Monte Carlo simulations of tBid association with the mitochondrial outer membrane

Bid, a BH3-only pro-apoptopic member of the BCL-2 protein family, regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 α-helices (H1–H8, respectively) and is soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid by caspase 8 in apoptosis yields activated “tBid” (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. The release of cytochrome c from mitochondria to the cytosol constitutes a critical control point in apoptosis that is regulated by interaction of tBid protein with mitochondrial membrane. tBid displays structural homology to channel-forming bacterial toxins, such as colicins or transmembrane domain of diphtheria toxin. By analogy, it has been hypothesized that tBid would unfold and insert into the lipid bilayer of the mitochondria outer membrane (MOM) upon membrane association. However, it has been shown recently that unlike colicins and the transmembrane domain of diphtheria toxin, tBid binds to the lipid bilayer maintaining α-helical conformation of its helices without adopting a transmembrane orientation by them. Here, the mechanism of the association of tBid with the model membrane mimicking the mitochondrial membrane is studied by Monte Carlo simulations, taking into account the underlying energetics. A novel two-stage hierarchical simulation protocol combining coarse-grained discretization of conformational space with subsequent refinements was applied which was able to generate the protein conformation and its location in the membrane using modest computational resources. The simulations show that starting from NMR-established conformation in the solution, the protein associates with the membrane without adopting the transmembrane orientation. The configuration (conformation and location) of tBid providing the lowest free energy for the system protein/membrane/solvent has been obtained. The simulations reveal that tBid upon association with the membrane undergoes significant conformational changes primarily due to rotations within the loops between helices H4 and H5, H6 and H7, H7 and H8. It is established that in the membrane-bound state of tBid-monomer helices H3 and H5 have the locations exposed to the solution, helices H6 and H8 are partly buried and helices H4 and H7 are buried into the membrane at shallow depth. The average orientation of tBid bound to the membrane in the most stable configuration reported here is in satisfactory agreement with the evaluations obtained by indirect experimental means. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Apoptosis-induced changes in mitochondrial lipids

Apoptosis is an active and tightly regulated form of cell death, which can also be considered a stress-induced process of cellular communication. Recent studies reveal that the lipid network within cells is involved in the regulation and propagation of death signalling. Despite the vast growth of our current knowledge on apoptosis, little is known of the specific role played by lipid molecules in the central event of apoptosis—the piercing of mitochondrial membranes. Here we review the information regarding changes in mitochondrial lipids that are associated with apoptosis and discuss whether they may be involved in the permeabilization of mitochondria to release their apoptogenic factors, or just lie downstream of this permeabilization leading to the amplification of caspase activation. We focus on the earliest changes that physiological apoptosis induces in mitochondrial membranes, which may derive from an upstream alteration of phospholipid metabolism that reverberates on the mitochondrial re-modelling of their characteristic lipid, cardiolipin. Hopefully, this review will lead to an increased understanding of the role of mitochondrial lipids in apoptosis and also help revealing new stress sensing mechanisms in cells. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

In vitro analysis of Bcl-2 proteins in mitochondria and endoplasmic reticulum: similarities in anti-apoptotic functions and differences in regulation

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.     

The interaction between tBid and cardiolipin or monolysocardiolipin

Bid, a BH3-only pro-apoptotic member of the Bcl-2 family, is cleaved by caspase 8 in apoptosis induced by death domain receptors. The carboxyl terminus of the cleavage product, tBid, remains associated with the amino terminal fragment (nBid) after cleavage. Dissociation of tBid from nBid occurs during targeting of tBid to mitochondria. We use an in vitro system and demonstrate that cardiolipin is sufficient for the dissociation. Monolysocardiolipin, a metabolite of cardiolipin that increases in mitochondria during apoptosis, has the same affinity to tBid as cardiolipin and is also capable of inducing dissociation of tBid from nBid. In contrast, phosphatidylethanolamine could not induce dissociation of tBid from nBid. To determine the site of tBid that interacts with cardiolipin, we performed mutational analysis by eliminating the positive-charged residues in helices 4-6. None of the single mutations can abolish the ability of tBid to target to mitochondria and to induce cytochrome c release, suggesting that positive-charged residues in helices 4-6 may not be required for mitochondrial targeting of tBid.     

钙离子信号与细胞凋亡

细胞凋亡的分子机制是什么?这个问题当前引起人们广泛的研究兴趣。作为重要的第二信使,钙信号在许多生理和细胞活动中都起到了十分重要的作用。钙信号是否也在凋亡的调控中起作用呢?虽然在过去十多年中,许多研究证据都表明钙信号参与凋亡的调控,但是,钙信号如何作用于凋亡过程的具体机理仍然是众说纷纭。事实上,许多研究结果仍存有争议。文章总结了近几年来大量关于钙信号与凋亡研究的成果,集中讨论了两个问题：1)在凋亡前期“决定阶段”有没有钙离子信号的参与?2)钙离子信号与哪些凋亡调控因子(包括Bcl-2族蛋白)相互作用及如何作用?这问题还牵涉到亚细胞结构中钙库的作用(包括细胞质、内质网和线粒体)。根据作者自己的实验结果,文章对这些文献中不同的说法作了一些具体的评估。最后,文章还提出了一个钙离子信号参与调控细胞凋亡的可能模型。     

