminally (ΔC) truncated prosurvival Bcl-2 protein with a BH3 peptide. The absence of the C-terminal segment that can anchor the Bcl-2 protein in a membrane apparently has little effect on the ensuing complex. That complex is believed to be responsible for the antiapoptotic function of Bcl-2, by sequestration of the BH3 motif either of the so-called BH3-only proteins such as Bim (''mode 1'') or of Bax or Bak (''mode 2'').³Although proapoptotic Bax and Bak have very similar three-dimensional structures to their prosurvival relatives,^{4, 5, 6} until recently^{7, 8} no structure of a complex of either Bax or Bak with a BH3 peptide had been captured, despite an accumulation of evidence that Bax and Bak could be activated directly by interaction with the BH3-only proteins Bid, Bim and possibly others.^{9, 10, 11, 12, 13}Unlike Bak, which is constitutively anchored in the mitochondrial outer membrane (MOM) via its C-terminal segment, Bax is largely cytosolic in healthy cells and accumulates at the MOM only upon a death signal.^{14, 15} There it is believed to display at least two different conformers,^{16, 17} one loosely associated with the MOM and another in which its membrane anchor (helix α9) is inserted into the MOM. In striking contrast to the antiapoptotic relatives of Bcl-2, a construct of Bax lacking its C-terminal membrane anchor, BaxΔC21, has no measurable interaction with BH3 peptides. However, in the presence of the detergent octylglucoside binding is detected by surface plasmon resonance (SPR) for the BH3 peptides of Bim, Bid, Bak and Bax itself with IC50s in the range of 0.1–1μM,^{7, 18} some 100-fold weaker compared with those measured similarly with (for example) Bcl-x_LΔC, where no detergent is required. Weaker interactions between BidBH3 or BimBH3 and BaxΔC as compared with Bcl-x_LΔC are not inconsistent with various models for the function of the Bcl-2 protein family whereby the prosurvival molecules sequester BH3 motifs with high affinity and long half-lives, but proapoptotic Bax and Bak are activated by transient (‘hit-and-run'') interactions with BH3 motifs.^{19, 20, 21}Complexes of BaxΔC21 bound to BH3 peptides from Bid and Bax have been prepared by coincubation of the protein with CHAPS and an excess of the peptides.⁷ Under these conditions, the protein undergoes a conformational change and dimerises via domain swapping of helical segments α2–α5 and α6–α8, dubbed ‘core'' and ‘latch'' domains, respectively. Although this ‘core/latch dimer'' is thought to be an in vitro artefact, its formation is diagnostic for the core and latch separation, which is required for membrane-associated Bax to dimerise via its core domains and then to permeabilise the MOM.⁷ If the latch domain is absent, as in a recombinant construct of GFP fused to Bax α2–α5, the core domain forms BH3:groove symmetric dimers,⁷ which, consistent with a wide body of evidence,^{21, 22, 23, 24, 25} are present in apoptotic pores.Previous work⁷ highlighted the importance of two hydrophobic ‘h0'' residues (Figure 1) in the peptide (I82/I83 in BidBH3) in governing Bid''s ability to activate Bax. Similar to Bid, Bim is also a potent direct activator of Bax, and the ‘h0'' amino acids in Bim are proline and glutamic acid. In the absence of a structure of BimBH3:BaxΔC, it remained unclear how these ‘h0'' residues were accommodated. Here we describe the crystal structures of BimBH3 26- and 20-mer peptides bound to BaxΔC26. Comparison with the structure of BidBH3:BaxΔC21 allows a dissection of the critical contacts between these two peptides and BaxΔC. The binding profiles of mutant BH3 peptides illustrate that BimBH3 binding to Bax is less dependent on the ‘h0'' residues compare with that in the case for BidBH3. The BimBH3 complex displays a similar cavity adjacent to Bax α1, α2, α5 and α8 as seen in the BidBH3 complex. We also describe a structure of BidBH3 bound to a BaxΔC21 mutant, I66A, which is more typical of the BH3 signature of antiapoptotic Bcl-2 family proteins^{7, 26}Open in a separate windowFigure 1BimBH3 binds BaxΔC. (a) BH3 peptide sequences used in this study, indicating the 5 hydrophobic amino-acid positions ‘h0''–‘h4''. (b) The core/latch dimer of BaxΔC26 bound to BimBH3. The two Bax polypeptides, shown here as cartoons, are coloured yellow and grey, and the two Bim peptides cyan and orange. A crystallographic dyad symmetry axis passes through the centre of this particle. (c) Structure of BimBH3:BaxΔC26 complex. The globular unit depicted comprises Bax residues 1–128 from one polypeptide and 129–166 from the other, together with the associated Bim peptide. Bax is represented by its surface and colour coded according to surface charge (blue, positive potential (4kT/e); red, negative potential (−2kT/e); calculated using the Adaptive Poisson–Boltzmann Solver.⁴¹ The trace of the Bim peptide (cyan) is shown with ‘h0'' (P144, E145), ‘h1'' (I148), ‘h2'' (L152), ‘h3'' (I155) and ‘h4'' (F159) represented as sticks. (d) Overlay of BimBH3:BaxΔC26 with BidBH3:BaxΔC21 (PDB:4BD2). Structures represented as cartoon ribbons, yellow for Bax in the Bim complex and magenta for Bax in the Bid complex. The peptides (Bim cyan and Bid blue) stand vertically in the foreground in this view (similar to Figure 1c), with their N termini at the bottom of the figure     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

