Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X_L, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X_L. However, inactivation of both Mcl-1 and Bcl-X_L was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X_L indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.The induction of cell death is a major mechanism by which many viruses cause disease in the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for therapeutic applications, such as the development of oncolytic viruses for the treatment of cancer (27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA viruses and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of multiple apoptotic pathways. It is important to determine how these pathways are activated and the role that they play in apoptosis induced by VSV in order to understand the virulence and oncolytic activity of the virus, as well as to provide a model to which other viruses can be compared.Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial (intrinsic) pathway through the initiator caspase caspase-9 (4, 19). This is due in part to the inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs primarily via the extrinsic pathway through the initiator caspase caspase-8 (12, 13). Infection with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the induction of apoptosis is most likely mediated by proteins already present in the host cell. Since it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt VSV.Bcl-2 family proteins function to either suppress or promote mitochondrial outer membrane permeabilization, thereby regulating the release of proapoptotic factors into the cytosol, such as cytochrome c, apoptosis-inducing factor (AIF), and Smac/Diablo (5). Bcl-2 family proteins are subdivided into three groups, depending on the conservation of Bcl-2 homology (BH) domains and function (reviewed in references 8 and 38). The multidomain antiapoptotic Bcl-2 proteins contain BH domains BH1 to BH4 and function to inhibit apoptosis by binding to proapoptotic Bcl-2 family members. Members of this group include Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and BFL-1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, containing BH domains BH1 to BH3. These proteins are primarily responsible for the permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology domain (BH3) and include Bid, Bad, Bim, Puma, Noxa, and Bik, among others. These proteins function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins by either binding to antiapoptotic proteins, causing the release of Bak and Bax, or binding to Bak and Bax, thereby causing their activation (6).The pathways leading to activation of Bak differ from those that activate Bax. Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-X_L, have been shown to interact with Bak, while Bax appears to be able to interact with all of the antiapoptotic proteins, with the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For some stimuli, such as the protein kinase inhibitor staurosporine (SSP), the topoisomerase II inhibitor etoposide, and UV radiation, Bak and Bax appear to be redundant, in that the deletion of both is required to render cells resistant to these agents (33). In contrast, Bak and Bax were nonredundant in the induction of apoptosis by Neisseria gonorrhoeae and cisplatin, such that both were required for apoptosis to occur (18).In the experiments reported here, the silencing of Bak or Bax expression with small interfering RNA (siRNA) showed that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt VSV. Overexpression of both of the antiapoptotic Bcl-2 family proteins known to interact with Bak, Mcl-1 and Bcl-X_L, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-X_L by siRNA transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition of new host gene expression led to the depletion of Mcl-1, enabling the rapid activation of apoptosis. However, inhibition of Bcl-X_L was also required for the initiation of apoptosis, indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, was shown to be important for induction of apoptosis by VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.     

Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1

Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax^−/−) MEFs but failed in Bak knockout (Bak^−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax^−/− MEFs, but not in Bak^−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x_L, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x_L in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x_L inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.Apoptosis is a mode of cell death utilized by multicellular organisms to remove unwanted cells. Also, many different cancer treatments, including chemotherapy and radiotherapy, induce apoptosis and result in the destruction of tumor cells. In some cases, apoptosis resistance can contribute to the failure of chemotherapy (14, 20, 24). Immunotoxins are a class of antitumor agents in which a powerful protein toxin is brought to the cancer cell by an antibody or an antibody fragment (for reviews, see references 28, 29, and 32). Several immunotoxins are currently in clinical trials, and one of these, BL22, targeting CD22, has shown excellent activity in drug-resistant hairy-cell leukemia (18, 19). Also, a fusion protein in which a fragment of diphtheria toxin is fused to the cytokine interleukin 2 (IL-2) (Ontak) is approved for the treatment of cutaneous T-cell lymphoma (26). Several studies carried out to determine how protein toxins and immunotoxins containing these toxins kill target cells have reported caspase activation (13, 16, 17, 30, 33). However, the steps leading up to caspase activation by these toxins that inhibit protein synthesis have not been elucidated.Bcl-2 family members are essential regulators of the mitochondrial (intrinsic) apoptosis pathway (1, 21). Proteins of this family have been divided into pro- and antiapoptotic proteins. Antiapoptotic proteins include the multi-Bcl-2 homology (BH) domain proteins Bcl-2, Bcl-x_L, Bcl-w, Mcl-1, Bcl-b, and Bcl2a1. Proapoptotic members can be further classified into two subfamilies, the multi-BH domain Bax homologues, including Bax, Bak, and Bok, and the BH3-only proteins, including Nbk/Bik, Noxa, Hrk, Bad, Bim, Puma, and Bmf. Bax and Bak are the most extensively studied central mediators in the mitochondrial apoptosis pathway (4, 6). Various stimuli, including pathogens, toxic drugs, irradiation, and starvation, induce a conformational change and activation of Bak/Bax, usually via BH3-only proapoptosis proteins. This results in the disruption of mitochondrial membranes and the release of apoptotic factors, such as cytochrome c, SMAC, and apoptosis-inducing factor, which lead to the activation of effector caspases (5, 37, 40, 42, 43).The roles of Bax and Bak can be redundant or nonredundant, depending on the apoptotic stimuli. Bak and Bax can compensate for each other in apoptosis induced by staurosporine, etoposide, UV irradiation, serum deprivation, tBid, Bim, Bad, or Noxa (37, 43). Bak plays an essential role for apoptosis induced by Semliki Forest virus, gliotoxin, Bcl-x_S, and vinblastine (22, 27, 34, 35), while Bax is favored for apoptosis induced by Nbk/Nik, a combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ionizing irradiation, or TRAIL and 5-fluorouracil (5-FU) (9, 10, 36, 38). Silencing of either Bak or Bax resulted in resistance to apoptosis induced by Neisseria gonorrhoeae and cisplatin (15). Sometimes the same stimulus may result in different outcomes in different cell types. NBK/Bik mediated Bax-dependent cell death in one study (9), while in another study, NBK/Bik activated BAK-mediated apoptosis (31).In the current study, we utilized mutant mouse embryo fibroblasts (MEFs) deficient in Bak, Bax, or both proteins and provided evidence for an essential role of Bak in apoptosis induced by Pseudomonas exotoxin A (PE) and other protein synthesis inhibitors. We found that Bak^−/− cells are resistant to killing by PE and that Mcl-1, which binds to Bak, controls apoptosis induced by PE.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of 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.     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

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.     

Mcl-1 Integrates the Opposing Actions of Signaling Pathways That Mediate Survival and Apoptosis

Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.Mcl-1 is an antiapoptotic member of the Bcl2 family. Gene knockout studies of mice demonstrate that Mcl-1 is essential for embryonic development and for the survival of hematopoietic cells (28-30). Studies of the stress response have demonstrated that Mcl-1 plays an important role in the sensitization of cells to apoptotic signals (1, 11, 25). Thus, exposure to UV radiation causes the rapid degradation of Mcl-1 and the release of proapoptotic partner proteins from Mcl-1 complexes (e.g., Bim). The mechanism of rapid Mcl-1 destruction is mediated by the combined actions of two different pathways. First, the exposure to stress causes phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) on the inhibitory site Ser-51 that prevents translation of Mcl-1 mRNA (1, 11, 25). Second, Mcl-1 is rapidly degraded by the ubiquitin-dependent proteasome pathway (27). Together, these pathways cause a rapid reduction in Mcl-1 expression. This loss of Mcl-1 may be a required initial response for the apoptosis of cells exposed to stress (25).The E3 ubiquitin protein ligase Mule/ARF-BP1 contains a BH3 domain that interacts with Mcl-1 and can initiate ubiquitin-dependent degradation of Mcl-1 (39). Recent studies have demonstrated that rapid stress-induced degradation of Mcl-1 is mediated by an alternative pathway involving the E3 ubiquitin protein ligase β-TrCP, which binds a stress-induced phosphodegron created by the phosphorylation of Mcl-1 by glycogen synthase kinase 3 (GSK3) (7, 21). How the exposure to stress causes GSK3-mediated phosphorylation of Mcl-1 is unclear, but GSK3 has been shown to directly phosphorylate Mcl-1 (7, 21). Mcl-1 phosphorylation and degradation may therefore be controlled by the prosurvival AKT pathway, which can negatively regulate GSK3 (7, 21).Mcl-1 is critically involved in the regulation of cell survival and is therefore subject to regulation by multiple mechanisms (26). Thus, Mcl-1 gene expression is regulated by many growth factors and cytokines (26), and Mcl-1 mRNA is regulated by microRNA pathways (24). The Mcl-1 protein is stabilized by binding TCTP (20) and the BH3-only protein Bim (4). In contrast, the BH3-only protein Noxa binds and destabilizes Mcl-1 (4, 36). Moreover, it is established that Mcl-1 is phosphorylated by several protein kinases on sites that may regulate Mcl-1 function. Phosphorylation of human Mcl-1 (hMcl-1) on Ser-64 (a site that is not conserved in other species) may enhance antiapoptotic activity by increasing the interaction of Mcl-1 with Bim, Noxa, and Bak (18). Phosphorylation on Ser-121 and Thr-163 may inhibit the antiapoptotic activity of hMcl-1 (15), and phosphorylation on Thr-163 may increase hMcl-1 protein stability (9). The conserved GSK3 phosphorylation site Ser-159 (and possibly Ser-155) can initiate rapid proteasomal degradation of hMcl-1 (7, 21). Together, these findings suggest that the function of Mcl-1 is very tightly regulated.The results of previous studies have implicated the c-Jun N-terminal protein kinase (JNK) in the regulation of Mcl-1 (15, 18). The purpose of this study was to test whether Mcl-1 is a target of signal transduction by JNK. We demonstrate that a key function of JNK is to prime Mcl-1 for phosphorylation by GSK3. JNK is required for GSK3-mediated degradation of Mcl-1 in response to stress. Coordinated regulation of the stress-activated JNK pathway and the AKT-inhibited GSK3 pathway is therefore required for stress-induced Mcl-1 degradation.     

GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product,Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein

KS-Bcl-2, encoded by Kaposi''s sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a gamma 2 herpesvirus implicated in several cancers, including KS, primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s disease. Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a tumorigenic gamma 1 herpesvirus known to be associated with lymphomas and nasopharyngeal carcinoma (10, 12).KSHV open reading frame 16 (orf16) encodes the KS-Bcl-2 protein, which shares sequence and functional homology with the Bcl-2 family (9, 31). Members of the Bcl-2 family are defined by the presence of up to four conserved domains known as the Bcl-2 homology (BH) domains. Several members also possess a carboxy-terminal transmembrane domain that mediates their association with intracellular membranes, such as the endoplasmic reticulum or mitochondria. Bcl-2 proteins are thought to serve primarily as cell death agonists or antagonists that integrate diverse survival and death signals, which are generated outside and within the cell (15, 37), yet Bcl-2 proteins also modulate cell cycle checkpoints, DNA repair/recombination pathways, calcium homeostasis, and cellular bioenergetics.All gammaherpesviruses encode Bcl-2 proteins that generally share 20 to 30% homology with one another and with their cellular counterparts (8, 11). The conservation of Bcl-2 homologues in these viruses indicates their importance for viral infection, with an evolutionarily conserved function of unknown nature. KS-Bcl-2, like most herpesvirus homologues of Bcl-2, contains a transmembrane domain and demonstrates conservation of sequences in both BH1 and BH2 but has only a low degree of homology with other regions of cellular Bcl-2 (18, 22). Still, KS-Bcl-2 shares 3-dimensional structural conservation with Bcl-2 family members and includes the conserved BH3 binding groove and a hydrophobic membrane anchor domain that also contains a mitochondrial outer membrane targeting signal (18). The BH3 binding cleft of KS-Bcl-2 binds with high affinity to peptides encoding BH3 domains present on the proapoptotic proteins Noxa, Bik, PUMA, Bak, Bax, Bid, Bim, and, to a much lesser extent, Bad (13, 18, 22). Based on these characteristics, KS-Bcl-2 has been suggested to have the closest resemblance to the cellular Bcl-2 family member Mcl-1 (13).Previous studies have demonstrated that KS-Bcl-2 protects various cell types from apoptosis mediated by the expression of BAX, tBid, or Bim through Sindbis virus infection or by ectopic expression of KSHV-cyclin-CDK6 (9, 13, 25, 31). However, unlike the cellular Bcl-2, KS-Bcl-2 is not a substrate for KSHV-cyclin-CDK6 phosphorylation (25) and cannot be converted into a proapoptotic protein via caspase cleavage (3). KS-Bcl-2 is able to form a stable complex with the cellular protein Aven, which binds Apaf-1 and is known as a regulator of caspase 9 and ataxia-telangiectasia (ATM) activation (7, 16). Like the cellular and other virus-encoded Bcl-2 proteins, KS-Bcl-2 binds Beclin and disrupts its lysosomal degradation pathway of autophagy (21, 29). However, since KS-Bcl-2 lacks the nonstructured loop located between the BH4 and BH3 domains, its binding to BH3-containing proapoptotic proteins and to the BH3-containing proautophagy protein Beclin is not modulated by phosphorylation (38).KS-Bcl-2 is transcribed during lytic virus infection (30, 31). Thus, inhibition of apoptosis and autophagy by KS-Bcl-2 may provide an attractive mechanism for prolonging the life span of KSHV-infected cells, which in turn enables increased virus production or establishment of latency. Whether the function of KS-Bcl-2 is necessary for KSHV-mediated oncogenesis is still unknown. Nevertheless, the KS-Bcl-2 protein is expressed in late-stage KS lesions but has not been detected in latent or in lytic KSHV-infected PEL cells (39).To explore the role of KS-Bcl-2 in cell signaling, we searched for its potential cellular-protein partners. In the present study, we describe a novel interaction between KS-Bcl-2 and the protein interacting with carboxyl terminus 1 (PICT-1) cellular protein, encoded by a candidate tumor suppressor gene, GLTSCR2. We show that this interaction specifically targets KS-Bcl-2 to the nucleolus and decreases its antiapoptotic activity.(Portions of this work were submitted to Bar Ilan Univeristy, Ramat Gan, Israel, by I. Kalt and T. Borodianskiy-Shteinberg in partial fulfillment of the requirements for the degree of Doctor of Philosophy.)     

