BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.     

Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell responses

Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear. Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses in vivo. We found, at the peak of the response to lymphocytic choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4⁺ and CD8⁺ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death, whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4⁺ and CD8⁺ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1

As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by three myeloma cell survival factors: interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and insulin-like growth factor (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from four cell lines were IL-6 starved and activated with IL-6 or IFN-alpha, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-alpha stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism. Cell Death and Differentiation (2000) 7, 1244 - 1252.     

Regulation of mitochondrial metabolism: yet another facet in the biology of the oncoprotein Bcl-2

The Bcl-2 (Bcl is B-cell lymphocytic-leukaemia proto-oncogene) family comprises two groups of proteins with distinct functional biology in cell-fate signalling. Bcl-2 protein was the first member to be discovered and associated with drug resistance in human lymphomas. Since then a host of other proteins such as Bcl-xL, Bcl-2A1 and Mcl-1 with similar anti-apoptotic functions have been identified. In contrast, the pro-apoptotic Bcl-2 proteins contain prototypic effector proteins such as Bax and Bak, and the BH3 (Bcl-2 homology)-only proteins comprising Bak, Bid, Bim, Puma and Noxa. A complex interplay between the association of pro-apoptotic and anti-apoptotic proteins with each other determines the sensitivity of cancer cells to drug-induced apoptosis. The canonical functional of Bcl-2 in terms of apoptosis inhibition is its ability to prevent mitochondrial permeabilization via inhibiting the translocation and oligomerization of pro-apoptotic proteins such as Bax; however, more recent evidence points to a novel mechanism of the anti-apoptotic activity of Bcl-2. Overexpression of Bcl-2 increases mitochondrial oxygen consumption and in doing so generates a slight pro-oxidant intracellular milieu, which promotes genomic instability and blocks death signalling. However, in the wake of overt oxidative stress, Bcl-2 regulates cellular redox status thereby preventing excessive build-up of ROS (reactive oxygen species), which is detrimental to cells and tissues. Taken together, the canonical and non-canonical activities of Bcl-2 imply a critical involvement of this protein in the processes of tumour initiation and progression. In the present paper we review these functionally distinct outcomes of Bcl-2 expression with implications for the chemotherapeutic management of cancers.     

Alpha 5/6 helix domains together with N-terminus determine the apoptotic potency of the Bcl-2 family proteins

A critical process in apoptosis is the permeabilization of the mitochondrial outer membrane (MOM). This process is known to be regulated by the multi-domain Bcl-2 family proteins. For example, the pro-apoptotic proteins Bax and Bak are responsible for forming pores at MOM. The anti-apoptotic proteins (including Bcl-2, Mcl-1 and Bcl-xL), on the other hand, can inhibit this pore-forming process. Interestingly, although these two subgroups of proteins perform opposite apoptotic functions, their structures are very similar. This raises two highly interesting questions: (1) Why do these structurally similar proteins play opposite roles in apoptosis? (2) What are the roles of different functional domains of a Bcl-2 family protein in determining its apoptotic property? In this study, we generated a series of deletion mutants and substitution chimera, and used a combination of molecular biology, bio-informatics and living cell imaging techniques to answer these questions. Our major findings are: (1) All of the Bcl-2 family proteins appear to possess an intrinsic pro-apoptotic property. (2) The N-termini of these proteins play an active role in suppressing their pro-apoptotic function. (3) The apoptotic potency is positively correlated with membrane affinity of the alpha 5/6 helix domains. (4) Charge distribution flanking the alpha 5/6 helices is also important for the apoptotic potency. These findings explain why different members of Bcl-2 family proteins with similar domain composition can function oppositely in the apoptotic process.     

Accumulation of the pro-apoptotic factor Bak is controlled by antagonist factor Mcl-1 availability

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to reinforce or downgrade signals leading to the irrevocable entry into apoptosis. Bak (Bcl-2-antagonist killer) is a mitochondrial pore-forming pro-apoptotic effector inhibited through titration by the anti-apoptotic protein Mcl-1 (Myeloid cell leukemia-1). Viruses have taken advantage of proteasome-dependent degradation of Bak as a mechanism to prevent apoptosis in infected cells. It is not clear however whether regulation of Bak protein level is involved in other physiological processes. In this report, we show that Mcl-1 level is paralleled by Bak while a Mcl-1 non-interacting mutant of Bak does not accumulate in cells. This mechanism is proteasome independent. Following serum withdrawal, Bak accumulation becomes independent of Mcl-1 level and cells are sensitized to pro-apoptotic stimuli. Based on these results, we propose that regulation of Mcl-1-Bak steochiometry is a control mechanism used as a checkpoint to prevent or allow entry into apoptosis.     