Puma strikes Bax

The commitment to programmed cell death via apoptosis is largely made upon activation of the proapoptotic mitochondrial proteins Bax or Bak. In this issue, Gallenne et al. (Gallenne, C., F. Gautier, L. Oliver, E. Hervouet, B. Noël, J.A. Hickman, O. Geneste, P.-F. Cartron, F.M. Vallette, S. Manon, and P. Juin. 2009. J. Cell Biol. 185:279–290) provide evidence that the p53 up-regulated modulator of apoptosis (Puma) protein can directly activate Bax.The Bcl-2 family of proteins participates in the control of the cell''s commitment to programmed cell death via the mitochondrial or intrinsic apoptotic pathway. Certain proteins in this family, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1/A1, inhibit apoptosis, whereas others in this family promote apoptosis. Proapoptotic Bax and Bak appear to be indispensible for apoptosis (Lindsten et al., 2000; Wei et al., 2001). How does the cell determine fate in the face of competing pro- and antiapoptotic proteins? The rheostat model proposed that when there were more antiapoptotic proteins than proapoptotic proteins, the cell survived and vice versa. However, in many cases, the conversion of a living cell to one committed to death occurs without significant change in the levels of pro- and antiapoptotic proteins. The participation of a third class of proapoptotic proteins largely explained this riddle. These proteins, so-called BH3-only as they share homology only in the proapoptotic Bcl-2 homology 3 domain, appear to act as sentinels of cell damage, which convert initial perturbations into death signals, that act in the mitochondrial pathway. Now, Gallenne et al. (see p. 279 of this issue) provide mechanistic insight into how the BH3-only protein Puma promotes apoptosis. The authors find that Puma, like the BH3-only proteins Bim and Bid, directly activates Bax.A key event in the commitment to apoptosis is Bax- and Bak-mediated permeabilization of the outer mitochondrial membrane. For this to occur, Bax and Bak alter their conformation from an inactive to an active form, form homo-oligomers in the membrane, and contribute to the formation of pores, which allows the egress of proapoptotic proteins to the cytosol (Fig. 1). Although there is consensus that Bax and Bak must shift from an inactive to an active state for this to occur, there is less consensus about what specific factors cause this crucial switch (Willis et al., 2007). Bid and Bim have been shown to cause activation (conformational change and oligomerization) of Bax and Bak in cellular, mitochondrial, and liposomal systems (Wei et al., 2000; Kuwana et al., 2002; Cartron et al., 2004; Certo et al., 2006). Direct interaction between these activators and Bax has been established experimentally (Gavathiotis et al., 2008; Lovell et al., 2008). Additional studies have suggested that p53 itself may translocate to the mitochondria and activate Bax after select stimuli (Mihara et al. 2003; Chipuk et al., 2004). Even heat has been indicted as a potential activating factor (Pagliari et al., 2005). It is quite possible that many activating factors remain to be discovered.Open in a separate windowFigure 1.Control of mitochondrial permeabilization by Bcl-2 family proteins. Activated Bax or Bak are available to oligomerize either when they are directly activated by activating factors, including activator BH3-only proteins (top), or when preactivated Bax or Bak are displaced from antiapoptotic proteins by either activator or sensitizer BH3-only proteins (bottom). Gallenne et al. (2009) provide evidence that Puma is an activator rather than a sensitizer. Oligomerized Bax or Bak participate in forming a pore that allows egress of proapoptotic factors like cytochrome c. Cytochrome c promotes formation of the apoptosome complex, which causes activation of effector caspases. These proteases cleave many key cellular proteins to bring about the apoptotic phenotype. Figure adapted with permission from the Journal of Cell Science (Brunelle, J.K., and A. Letai. 