Cardiolipin: Setting the beat of apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid which is known to be intimately linked with the mitochondrial bioenergetic machinery. Accumulating evidence now suggests that this unique lipid also has active roles in several of the mitochondria-dependant steps of apoptosis. CL is closely associated with cytochrome c at the outer leaflet of the mitochondrial inner membrane. This interaction makes the process of cytochrome c release from mitochondria more complex than previously assumed, requiring more than pore formation in the mitochondrial outer membrane. While CL peroxidation could be crucial for enabling cytochrome c dissociation from the mitochondrial inner membrane, cytochrome c itself catalyzes CL peroxidation. Moreover, peroxy-CL directly activates the release of cytochrome c and other apoptogenic factors from the mitochondria. CL is also directly involved in mitochondrial outer membrane permeabilization by enabling docking and activation of pro-apoptotic Bcl-2 proteins. It appears therefore that CL has multiple roles in apoptosis and that CL metabolism contributes to the complexity of the apoptotic process.     

促细胞凋亡因子Bid蛋白的研究进展

Bid蛋白是Bcl-家族中促凋亡类的蛋白。它具有可被caspase8酶切高控、高效地诱民细胞色素c从线粒体泄漏到胞浆中的功能,从而在细胞凋亡中起着重要的作用,因而倍受人们的重视。Bid蛋白的功能被发现以来,短短几年间,人们从分子生物学、细胞学、结构分析以及利用脂质体模型膜体系等各方面对Bid蛋白进行研究,取得了很大的进展。     

Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca²⁺) transfer from the endoplasmic reticulum (ER) to the mitochondria in several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylated AKT1 (m/p-AKT1), were found to have a reduced Ca²⁺ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca²⁺ response after Ca²⁺ release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca²⁺ from intracellular stores.Most importantly, this alteration of ER Ca²⁺ content and release, reduces significantly cellular sensitivity to Ca²⁺ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca²⁺ homeostasis, that may underlie its protective role against some proapoptotic stimuli.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.     

Bax activation and mitochondrial insertion during apoptosis

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.     

Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.     

Cardiolipin-enriched raft-like microdomains are essential activating platforms for apoptotic signals on mitochondria

Cardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase-8 on mitochondria. We hypothesize that these platforms may correspond to “raft-like” microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in “raft-like” microdomains could be to anchor caspase-8 at contact sites between inner and outer membranes, facilitating its self-activation, Bid cleavage and apoptosis execution. The role played by “raft-like” microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Ruthenium methylimidazole complexes induced apoptosis in lung cancer A549 cells through intrinsic mitochondrial pathway

Ruthenium(II) methylimidazole complexes, with the general formula [Ru(MeIm)₄(N?N)]²⁺ (N?N = tip (RMC1), iip (RMC2), dppz (RMC3), dpq (RMC4); MeIm = 1-methylimidazole, tip = 2-(thiophene-2-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, iip = 2-(1H-imidazol-4-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, dpq = pyrazino [2,3-f] [1,10]phenanthroline), were synthesized and characterized. As determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, these complexes displayed potent anti-proliferation activity against various cancer cells. RMC1 inhibited the growth of A549 (human lung adenocarcinoma) lung cells through induction of apoptotic cell death, as evidenced by the accumulation of cell population in sub-G1 phase. RMC1 also induced the depletion of mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Another experiment showed that Bid protein was also activated by RMC1, which implied that RMC1 could existed two pathways crosstalk, namely, have exogenous death receptor signaling pathway. These results demonstrated that RMC1 induced cancer cell death by acting on both mitochondrial and death receptor apoptotic pathways, suggesting that RMC1 could be a candidate for further evaluation as a chemotherapeutic agent against human cancers.     

VDAC regulation: role of cytosolic proteins and mitochondrial lipids

It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006). Indeed, VDAC, positioned on the interface between mitochondria and the cytosol (Colombini, Mol Cell Biochem 256:107–115, 2004), is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. VDAC gating has been studied extensively for the last 30 years on reconstituted VDAC channels. In this review we focus exclusively on physiologically relevant regulators of VDAC gating such as endogenous cytosolic proteins and mitochondrial lipids. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. We also discuss how characteristic mitochondrial lipids, phospatidylethanolamine and cardiolipin, could regulate VDAC gating. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.     

Mitochondrial dysfunction in rat with nonalcoholic fatty liver: Involvement of complex I, reactive oxygen species and cardiolipin

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H₂O₂) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.     

Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane

In many apoptotic responses, pro-apoptotic members of the Bcl-2 family trigger the permeabilization of the outer mitochondrial membrane, thereby allowing the release of mitochondrial apoptogenic factors that contribute to caspase activation in the cytosol. The mechanisms that lead to the activation of pro-apoptotic Bcl-2 family members and to the permeabilization of the outer mitochondrial membrane are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to these events, regarding both additional proteins that were recently suggested to be involved, and the roles of lipids.