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.     

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.     

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.     

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.     

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.     

Calcium signaling in osteoclasts

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca²⁺) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca²⁺ oscillations occur during RANKL-mediated osteoclastogenesis. Ca²⁺ oscillations provide a digital Ca²⁺ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca²⁺/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca²⁺ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca²⁺ oscillations, and we discuss possible molecular players in this specialized Ca²⁺ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

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.     

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer.     

Role of apoptosis in cardiovascular disease

Apoptosis plays a key role in the pathogenesis in a variety of cardiovascular diseases due to loss of terminally differentiated cardiac myocytes. Cardiac myocytes undergoing apoptosis have been identified in tissue samples from patients suffering from myocardial infarction, diabetic cardiomyopathy, and end-stage congestive heart failure. Apoptosis is a highly regulated program of cell death and can be mediated by death receptors in the plasma membrane, as well as the mitochondria and the endoplasmic reticulum. The cell death program is activated in cardiac myocytes by various stressors including cytokines, increased oxidative stress and DNA damage. Many studies have demonstrated that inhibition of apoptosis is cardioprotective and can prevent the development of heart failure. This review provides a current overview of the evidence of apoptosis in cardiovascular diseases and discusses the molecular pathways involved in cardiac myocyte apoptosis.     

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.     

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.     

Calcium signaling in lacrimal glands

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca²⁺-mobilizing messenger, IP₃, and release of Ca²⁺ stored in the endoplasmic reticulum. The loss of Ca²⁺ from the endoplasmic reticulum then triggers a process known as store-operated Ca²⁺ entry, involving a Ca²⁺ sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

Regulation of T-cell apoptosis by reactive oxygen species

To ensure that a constant number of T cells are preserved in the peripheral lymphoid organs, the production and proliferation of T cells must be balanced out by their death. Newly generated T cells exit the thymus and are maintained as resting T cells. Transient disruption of homeostasis occurs when naïve T cells undergo antigen-induced expansion, a process involving intracellular signaling events that lead to T cell proliferation, acquisition of effector functions, and, ultimately, either apoptosis or differentiation into long-lived memory cells. The last decision point (death vs. differentiation) is a crucial one: it resets lymphoid homeostasis, promotes protective immunity, and limits autoimmunity. Despite its importance, relatively little is known about the molecular mechanisms involved in this cell fate decision. Although multiple mechanisms are likely involved, recent data suggest an underlying regulatory role for reactive oxygen species in controlling the susceptibility of T cells to apoptosis. This review focuses on recent advances in our understanding of how reactive oxygen species modulate T-cell apoptosis.     

Polycystin and calcium signaling in cell death and survival

Mutations in polycystin-1 (PC1) and polycystin-2 (PC2) result in a commonly occurring genetic disorder, called Autosomal Dominant Polycystic Kidney Disease (ADPKD), that is characterized by the formation and development of kidney cysts. Epithelial cells with loss-of-function of PC1 or PC2 show higher rates of proliferation and apoptosis and reduced autophagy. PC1 is a large multifunctional transmembrane protein that serves as a sensor that is usually found in complex with PC2, a calcium (Ca²⁺)-permeable cation channel. In addition to decreased Ca²⁺ signaling, several other cell fate-related pathways are de-regulated in ADPKD, including cAMP, MAPK, Wnt, JAK-STAT, Hippo, Src, and mTOR. In this review we discuss how polycystins regulate cell death and survival, highlighting the complexity of molecular cascades that are involved in ADPKD.