2009. J. Cell Sci. 122:437–441).Antiapoptotic proteins inhibit apoptosis by binding proapoptotic factors. In many cases, the proapoptotic factors are activator BH3-only proteins like Bid and Bim. However, in some cases, the proapoptotic factors may also include activated monomeric Bax and Bak, which are intercepted before they can oligomerize and form pores. Cells have been described in which antiapoptotic proteins are loaded with abundant prodeath proteins as being “primed for death.” Such cells are particularly sensitive to treatment with chemotherapy and antagonists of antiapoptotic proteins like ABT-737 (Certo et al., 2006; Deng et al., 2007). In most cells, the vast majority of Bax and Bak are in the inactive form, and activated Bax and Bak can be difficult to detect in the absence of toxic perturbation. Nonetheless, BH3-only molecules, which lack the ability to directly activate Bax or Bak, can cause apoptosis by competing for binding to antiapoptotic proteins (Fig. 1). If this competition frees sufficient activator proteins (or activated Bax and Bak), oligomerization of Bax and Bak ensues, committing the cell to death. Based on performance in assays on mitochondria and artificial liposomes spiked with Bax, the BH3-only family has thus been segregated into two subfamilies: the sensitizers and the activators.Where does Puma fit in? Puma was initially identified as a p53-regulated gene that was induced after DNA damage (Nakano and Vousden, 2001). It has subsequently been found that Puma is responsible for much of the proapoptotic effect of p53 induction but that Puma can also cause apoptosis in a p53-independent fashion (Jeffers et al., 2003; Villunger et al., 2003). The assignment of Puma as either a sensitizer or an activator has been somewhat contentious. The BH3 domains of BH3-only proteins are both necessary and sufficient to interact with Bcl-2 family members and seem to largely recapitulate function of the entire protein. For instance, the BH3 domains of Bid and Bim can activate Bax and Bak in liposomal or mitochondrial settings. The Puma BH3 domain lacked this function in several studies, leading many to classify Puma as a sensitizer (Kuwana et al., 2005; Certo et al., 2006). However, experiments with the full-length protein translated in vitro show an ability to activate Bax comparable with that of Bim and Bid (Kim et al., 2006).Cartron et al. (2004) has previously found that the BH3 domains of Bim and Puma but not the sensitizer Bad interact with Bax and cause its activation. In Gallenne et al. (2009), the role of Puma as an activator is further supported by three main pieces of evidence. First, Bax preincubated with the Puma BH3 peptide is more toxic to microinjected cells than is Bax alone. This enhancement is blocked by coincubation with a peptide mimicking the putative interaction site on Bax, the Hα1 C-terminal peptide. This suggests that the interaction of the Puma BH3 domain with a site on the first α helix of Bax is necessary for Puma''s enhancement of Bax killing. It is worth noting that this interaction site on Bax, first identified by this group 4 yr ago, overlaps with an interaction site of the activator Bim BH3 peptide with Bax recently demonstrated by nuclear magnetic resonance in solution (Gavathiotis et al., 2008). The fact that two groups independently identified a similar and unexpected site for interaction of activating BH3 domains with Bax lends some confidence to this finding.Additionally, because the Bcl-2 family is absent from the yeast genome, the authors exploit yeast to study Puma and Bax in a setting uncontaminated by the contribution of unmeasured Bcl-2 family proteins. Again, they find that coexpression of Puma is necessary for efficient killing by Bax. Finally, the authors investigate the participation of Puma in killing human colorectal cancer cells with ABT-737. ABT-737 is a BH3 mimetic that promotes apoptosis by binding antiapoptotic proteins and displacing select prebound prodeath proteins. Thus, ABT-737 can only kill cells that are primed with either activators or preactivated Bax or Bak. They find that ABT-737 treatment results in the freeing of Puma, which then interacts with Bax, correlating with the death of the cell. This finding suggests that Puma can play the priming function that is likely critical to sensitivity to many chemotherapeutic agents as well as ABT-737 (Deng et al., 2007). This role may be particularly important in cells in which Bim and Bid are not expressed at high levels.Some questions remain. It is not clear why several laboratories have consistently failed to observe an activating function for the BH3 domain of Puma in either mitochondrial or liposomal systems. It is possible that even if Puma can play an activating role, the efficiency of this function may vary considerably according to context and perhaps be much less in many contexts than that of Bid or Bim. In a full-length Puma protein, perhaps interactions of the Puma BH3 domain with Bax are enhanced. It is also possible that unknown posttranslational modifications of Puma or Bax, varying according to cellular context, significantly influence the ability of Puma to activate Bax. In any case, Gallenne et al. (2009) have strengthened the case for Puma as an activator so that its potential contribution to this function cannot be ignored. One must now wonder: what other activators might still be out there waiting to be discovered?     

JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1–3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.BH3 interacting-domain death agonist (Bid), a member of BH3-only group of proteins in the Bcl-2 family, functions as a sensor of cellular damage and activator of pro-apoptotic Bax and Bak.^{1, 2} Bid is a 23 kDa protein localized primarily in the cytosol, but upon apoptotic stimuli it is cleaved to yield a truncated 15 kDa C-terminal fragment tBid. tBid translocates to the mitochondrial membrane, where it interacts with Bax and Bak, enhancing their oligomerization and leading to outer membrane permeabilization, loss of membrane potential and release of mitochondrial apoptogens.^{3, 4} The canonical example of the activation of Bid cleavage is the FAS-mediated apoptosis, and Bid is viewed as the key molecule in the integration of death receptor and mitochondrial apoptotic pathways.^{5, 6} The interaction of tBid with Bax or Bak proceeds through the BH3 domain of Bid and occurs only after the protein is localized to mitochondria.⁷ In the full-length Bid, the BH3 domain can be masked by the N-terminal portion of the protein through the interaction with an α-helical BH-3-like region, the BH3-B domain.^{5, 8} The caspase-8 cleavage in the middle of the large flexible loop connecting the BH3 and BH3-B domains leads to structural rearrangements of the C-terminal portion of Bid enabling its insertion into mitochondrial membrane.⁹ The dissociation of the N-terminal fragment in the presence of the mitochondrial membrane and conformational changes of tBid molecule make the BH3 domain accessible for Bax or Bak.¹⁰ Other proteolytic enzymes can cleave Bid within the loop but caspase-8 appears to be a major factor generating tBid.⁸ Full-length Bid can also translocate to mitochondria and induce apoptosis^{11, 12, 13, 14} but its pro-apoptotic activity is weaker than the activity of tBid.¹⁵ It has been hypothesized that in contrast to tBid, the conformational changes enabling the translocation of full-length Bid to mitochondria are reversible.⁹Several studies have also indicated the cleavage-independent pro-survival function of Bid in S-phase checkpoint and highlighted the regulation of Bid by phosphorylation at several residues.^{16, 17} Thus, ATM/ATR protein kinases can phosphorylate Bid at Ser61, Ser64 and Ser78, which protects from caspase-8 cleavage.¹⁷ In response to DNA damage, Bid is phosphorylated by ATM protein kinase and translocates to the nucleus to contribute to the decision of cell fate.^{16, 17} Interestingly, the ablation of phosphorylation at Ser61 and Ser78 ATM sites caused accumulation of full-length Bid in the mitochondria of hematopoetic stem cells and increased cellular proliferation.¹⁸ Furthermore, the phosphorylation of murine Bid at Thr58, Ser61 and Ser64 near the caspase-8 cleavage site by casein kinase I and II protected the protein from cleavage, thus making it less active towards the induction of apoptosis.¹⁹ Moreover, the pro-survival function of Bid was suggested by the finding that its loss inhibited tumorigenesis of T cells.²⁰ Overall, phosphorylation of Bid can serve as a switch between the pro-apoptotic and pro-survival functions of the protein.Although phosphorylation of Bid by c-Jun N-terminal kinase (JNK) has not been demonstrated so far, it has been reported that the alternative processing of Bid, which generates jBid, is JNK-dependent.²¹ Interestingly, the accumulation of full-length Bid and its translocation to mitochondria was observed in HeLa cells in response to staurosporine,²² a known JNK activator.²³ Tight relationships between JNK and Bid have been also demonstrated in mouse models of TNFα-induced liver injury.²⁴ This study indicated that Bid is downstream of JNK in TNFα-induced apoptosis and the pro-apoptotic activity of JNK2 is mainly mediated by Bid. Here we report that in PC-3 cells, JNK1/2 phosphorylate Bid at Thr59 in response to folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), thus protecting Bid from caspase-8 cleavage. This leads to apoptosis owing to a strong accumulation and mitochondrial translocation of full-length Bid.     

CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition

The CD300 receptor family members are a group of molecules that modulate a variety of immune cell processes. We show that mouse CD300b (CLM7/LMIR5), expressed on myeloid cells, recognizes outer membrane-exposed phosphatidylserine (PS) and does not, as previously reported, directly recognize TIM1 or TIM4. CD300b accumulates in phagocytic cups along with F-actin at apoptotic cell contacts, thereby facilitating their engulfment. The CD300b-mediated activation signal is conveyed through CD300b association with the adaptor molecule DAP12, and requires a functional DAP12 ITAM motif. Binding of apoptotic cells promotes the activation of the PI3K-Akt kinase pathway in macrophages, while silencing of CD300b expression diminishes PI3K-Akt kinase activation and impairs efferocytosis. Collectively, our data show that CD300b recognizes PS as a ligand, and regulates the phagocytosis of apoptotic cells via the DAP12 signaling pathway.In both developing and mature multicellular organisms, large numbers of apoptotic cells are continually generated and must be cleared by neighboring cells or ‘professional'' phagocytes.^{1, 2, 3, 4} If not properly cleared, they become necrotic, pro-inflammatory and immunogenic, potentially leading to the development of autoimmune diseases, such as systemic lupus erythematous (SLE).^{5, 6, 7, 8} Therefore, phagocytes possess sensing systems to facilitate the clearance of apoptotic cells.^{1, 2, 3} Once guided to their location by diffusible ‘find me'' signals, phagocytes recognize apoptotic cells through their display of characteristic cell surface molecules (‘eat me'' signals).^{4, 7} The most common signal promoting phagocytosis is the recognition of phosphatidylserine (PS), which when exposed on the outer leaflet of the plasma membrane signals phagocytes to engulf apoptotic cells.² Multiple receptors for PS exist on phagocytic cells, although not necessarily simultaneously; these include stabilins,^{9, 10} T cell Ig mucin (TIM) 1 and TIM4,^{11, 12} BAI1,¹³ MFGE8, which bridges PS to integrin αvβ3,¹⁴ and Protein S and Gas6, which bridge PS to TAM receptors.¹⁵ Recently, we and others demonstrated that the CD300 family members, human and mouse CD300a,^{16, 17} and mouse CD300f,^{18, 19} also bind PS, and their expression regulates apoptotic cell phagocytosis.The CD300 family contains both activating and inhibitory receptor members.²⁰ CD300b has a short intracellular tail and gains activation potential by association with DNAX activating protein of 12 kDa (DAP12) or DAP10 adaptor molecules.²¹ CD300b is predominantly expressed on myeloid cells, including neutrophils, macrophages and mast cells. Antibody cross-linking of human and mouse CD300b has been shown to induce the release of inflammatory cytokines from mast cells.²¹ The ligand for CD300b remains a matter of debate. A recent study found that a soluble form of CD300b, released in response to Toll-like receptor ligation, recognizes unknown ligands on the surface of macrophages, resulting in the release of inflammatory cytokines.²² Others have identified the PS-binding receptors TIM1 and TIM4 as endogenous ligands for CD300b, but not PS itself.²³Here, we show that CD300b binds to PS, and recognizes PS on TIM1 or TIM4 expressing cells rather than TIM1 or TIM4 alone. We found that CD300b promotes PS-dependent apoptotic cell phagocytosis upon ectopic expression in cell lines, without the need for additional PS receptors. In addition, CD300b-mediated phagocytosis requires the association of the adaptor protein DAP12 for effective signaling. Inhibition of CD300b function by either anti-CD300b antibody treatment or siRNA transfection significantly decreases macrophage-dependent phagocytosis of apoptotic cells. Furthermore, CD300b silencing in macrophages severely impairs the apoptotic cell-induced phosphorylation of PI3K, Akt and Syk, but not Erk. Thus, our data show that CD300b is an activating receptor that has an important role in macrophage-mediated clearance of apoptotic cells.     

Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca²⁺ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca²⁺-dependent ROS production.The Bcl-2 proteins are a family of molecules comprised of both pro- and antiapoptotic members essential for the regulation of apoptotic cell death. In the classical paradigm, the antiapoptotic proteins Bcl-2, Bcl-x_L and Mcl-1, inhibit cell death during receipt of apoptotic stimuli by binding and sequestering the proapoptotic members.¹ It is now appreciated, however, that in the absence of apoptotic stimuli, Bcl-2 proteins have numerous non-canonical interactions that influence diverse cellular functions, although the precise mechanisms are poorly understood.² Since antiapoptotic Bcl-2 family members are frequently upregulated in cancer, determining if and how these non-canonical interactions confer survival or other advantages to the cancer cell, will be an important step toward identifying new therapeutic targets. One such interaction is with the outer mitochondrial membrane-localized voltage-dependent anion channel (VDAC), a porin channel with three isoforms that serves as a major diffusion pathway for ions and metabolites,³ and whose gating properties are affected by either Bcl-2 or Bcl-x_L binding.^{4, 5, 6}We recently identified an important role for Bcl-x_L/VDAC interactions in the regulation of mitochondrial [Ca²⁺].⁷ Moving Ca²⁺ from the cytoplasm to the mitochondrial matrix requires transfer across the outer membrane by VDAC^3,8 and across the inner membrane by the Ca²⁺ uniporter.⁹ Our studies showed that Bcl-x_L interacts with VDAC to facilitate Ca²⁺ uptake into the mitochondrial matrix. It is not known if other Bcl-2 family members, particularly Bcl-2 and Mcl-1, which are also known VDAC binding partners impart the same physiological regulation on mitochondrial [Ca²⁺]. Furthermore, the specific physiological consequences and significance of this regulation remain to be determined.Increased production and reduced scavenging of reactive oxygen species (ROS) is frequently observed in cancer cells.¹⁰ While excessive ROS levels are toxic, sub-lethal production serves an important signaling function, particularly in cancers, were ROS promote cell proliferation, migration and invasion.^{11, 12, 13, 14, 15} A primary source of ROS are the mitochondria, and a number of mitochondrial signaling pathways are known to be remodeled and contribute to elevated ROS in cancer cells, including those involved in regulating the electron transport chain (ETC) function and metabolic activity.^{11,16, 17, 18} It is recognized that upregulation of antiapoptotic Bcl-2 proteins are also associated with a pro-oxidant intracellular environment.^{19, 20, 21, 22} Mechanistically, they are thought to act at the level of the mitochondria to affect the respiratory chain and increase production of ROS. Since matrix [Ca²⁺] is an important regulator of mitochondrial metabolism,^23,24 and as such, contributes to the regulation of mitochondrial ROS production,²⁵ we reasoned that antiapoptotic Mcl-1/VDAC interactions could promote ROS generation by facilitating matrix Ca²⁺ uptake.Understanding non-canonical roles of Mcl-1 is an important step toward identifying novel therapeutic targets, particularly in cancers where it is highly expressed, such as in non-small cell lung cancer (NSCLC).^26,27 Therefore, we hypothesized that Mcl-1 binding to VDAC promotes mitochondrial Ca²⁺ uptake and ROS production in NSCLC cells and that this is essential in maintaining the cancer cell phenotype. To test this, we assessed the biochemical interaction between Mcl-1 and VDAC and examined the effects of manipulating Mcl-1 expression levels and Mcl-1/VDAC interactions on mitochondrial Ca²⁺ uptake, ROS generation and NSCLC cell proliferation and migration.     

