Regulating cell death at,on, and in membranes

Bcl-2 family proteins are central regulators of apoptosis. Various family members are located in the cytoplasm, endoplasmic reticulum, and mitochondrial outer membrane in healthy cells. However during apoptosis most of the interactions between family members that determine the fate of the cell occur at the membranes of intracellular organelles. It has become evident that interactions with membranes play an active role in the regulation of Bcl-2 family protein interactions. Here we provide an overview of various models proposed to explain how the Bcl-2 family regulates apoptosis and discuss how membrane binding affects the structure and function of each of the three categories of Bcl-2 proteins (pro-apoptotic, pore-forming, and anti-apoptotic). We also examine how the Bcl-2 family regulates other aspects of mitochondrial and ER physiology relevant to cell death.     

Bcl-2 family members: integrators of survival and death signals in physiology and pathology [corrected

The members of the Bcl-2 family of proteins are crucial regulators of apoptosis. In order to determine cell fate, these proteins must be targeted to distinct intracellular membranes, including the mitochondrial outer membrane (MOM), the membrane of the endoplasmic reticulum (ER) and its associated nuclear envelope. The targeting sequences and mechanisms that mediate the specificity of these proteins for a particular cellular membrane remain poorly defined. Several Bcl-2 family members have been reported to be tail-anchored via their predicted hydrophobic COOH-terminal transmembrane domains (TMDs). Tail-anchoring imposes a posttranslational mechanism of membrane insertion on the already folded protein, suggesting that the transient binding of cytosolic chaperone proteins to the hydrophobic TMD may be an important regulatory event in the targeting process. The TMD of certain family members is initially concealed and only becomes available for targeting and membrane insertion in response to apoptotic stimuli. These proteins either undergo a conformational change, posttranslational modification or a combination of these events enabling them to translocate to sites at which they are functional. Some Bcl-2 family members lack a TMD, but nevertheless localize to the MOM or the ER membrane during apoptosis where they execute their functions. In this review, we will focus on the intracellular targeting of Bcl-2 family members and the mechanisms by which they translocate to their sites of action. Furthermore, we will discuss the posttranslational modifications which regulate these events.     

线粒体在细胞凋亡中的介导作用

线粒体是细胞内产生能量的重要细胞器,被认为是细胞生存与死亡的调节中心。Bcl-2家族蛋白、内质网和溶酶体能引起线粒体膜通透性的改变,造成线粒体功能损伤,诱导细胞凋亡。本文主要综述线粒体在Bcl-2家族蛋白、内质网和溶酶体诱导细胞凋亡中作用的研究进展。     

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

Interaction of the C-terminal domain of Bcl-2 family proteins with model membranes

Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.     

Interaction of the C-terminal domain of Bcl-2 family proteins with model membranes

Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.     

The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis

The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.     

The apoptotic regulator Nrz controls cytoskeletal dynamics via the regulation of Ca2+ trafficking in the zebrafish blastula

Bcl-2 family members are key regulators of apoptosis. Their involvement in other cellular processes has been so far overlooked. We have studied the role of the Bcl-2 homolog Nrz in the developing zebrafish. Nrz was found to be localized to the yolk syncytial layer, a region containing numerous mitochondria and ER membranes. Nrz knockdown resulted in developmental arrest before gastrulation, due to free Ca(2+) increase in the yolk cell, activating myosin light chain kinase, which led to premature contraction of actin-myosin cables in the margin and separation of the blastomeres from the yolk cell. In the yolk syncytial layer, Nrz appears to prevent the release of Ca(2+) from the endoplasmic reticulum by directly interacting with the IP3R1 Ca(2+) channel. Thus, the Bcl-2 family may participate in early development, not only by controlling apoptosis but also by acting on cytoskeletal dynamics and cell movements via Ca(2+) fluxes inside the embryo.     

Oxysterol binding protein and its homologues: new regulatory factors involved in lipid metabolism

PURPOSE OF REVIEW: Oxysterol binding protein was discovered in the 1980s as a cytosolic high-affinity receptor for oxysterols, but its function has remained enigmatic. Families of genes/proteins with sequence homology to oxysterol binding protein have been identified in eukaryotes from yeast to man, indicating that these proteins, denoted as oxysterol binding protein-related proteins (ORPs), serve a fundamental purpose conserved in evolution. This review discusses recent findings that provide important clues to the mode of action of these proteins. RECENT FINDINGS: The long variant of ORP1 is induced upon differentiation of monocytes to macrophages and has capacity to enhance the trans-activation potential of liver X receptors, indicating a function in macrophage lipid metabolism. Important clues to ORP function were provided by the finding that most family members carry an endoplasmic reticulum targeting motif, while the amino-terminal regions of the proteins have targeting specificities for other organelles. Extensive splice variation occurs within the gene family, suggesting that a large number of distinct protein products are encoded. Further implications were obtained for a possible role of a family member in tumor cell metastasis. SUMMARY: ORPs constitute a novel family of proteins implicated in cellular lipid metabolism and different aspects of cell regulation. The function of several family members is connected with cellular sterol metabolism, and there is evidence for a role of oxysterol binding protein in lipid transport from the endoplasmic reticulum. Recently, a model on the function of these proteins at membrane contact sites, specialized zones of communication between two different organelles, has been presented.     

