Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax^-/-Bak^-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.     

Deficiency in Apoptotic Effectors BAX and BAK Reveals an Autophagic Cell Death Pathway Initiated by Photodamage to the Endoplasmic Reticulum

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca²⁺-ATPase (SERCA) pump and consequent loss in the ER-Ca²⁺ homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca²⁺ emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic BAX and BAK proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax^-/-/Bak^-/- cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of BAX-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells.

Addendum to:

Role of Endoplasmic Reticulum Depletion and Multidomain Proapoptotic BAX and BAK Proteins in Shaping Cell Death After Hypericin-Mediated Photodynamic Therapy

E. Buytaert, G. Callewaert, N. Hendrickx, L. Scorrano, D. Hartmann, L. Missiaen, J.R. Vandenheede, I. Heirman, J. Grooten and P. Agostinis

FASEB J 2006; In press     

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.     

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.     

线粒体在控制细胞死亡中的作用

细胞死亡由细胞坏死或细胞凋亡所致。细胞坏死时 ,细胞质膜形成疱 (突起 ) ,疱破裂释放细胞内容物 ;细胞凋亡时 ,细胞内容物不释放到细胞外。细胞坏死时 ,细胞内ＡＴＰ耗竭 ;凋亡时 ,细胞需利用ＡＴＰ完成凋亡过程。1.线粒体外膜释放凋亡活性物质细胞凋亡过程中 ,原先位于线粒体膜间隙的某些与凋亡有关的活性物质释放到胞液中 ,这些物质包括细胞色素ｃ(Ｃｙｔｃ)、凋亡诱导因子 (ａｐｏｐｔｏｓｉｓ ｉｎｄｕｃｉｎｇｆａｃｔｏｒ ,ＡＩＦ)、线粒体胱天蛋白酶 (ｃａｓｐａｓｅ)2 ,3,9、ｈｓｐ10、ｈｓｐ6 0、Ｂｃｌ 2家族成员等。细胞受到凋…     

Mitochondrial Regulation of Cell Death

Although required for life, paradoxically, mitochondria are often essential for initiating apoptotic cell death. Mitochondria regulate caspase activation and cell death through an event termed mitochondrial outer membrane permeabilization (MOMP); this leads to the release of various mitochondrial intermembrane space proteins that activate caspases, resulting in apoptosis. MOMP is often considered a point of no return because it typically leads to cell death, even in the absence of caspase activity. Because of this pivotal role in deciding cell fate, deregulation of MOMP impacts on many diseases and represents a fruitful site for therapeutic intervention. Here we discuss the mechanisms underlying mitochondrial permeabilization and how this key event leads to cell death through caspase-dependent and -independent means. We then proceed to explore how the release of mitochondrial proteins may be regulated following MOMP. Finally, we discuss mechanisms that enable cells sometimes to survive MOMP, allowing them, in essence, to return from the point of no return.In most organisms, mitochondria play an essential role in activating caspase proteases through a pathway termed the mitochondrial or intrinsic pathway of apoptosis. Mitochondria regulate caspase activation by a process called mitochondrial outer membrane permeabilization (MOMP). Selective permeabilization of the mitochondrial outer membrane releases intermembrane space (IMS) proteins that drive robust caspase activity leading to rapid cell death. However, even in the absence of caspase activity, MOMP typically commits a cell to death and is therefore considered a point of no return (Fig. 1). Because of this pivotal role in dictating cell fate, MOMP is highly regulated, mainly through interactions between pro- and antiapoptotic members of the Bcl-2 family. In this article, we begin by discussing how mitochondria may have evolved to become central players in apoptotic cell death. We then provide an overview of current models addressing the mechanics of MOMP, outlining how this crucial event leads to cell death through both caspase-dependent or -independent mechanisms. Finally, we discuss how caspase activity may be regulated post-MOMP and define other processes that allow cells to survive MOMP and, in effect, return from the point of no return.Open in a separate windowFigure 1.Mitochondrial regulation of cell death. Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) can lead to caspase-dependent apoptosis (left) or caspase-independent cell death (right). Following MOMP, soluble proteins are released from the mitochondrial intermembrane space into the cytoplasm. Cytochrome c binds to monomeric Apaf-1 leading to its conformational change and oligomerization. Procaspase-9 is recruited to heptameric Apaf-1 complexes forming the apoptosome. This leads to activation of caspase-9 and, through caspase-9-mediated cleavage, activation of the executioner caspases-3 and -7. Release of Smac and Omi from the mitochondrial intermembrane space facilitates caspase activation by neutralizing the caspase inhibitor XIAP. MOMP can also lead to nonapoptotic cell death through a gradual loss of mitochondrial function and/or release of mitochondrial proteins that kill the cell in a caspase-independent manner.     

A stapled BID BH3 helix directly binds and activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.     

细胞程序性死亡的非Caspase依赖型途径

程序性细胞死亡是一种程序化的主动性细胞死亡,半胱胺酸天冬氨酸特异性蛋白酶家族(在该过程中起着不可忽视的作用.基于Caspase在程序性细胞死亡过程中所起的作用,将程序性细胞死亡分为两大类:Caspase依赖型和Caspase非依赖型.前者即典型的凋亡,后者包括自体吞噬、副凋亡、有丝分裂灾变、凋亡样程序性死亡、坏死样程序性死亡等.这些Caspase非依赖型的细胞程序性死亡途径与生理及病理现象密切相关.     

BH3-triggered structural reorganization drives the activation of proapoptotic BAX

BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.     