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.Apoptosis is an important regulatory mechanism of tissue homeostasis. It is triggered by the extrinsic pathway through the activation of proapoptotic receptors or by the intrinsic pathway through the destabilization of mitochondria in response to various forms of cell injury or stress.¹ Notably, stressed cells are also strongly influenced by intercellular communicative networks. This includes diffusible growth factors, cytokines and other small molecules secreted from neighbouring cells, which can modulate the fate of distressed cells. For example, stem cells release growth factors to protect dysfunctional neurons in the brain.² In tumour stroma, activated fibroblasts are thought to promote tumour progression by secreting growth factors that act in a paracrine manner.³ Moreover, contact-dependent signalling, for example, via adhesion molecules, can trigger contact inhibition or protection of endothelial cells.⁴ In addition, gap junctions have been shown to be involved in the transfer of death or survival molecules in different cell types.⁵ Therefore, the signals transferred from neighbouring cells influence the viability of target cells through different pathways.In 2004, our group described a previously unrecognized form of cell-to-cell interaction based on nanoscaled, F-actin-containing membrane tubes.^{6, 7} These tubes, referred to as membrane or tunneling nanotubes (TNTs), were subsequently found in numerous cell types in culture and in tissues.^{8, 9, 10, 11} Importantly, TNTs facilitate the intercellular exchange of diverse cellular signals and components ranging from electrical signalling to organelles.^{12, 13, 14, 15} Moreover, pathogens such as human immunodeficiency virus (HIV) and prions can spread between cells along TNTs.^{16, 17} Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells to alter the fate of recipient cells. Indeed, it has been shown that TNTs can propagate the death signal Fas ligand between T lymphocytes to induce cell death.^{18, 19} TNTs have been also proposed to participate in the rescue of injured cardiomyoblasts or endothelial cells by mesenchymal stem cells (MSCs) through transferred mitochondria.^{20 ,21} However, the rescue mechanism by how and when this event was accomplished remains elusive.In this study, we found that PC12 cells stressed by ultraviolet (UV) radiation were rescued from apoptosis when cocultured with untreated, healthy PC12 cells. Single-cell analysis showed that stressed cells in the early stages of apoptosis form a new type of TNT to interact with untreated cells. These TNTs have a distinct cytoskeletal composition and biophysical properties when compared with TNTs interconnecting normal PC12 cells. We also observed the presence and transport of mitochondria in the TNTs formed by stressed cells. Notably, the rescue effect was inhibited when the formation of TNTs were impaired by incubating with an F-actin-depolymerizing drug, or when the mitochondria of rescuer cells were damaged. Our results suggest that the delivery of functional mitochondria via TNTs mediates the recovery of PC12 cells in the early stages of apoptosis.     

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, Bim_EL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.The intrinsic mitochondrial apoptotic pathway is required for efficient chemotherapeutic killing of cancer cells,¹ and is initiated through BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). MOMP releases cytochrome c to activate effector caspases.² Conversely, inhibitor of apoptosis protein (IAP) family members suppress initiator and effector caspases via direct binding and E3 ligase activities.^{3, 4, 5} Consequently, MOMP-induced release of Smac (second mitochondria-derived activator of caspases) from the mitochondria, to inhibit XIAP (X-chromosome-linked IAP)-mediated caspase suppression, can be required for apoptosis.⁶Autophagy, a lysosomal degradative mechanism undergoing extensive crosstalk with cell death and survival pathways,⁷ degrades damaged mitochondria in a process termed mitophagy.^{8, 9} Damaged mitochondria are targeted by lysosomal degradation through the recruitment of autophagy receptors to the outer mitochondrial membrane (OMM),⁸ or via delivery of mitochondrial-derived vesicles (MDVs) directly to the lysosome.¹⁰ The E3 ubiquitin ligase Parkin targets and ubiquitylates mitochondria, mediating both MDV degradation¹¹ and autophagy receptor-dependent mitophagy.^{12, 13} Alternatively, Fundc1¹⁴ and atypical BH3-only Nip family proteins Bnip3 and Bnip3L/Nix localize to the OMM and act as mitophagy receptors via their LC3-interacting region (LIR).^{15, 16, 17, 18} While targeting of damaged mitochondria suggests that mitophagy may counter apoptotic mitochondria, mitophagy occurs progressively over days,^{12, 14, 16, 17, 19} a rate that is likely insufficient to alter intracellular propagation of mitochondrial apoptosis, which can occur within minutes.^{20, 21} Indeed, Bnip3- and Fundc1-induced mitophagy have no direct effect on apoptosis,^{14, 18} and we determined that Bnip3-mediated mitophagy was cytoprotective if activated before apoptosis.¹⁷ While MDV delivery of mitochondria to lysosomes operates at a higher rate, minutes to hours,¹⁰ this process is regulated by Parkin and restricted to specific mitochondrial components.¹¹ Overall, for most intrinsic apoptosis scenarios it remains unknown whether lysosomal processing of mitochondria influences their capacity to activate or enhance apoptosis.Here, we used high-resolution imaging to evaluate the behavior of apoptosis, autophagy, lysosomal and ubiquitylation pathways in response to canonical (tBid, Bim_EL, Bik, Bad) and atypical (Bnip3, Bnip3L/Nix) BH3-only protein expression. We report that, in parallel to intrinsic apoptosis signaling, canonical BH3-only proteins induce the recruitment of endolysosomal machinery, in the absence of mitophagy. We determined that mitochondrial depolarization rapidly translocates the caspase inhibitor XIAP to the mitochondria. There, XIAP actuates MOMP within all mitochondria, concomitant with ubiquitylation at the OMM and inside OMM-bound regions, and triggers ubiquitin-dependent recruitment of Rab5 and its binding partners, as well as late endosomes into the mitochondria. Consequently, in a manner dependent on lysosome- and proteasome-activities, XIAP degrades its inhibitor Smac. We propose that in response to bioenergetic stress, the functional integration between lysosomes and mitochondria, mediated by XIAP and independent of autophagy, offers a novel mechanism to modulate mitochondrial apoptosis.     