Bcl-2-family proteins and the role of mitochondria in apoptosis

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.     

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca²⁺) signals. Since the proper supply of Ca²⁺ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca²⁺ signaling. The endoplasmic reticulum (ER) is the main Ca²⁺ storage for the cell and Bcl-2 family proteins competitively regulate its Ca²⁺ concentration. Bcl-2 family proteins also regulate the flux of Ca²⁺ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP₃Rs) to mediate their opening. Type 1 IP₃Rs reside at the bulk ER to coordinate cytosolic Ca²⁺ signals, while type 3 IP₃Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca²⁺ uptake. In healthy cells, mitochondrial Ca²⁺ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca²⁺ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca²⁺ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca²⁺ signaling and how mitochondrial Ca²⁺ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.     

Cyclin-dependent kinase inhibitors,roscovitine and purvalanol,induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.     

Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction

Diabetes is a metabolic disease affecting nearly 300 million individuals worldwide. Both types of diabetes (1 and 2) are characterized by loss of functional pancreatic β-cell mass causing different degrees of insulin deficiency. The Bcl-2 family has a double-edged effect in diabetes. These proteins are crucial controllers of the mitochondrial pathway of β-cell apoptosis induced by pro-inflammatory cytokines or lipotoxicity. In parallel, some Bcl-2 members also regulate glucose metabolism and β-cell function. In this review, we describe the role of Bcl-2 proteins in β-cell homeostasis and death. We focus on how these proteins interact, their contribution to the crosstalk between endoplasmic reticulum stress and mitochondrial permeabilization, their context-dependent usage following different pro-apoptotic stimuli, and their role in β-cell physiology.     

The critical role of JNK in the ER-mitochondrial crosstalk during apoptotic cell death

Apoptosis or programmed cell death is an extremely coordinated phenomenon that involves the participation of a complex interacting crosstalk between the endoplasmic reticulum and mitochondria. This involves a series of signaling molecules like stress kinases, caspases, Bcl-2 family of proteins, etc. that coordinately induce apoptosis by releasing apoptotic proteins from the mitochondria and mediate DNA damage of the cell. Among the stress kinases, JNK, a member of the MAPK family has been believed to be critically mediating these apoptotic phenomena. The involvement of JNK has been clouded by controversies because of its role both as a pro-apoptotic and an anti-apoptotic mediator. A very significant initiator of JNK activation is the pro-inflammatory cytokine, IL-1β, levels of which are significantly elevated in varied diseases especially diabetes where it is believed to significantly contribute to pancreatic β-cell death. During apoptotic cell death, the endoplasmic reticulum and the mitochondrion participate in a relay of cellular events that determine the onset of the classical apoptotic pathways. Here we discuss the details of this ER-mitochondrial crosstalk and the role of JNK herein that ultimately culminates into apoptotic cell death that is evident in various pathophysiological conditions.     

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.     

BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L)

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 alpha-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 (reticulophagy). 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.     

The daily job of night killers: alternative roles of the BCL-2 family in organelle physiology

Apoptosis is essential for maintenance of tissue homeostasis and its deregulation underlies many disease conditions. The BCL-2 family of proteins is a group of evolutionarily conserved regulators of cell death, comprising both anti- and pro-apoptotic members, which operate at the mitochondrial membrane to control caspase activation. Different BCL-2-related proteins are also located in multiprotein complexes at the endoplasmic reticulum (ER), which are involved in the control of diverse cellular processes, including calcium homeostasis, autophagy, the unfolded protein response and ER morphogenesis. Here, we describe the emerging concept that BCL-2-related proteins have alternative functions beyond apoptosis to control the essential functions of the cell.     

Developmental expression patterns of Bcl-2, Bcl-x, Bax, and Bak in teeth

The ontogenic profile of expression of four members of the Bcl-2 family (Bcl-2, Bcl-x, Bax and Bak) was examined in the mouse by immunohistochemistry using paraffin sections. All four members were expressed in changing patterns during critical stages of tooth morphogenesis. Expression was detected in epithelial cell populations including the dental lamina, internal dental epithelium (IDE; differentiating ameloblasts), stratum intermedium and stellate reticulum cells, as well as in the condensed dental mesenchyme. The temporo-spatial localization of the various members of the Bcl-2 family in dental epithelium and mesenchyme showed striking overlapping areas but often their expression patterns differed. In general, contemporaneous co-expression of the Bcl-2 and Bax proteins, and of the Bcl-x and Bak proteins was noted in various types of cells during the developmental process, with the intensity of Bcl-2>Bax and of Bak>Bcl-x. Expression was pronounced at sites where interaction between surface ectoderm and induced mesenchyme takes place, and at the enamel knot, which is regarded as organization/regulating center for tooth development. Around birth, after the structural maturation was accomplished, the expression was down-regulated. The absence of elevated expression of each of these four members of the Bcl-2 family after birth in the teeth suggests that these proteins are relevant during the accomplishment of the basic architecture but not once the structure of the tooth is established.     

The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect

Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2.