鱼类和哺乳类Bcl-2家族蛋白的研究进展

细胞凋亡, 即细胞程序性死亡, 在多细胞生物的发育和稳态调控过程中发挥关键作用。Bcl-2家族蛋白是凋亡过程中的主要调控因子, 关于Bcl-2家族蛋白在凋亡过程中的功能及其作用机制一直是研究的热点。已有研究显示Bcl-2家族蛋白不仅作用于线粒体引发凋亡, 并且参与了包括对细胞内质网Ca2+的调控、DNA损伤的修复及与自噬的相互作用等多种反应, 从多方面对细胞的生存状态进行调控。Bcl-2家族蛋白保守存在于脊椎动物和无脊椎动物中, 其功能在进化中存在异同。文章以高等脊椎动物(哺乳动物)和低等脊椎动物(硬骨鱼类)为代表, 总结了近年来Bcl-2家族蛋白在调控宿主凋亡与自噬、DNA损伤及新陈代谢等方面取得的最新进展。该研究为深入了解鱼类和哺乳类Bcl-2家族蛋白的功能和作用机制提供了重要参考。     

BcL2蛋白质家族——定位与转位

Bcl-2蛋白质家族的抗凋亡和促凋亡成员,在线粒体水平上决定细胞的存活或死亡.在正常细胞中,这些成员呈现功能适应性的细胞内分布;抗凋亡成员主要定位于细胞内膜系特别是线粒体外膜上：但绝大多数促凋亡成员主要分布于细胞浆中.细胞接受死亡信号后,Bcl-2家族成员本身受到一系列的调节,如磷酸化、裂解、蛋白质-蛋白质相互作用等,结果之一是促凋亡成员发生细胞内定位的改变,从细胞浆转位于线粒体膜上,并引发线粒体功能异常及其内外膜间致凋亡因子的释放,最终导致细胞凋亡.     

Bcl-2 proteins and mitochondria—Specificity in membrane targeting for death

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Mcl-1 rescues a glitch in the matrix

Bcl-2 family proteins are known to control cell death and influence mitochondrial function. The function of Mcl-1, an anti-apoptotic Bcl-2 protein, is now shown to depend on its subcellular localization. Mcl-1 at the mitochondrial outer membrane inhibits mitochondrial permeabilization to block apoptosis. However, a cleaved form of Mcl-1 localizes to the mitochondrial matrix and controls inner mitochondrial morphology and oxidative phosphorylation, without directly modulating apoptosis.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.     

OBATOCLAX and ABT-737 Induce ER Stress Responses in Human Melanoma Cells that Limit Induction of Apoptosis

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.     

Completing the family portrait of the anti-apoptotic Bcl-2 proteins: crystal structure of human Bfl-1 in complex with Bim

Evasion of apoptosis is recognized as a characteristic of malignant growth. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members have therefore emerged as potential therapeutic targets due to their critical role in proliferating cancer cells. Here, we present the crystal structure of Bfl-1, the last anti-apoptotic Bcl-2 family member to be structurally characterized, in complex with a peptide corresponding to the BH3 region of the pro-apoptotic protein Bim. The structure reveals distinct features at the peptide-binding site, likely to define the binding specificity for pro-apoptotic proteins. Superposition of the Bfl-1:Bim complex with that of Mcl-1:Bim reveals a significant local plasticity of hydrophobic interactions contributed by the Bim peptide, likely to be the basis for the multi specificity of Bim for anti-apoptotic proteins.     

Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma’s well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.     

Glucose metabolism attenuates p53 and Puma-dependent cell death upon growth factor deprivation

Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation, DNA fragmentation, and loss of clonogenic survival. Importantly, although p53 levels did not change or were slightly reduced, p53 activity was suppressed by elevated glucose metabolism to inhibit Puma induction after growth factor withdrawal. These data show that p53 is metabolically regulated and that glucose metabolism initiates a signaling mechanism to inhibit p53 activation and suppress Puma induction, thus promoting an anti-apoptotic balance to Bcl-2 family protein expression that supports cell survival.