Stabilization of apoptotic cells: generation of zombie cells

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn²⁺ (a caspase inhibitor) and coenzyme Q₁₀ (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.Apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in multicellular organisms, including humans.¹ The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (increased apoptosis) or cancer (reduced apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of apoptosis by radiation, chemotherapy or both. In particular, many antineoplasic drugs such as campothecin, a topoisomerase I inhibitor, kill tumor cells by inducing apoptosis.Apoptosis is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells and potential harmful secondary effects. However, when massive cell death overwhelms macrophage clearance, as for example in early postchemotherapy or viral infection,² apoptotic cells may progress to secondary necrosis characterized by cell membrane degradation with spillage of intracellular contents to the extracellular milieu.³ Similarly, cells undergoing apoptosis in vitro cannot usually be cleared by phagocytes and undergo a late process of secondary necrosis.⁴In the execution phase of apoptosis, effector caspases cleave vital cellular proteins, leading to the morphological changes that characterize apoptosis. These changes include destruction of the nucleus and other organelles, DNA fragmentation, chromatin condensation, cell shrinkage, cell detachment and membrane blebbing.⁵ In apoptosis, all the degradative processes are isolated from the extracellular space by the plasma membrane that remains impermeable. However, the mechanisms involved in plasma membrane and associated protein protection from the action of caspases are not completely understood. In contrast, necrosis is accompanied by disruption of plasma membrane integrity with the subsequent release of all intracellular compounds to the intercellular space, thus inducing inflammation and more toxic effects to adjacent cells.^{6, 7}To allow the dramatic morphological changes that accompany the execution phase, an apoptotic cell undergoes a series of profound cytoskeletal breakdowns/rearrangements. Previous evidence suggests that the actomyosin cytoskeleton plays an essential role in apoptotic cell remodeling during the early events of the execution phase, whereas all other cytoskeleton elements (microtubules and intermediate filaments) are dismantled.⁸ However, during the course of the execution phase and after actininomyosin ring contraction, the actomyosin filaments are also depolymerized by a caspase-dependent mechanism. In this situation, the apoptotic cell forms a network of apoptotic microtubules that becomes the main cytoskeleton element of the apoptotic cell. The presence of microtubules in apoptotic cells has previously been reported.^{9, 10} Moreover, more recent results indicate that microtubules during apoptosis assist in the dispersal of nuclear and cellular fragments,^{11, 12} and may help to preserve the integrity of plasma membrane of the dying cell.¹³Reactive oxygen species (ROS) are also important mediators of apoptosis. ROS have been shown to play a major role in apoptosis signaling.^{14, 15, 16} Electron leak in the presence of oxygen during the process of oxidative phosphorylation make mitochondria the major endogenous source of ROS in the cell. Although mitochondria have been identified as a key player, the mechanism connecting ROS and apoptosis remains unclear.¹⁷ It has been debated whether increased ROS during apoptosis is a cause or a consequence of impaired mitochondrial function, and whether ROS are a death signal to the mitochondria or are produced as effector molecules by the mitochondria in response to apoptosis signal.^{18, 19} Hyperproduction of ROS in execution stages of apoptosis is thought to be caused by the disruption of the mitochondrial respiratory chain after release of cytochrome c into the cytosol.²⁰The main objective of this work was to develop a method for the stabilization of apoptotic cells for proper apoptosis detection or safer potential therapeutic applications. Our results show that apoptotic cells can be stabilized by a cocktail of a microtubule stabilizer (taxol), a caspase inhibitor such (Zn²⁺) and an antioxidant (coenzyme Q₁₀ (CoQ)).