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.     

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?     

Functional Cooperation of the Proapoptotic Bcl2 Family Proteins Bmf and Bim In Vivo

Bcl2-modifying factor (Bmf) is a member of the BH3-only group of proapoptotic proteins. To test the role of Bmf in vivo, we constructed mice with a series of mutated Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation by the c-Jun NH₂-terminal kinase (JNK) on Ser⁷⁴, or mimic Bmf phosphorylation on Ser⁷⁴. We report that the loss of Bmf causes defects in uterovaginal development, including an imperforate vagina and hydrometrocolpos. We also show that the phosphorylation of Bmf on Ser⁷⁴ can contribute to a moderate increase in levels of Bmf activity. Studies of compound mutants with the related gene Bim demonstrated that Bim and Bmf exhibit partially redundant functions in vivo. Thus, developmental ablation of interdigital webbing on mouse paws and normal lymphocyte homeostasis require the cooperative activity of Bim and Bmf.Bmf is a proapoptotic BH3-only member of the Bcl2-related protein family that is implicated in cell death caused by anoikis (23, 26, 27), arsenic trioxide (19), histone deacetylase inhibitors (33, 34), transforming growth factor β (24), and tumor necrosis factor alpha (8). Mice with a loss of Bmf expression exhibit B-cell hyperplasia and increased sensitivity to γ-radiation-induced B-cell lymphoma (14). These observations indicate that Bmf represents an important mediator of cell death signaling pathways.The structure of Bmf includes a BH3 domain that is essential for apoptosis induction. In addition, Bmf contains a sequence motif that is required for interactions with dynein light chain 2 (DLC2), a component of the myosin V motor complex (23). The interaction of Bmf with DLC2 is required for the recruitment of Bmf to the cytoskeleton. The release of Bmf from complexes sequestered on the cytoskeleton may contribute to anoikis (23). Interestingly, this regulatory mechanism is shared by the related proapoptotic BH3-only protein Bim, which interacts via a similar sequence motif with dynein light chain 1 (DLC1), a component of the dynein motor complex (22).The similarities between Bmf and Bim include the presence of a conserved phosphorylation site (Bmf Ser⁷⁴ and Bim Thr¹¹²) that is a substrate for the c-Jun NH₂-terminal kinase (JNK) (15). Data from biochemical studies indicate that the JNK-mediated phosphorylation of Bmf and Bim may increase apoptotic activity (15). Indeed, mice with a germ line point mutation in the Bim gene (Thr¹¹² replaced with Ala) exhibit decreased apoptosis (10). These studies indicate that Bmf and Bim may mediate, in part, proapoptotic signaling by JNK (3, 30).The purpose of this study was to examine the role of Bmf using mouse models with germ line defects in the Bmf gene, including mice with Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation, or mimic Bmf phosphorylation. We examined the effects of these mutations in mice with both wild-type and mutant alleles of the related gene Bim. The results of our analysis demonstrate that Bmf and Bim exhibit partially redundant functions, that phosphorylation on Ser⁷⁴ is not essential for Bmf activity, and that phosphorylation on Ser⁷⁴ can contribute to increased levels of Bmf activity in vivo.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).