Native, not nitrated, cytochrome c and mitochondria-derived hydrogen peroxide drive osteoclast apoptosis

Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H₂O₂ production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H₂O₂ production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H₂O₂. Trapping H₂O₂-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts. reactive oxygen species; nitric oxide; free radicals; caspase     

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.     

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins.     

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.     

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.     

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.     

Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulfide

The present study investigated the mechanism of mouse pancreatic acinar cell apoptosis induced by H₂S in an in vitro system, using isolated pancreatic acini. Treatment of pancreatic acini with 10 µM NaHS (a donor of H₂S) for 3 h caused phosphatidylserine externalization as shown by annexin V binding, an indicator of early stages of apoptosis. This treatment also resulted in the activation of the caspase cascade and major changes at the mitochondrial level. Caspase-3, -8, and -9 activities were stimulated by H₂S treatment. Treatment with inhibitors of caspase-3, -8, and -9 significantly inhibited H₂S-induced phosphatidylserine externalization as shown by reduced annexin V staining. The mitochondrial membrane potential was collapsed in H₂S-treated acini as evidenced by fluorescence microscopy and quantitative analysis. Furthermore, the treatment of acini with H₂S caused the release of cytochrome c by the mitochondria. To investigate the mechanism underlying pancreatic acinar cell apoptosis, we also characterized the protein expression of a range of molecules that are each known to influence the apoptotic pathway. Among proapoptotic proteins, Bax expression was activated in H₂S-treated cells but not Bid, and the antiapoptotic proteins Bcl-X_L and Bcl-2 did not show any activation in pancreatic acinar cell apoptosis. The death effector domain-containing protein Flip is downregulated in H₂S-treated acini. These results demonstrate the induction of pancreatic acinar cell apoptosis in vitro by H₂S and the involvement of both mitochondrial and death receptor pathways in the process of apoptosis. caspase; H₂S; mitochondria; Bcl-2; Flip     

Specific interaction of the antiapoptotic protein Nr-13 with phospholipid monolayers is prevented by the BH3 domain of Bax

Members of the Bcl-2 protein family regulate apoptosis by controlling the release of apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. Proapoptotic members induce release by increasing outer membrane permeability, while antiapoptotic members prevent this. The activity of Bcl-2 proteins depends mostly on their insertion into the mitochondrial membrane, which is reported to occur via putative channels formed by the two central hydrophobic helices. The pro- and antiapoptotic activity of Bcl-2 proteins can also be modulated by heterodimerization between antagonists through the BH3 domain of proapoptotic members, though the position of the heterodimer with respect to the membrane has never been elucidated. In this work, the membrane insertion capacity of the antiapoptotic Bcl-2 related protein Nr-13 was explored, using monolayer expansion measurements. Nr-13 penetrates into the monolayer with a molecular cross-section of 1100A(2), thereby implicating almost all alpha-helical domains of the molecule in this process. A mutant protein, bearing neutral instead of acidic residues in the loop between the two putative channel-forming fifth and sixth alpha-helices, retained the ability to interact with the lipid monolayer, suggesting that the membrane insertion of Nr-13 is not exclusively alpha5-alpha6-dependent. In contrast, the specific interaction of Nr-13 with the monolayer was prevented by heterodimer formation with the BH3 domain of proapoptotic Bax. These findings are discussed in terms of a model for monolayer insertion in which the antiapoptotic Nr-13 and proapoptotic proteins exert their antagonistic effects by preventing each other from reaching the membrane.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

The Role of the Mitochondrial Apoptosis Induced Channel MAC in Cytochrome <Emphasis Type="Italic">c</Emphasis> Release

Permeabilization of the mitochondrial outer membrane is a crucial event during apoptosis. It allows the release of proapoptotic factors, like cytochrome c, from the intermembrane space, and represents the commitment step in apoptosis. The mitochondrial apoptosis-induced channel, MAC, is a high-conductance channel that forms during early apoptosis and is the putative cytochrome c release channel. Unlike activation of the permeability transition pore, MAC formation occurs without loss of outer membrane integrity and depolarization. The single channel behavior and pharmacology of reconstituted MAC has been characterized with patch-clamp techniques. Furthermore, MAC’s activity is compared to that detected in mitochondria inside the cells at the time cytochrome c is released. Finally, the regulation of MAC by the Bcl-2 family proteins and insights concerning its molecular composition are also discussed.     

The role of the interaction between signaling protein domains and of the complexes of signaling proteins in apoptosis initiation

The data of recent years on apoptosis were revisited to demonstrate that the functioning of signaling proteins during apoptosis depends on their localization on mitochondria or in the cytosol. The major effect of signaling proteins depends on the number of pro-and antiapoptotic domains in their structure, which is observed after cleavage, oligomerization, and complexing with other proteins. The structure of known signaling proteins was analyzed. The effect of complexing with phosphatases and 14-3-3 proteins was demonstrated by the example of Bad protein. Detailed data on the proapoptotic factors and their inhibitors affecting caspase activation and released from mitochondria with cytochrome c are given.     

Contribution of apoptotic cell death to renal injury

Cell number abnormalities are frequent in renal diseases, and range from the hypercellularity of postinfectious glomerulonephritis to the cell depletion of chronic renal atrophy. Recent research has shown that apoptosis and its regulatory mechanisms contribute to cell number regulation in the kidney. The role of apoptosis ranges from induction to repair and progression of renal injury. Death ligands and receptors, such as TNF and FasL, proapoptotic and antiapoptotic Bcl-2 family members and caspases have all been shown to participate in apoptosis regulation in the course of renal injury. These proteins represent potential therapeutic targets, which should be further explored.     

ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus

Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax

Mitochondria act as a focal point for upstream apoptosis signals by releasing cytochrome c into the cytosol, leading to the activation of caspases and subsequent cell death. Members of the Bcl-2 protein family regulate this phenomenon by heterodimerization via the BH3 domain of proapoptotic members opposing their pro- and antiapoptotic functions. The mechanism of cytochrome c release from mitochondria and of its regulation remains controversial. In vitro binding studies of purified and biologically active proteins should help in understanding the molecular mechanism of interactions and protein functions. In this work, the Bcl-2-related antiapoptotic chicken protein Nr-13 was overexpressed as a highly soluble recombinant protein which showed correct folding as judged by circular dichroism and fluorescence spectroscopy. Purified Nr-13 inhibits caspase-3 activation in a Xenopus egg-derived cell-free system, and neutralizes the proapoptotic activity of a synthetic peptide containing the BH3 domain of Bax. The latter effect correlates with the high-affinity binding of the BH3 peptide to Nr-13 as monitored by the intrinsic tryptophan fluorescence. On the basis of the structural similarity with Bcl-x(L), putative residues involved in this interaction were identified. Nr-13 exhibits a high-affinity interaction with cytochrome c which is prevented by preincubation with the BH3-Bax peptide. These findings are discussed with respect to a model for the regulation of apoptosis in which a direct interaction between the antiapoptotic protein and cytochrome c may prevent the apoptosis.