Intersection between Mitochondrial Permeability Pores and Mitochondrial Fusion/Fission

The goal of this review is to highlight recent developments in the field of mitochondrial membrane processes, which provide new insights into the relation between mitochondrial fission/fusion events and the mitochondrial permeability transition (MPT). First, we distinguish between pore opening events at the inner and outer mitochondrial membranes. Inner membrane pore opening, or iMPT, leads to membrane depolarization, release of low molecular weight compounds, cristae reorganization and matrix swelling. Outer membrane pore opening, or oMPT, allows partial release of apoptotic proteins, while complete release requires additional remodeling of inner membrane cristae. Second, we summarize recent data that supports a similar temporal and physical separation between inner and outer mitochondrial membrane fusion events. Finally, we focus on cristae remodeling, which may be the intersection between oMPT and iMPT events. Interestingly, components of fusion machinery, such as mitofusin 2 and OPA1, appear to play a role in cristae remodeling as well. Special issue dedicated to John P. Blass.     

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax''s anticancer efficacy.     

BH3-Only Proteins and BH3 Mimetics Induce Autophagy by Competitively Disrupting the Interaction between Beclin 1 and Bcl-2/Bcl-XL

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X_L) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X_L. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X_L. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X_L can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X_L complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X_L complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X_L at the level of the endoplasmic reticulum.

Addendum to:

Functional and Physical Interaction Between Bcl-X_L and a BH3-Like Domain in Beclin-1

M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. Kroemer

EMBO J 2007; In press     

Mitochondrial Genome Mutation in Cell Death and Aging

This article reviews the concept, molecular genetics, and pathology of cell death and agingin relation to mitochondrial genome mutation. Accumulating evidence emphasizes the role ofgenetic factors in the development of naturally occurring cell death and aging. The ATPrequired for a cell's biological activity is almost exclusively produced by mitochondria. Eachmitochondrion possesses its own DNA (mtDNA) that codes essential subunits of themitochondrial energy-transducing system. Recent studies confirm that mtDNA is unexpectedly fragileto hydroxyl radical damage, hence to the oxygen stress. Cellular mtDNA easily fragmentsinto over a hundred-types of deleted mtDNA during the life of an individual. Cumulativeaccumulation of these oxygen damages and deletions in mtDNA results in a defective energytransducing system and in bioenergetic crisis. The crisis leads cells to the collapse ofmitochondrial trans-membrane potential, to the release of the apoptotic protease activating factors intocytosol, to uncontrolled cell death, to tissue degeneration and atrophy, and to aging. Thetotal base sequencing of mtDNA among individuals revealed that germ-line point mutationstransmitted from ancestors accelerate the somatic oxygen damages and mutations in mtDNAleading to phenotypic expression of premature aging and degenerative diseases. A practicalsurvey of point mutations will be useful for genetic diagnosis in predicting the life-span ofan individual.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

Targeting the PI3K/Akt Cell Survival Pathway to Induce Cell Death of HIV-1 Infected Macrophages with Alkylphospholipid Compounds

Background

HIV-1 infected macrophages and microglia are long-lived viral reservoirs persistently producing viral progenies. HIV-1 infection extends the life span of macrophages by promoting the stress-induced activation of the PI3K/Akt cell survival pathway. Importantly, various cancers also display the PI3K/Akt activation for long-term cell survival and outgrowth, and Akt inhibitors have been extensively searched as anti-cancer agents. This led us to investigate whether Akt inhibitors could antagonize long-term survival and cytoprotective phenotype of HIV-1 infected macrophages.

Principal Findings

Here, we examined the effect of one such class of drugs, alkylphospholipids (ALPs), on cell death and Akt pathway signals in human macrophages and a human microglial cell line, CHME5, infected with HIV-1 BaL or transduced with HIV-1 vector, respectively. Our findings revealed that the ALPs, perifosine and edelfosine, specifically induced the death of HIV-1 infected primary human macrophages and CHME5 cells. Furthermore, these two compounds reduced phosphorylation of both Akt and GSK3β, a downstream substrate of Akt, in the transduced CHME5 cells. Additionally, we observed that perifosine effectively reduced viral production in HIV-1 infected primary human macrophages. These observations demonstrate that the ALP compounds tested are able to promote cell death in both HIV-1 infected macrophages and HIV-1 expressing CHME5 cells by inhibiting the action of the PI3K/Akt pathway, ultimately restricting viral production from the infected cells.

Significance

This study suggests that Akt inhibitors, such as ALP compounds, may serve as potential anti-HIV-1 agents specifically targeting long-living HIV-1 macrophages and microglia reservoirs.     

The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function

The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway.     

线粒体融合/分裂对线粒体的质量控制作用

线粒体是一种结构和功能复杂而敏感的细胞器,拥有独立于细胞核的基因组,在细胞的不同时相,生理过程和环境条件下,线粒体的形态,数量和质量,具有高度的可塑性。线粒体是细胞和生物体内最主要的能量供应场所,几乎存在于所有种类的细胞中,是一种动态变化的细胞器。正常情况下,线粒体的数量、形态以及功能维持相对稳定的状态,称之为线粒体稳态。当上述状态发生紊乱时,细胞乃至生物体形态、功能也将受到影响甚至死亡。线粒体质量控制是在细胞中维持正常状态的关键机制,决定着线粒体的命运。近年,随着线粒体研究的深入和具体,逐渐发现融合/分裂在其形态、数量、遗传物质等质量控制相关的方面挥了重要作用。本文通过探讨融合/分裂对线粒体质量控制的作用机制,总结和讨论相关前沿研究,为后期研究提供一定的理论依据。