p53上调凋亡调制物的促凋亡作用

p53上调凋亡调制物(p53up-regulated modulator of apoptosis,PUMA)是Bcl-2家族中BH3-only(Bcl-2 homology 3-only)蛋白质家族成员,通过其BH3结构域与所有的Bcl-2抗凋亡蛋白质结合,引发线粒体功能障碍和胱天蛋白酶(caspase)级联反应,诱导细胞凋亡。PUMA被证实在多种病理性应激介导的细胞凋亡中发挥着至关重要的作用,因而成为近年研究的热点。     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Apoptosis: why and how does it occur in biology?

The literature on apoptosis has grown tremendously in recent years, and the mechanisms that are involved in this programmed cell death pathway have been enlightened. It is now known that apoptosis takes place starting from early development to adult stage for the homeostasis of multicellular organisms, during disease development and in response to different stimuli in many different systems. In this review, we attempted to summarize the current knowledge on the circumstances and the mechanisms that lead to induction of apoptosis, while going over the molecular details of the modulator and mediators of apoptosis as well as drawing the lines between programmed and non-programmed cell death pathways. The review will particularly focus on Bcl-2 family proteins, the role of different caspases in the process of apoptosis, and their inhibitors as well as the importance of apoptosis during different disease states. Understanding the molecular mechanisms involved in apoptosis better will make a big impact on human diseases, particularly cancer, and its management in the clinics.     

Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell death mechanisms

Genetic analysis of programmed cell death in Drosophila reveals many similarities with mammals. Heretofore, a missing link in the fly has been the absence of any Bcl-2/Bax family members, proteins that function in mammals as regulators of mitochondrial cytochrome c release. A Drosophila homologue of the human killer protein Bok (DBok) was identified. The predicted structure of DBok is similar to pore-forming Bcl-2/Bax family members. DBok induces apoptosis in insect and human cells, which is suppressible by anti-apoptotic human Bcl-2 family proteins. A caspase inhibitor suppressed DBok-induced apoptosis but did not prevent DBok-induced cell death. Moreover, DBok targets mitochondria and triggers cytochrome c release through a caspase-independent mechanism. These characteristics of DBok reveal evolutionary conservation of cell death mechanisms in flies and humans.     

Regulation of apoptosis by BH3 domains in a cell-free system

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins.Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system.Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins

Programmed cell death or apoptosis is central to many physiological processes and pathological conditions such as organogenesis, tissue homeostasis, cancer, and neurodegenerative diseases. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other pro-apoptotic factors. Control of the formation of the mitochondrial apoptosis-induced channel, or MAC, is central to the regulation of apoptosis by Bcl-2 family proteins. MAC is detected early in apoptosis by patch clamping the mitochondrial outer membrane. The focus of this review is on the regulation of MAC activity by Bcl-2 family proteins. The role of MAC as the putative cytochrome c release channel during early apoptosis and insights concerning its molecular composition are also discussed.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.     

Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3

The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Here, we show that Bcl-2 interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

The established and the predicted roles of dynein light chain in the regulation of mitochondrial apoptosis

The mitochondrial pathway of apoptosis is regulated by the interplay between the members of Bcl-2 family. Within this family, BH3-only proteins are the sensors of apoptotic stimuli and can trigger apoptosis either by inhibiting the anti-apoptotic Bcl-2-family proteins or by directly activating the effectors Bax and Bak. An expanding body of research suggests that a number of non-Bcl-2 proteins can also interact with Bcl-2 proteins and contribute to the decision of cell fate. Dynein light chain (LC8, DYNLL or DLC), a hub protein and a dimerizing engine has been proposed to regulate the pro-apoptotic activity of two BH3-only proteins, Bim and Bmf. Our recent work has provided insight into the mechanisms through which DLC1 (DYNLL1) modulates Bim activity. Here we discuss the present day understanding of Bim-DLC interaction and endeavor to evaluate this interaction in the light of information from studies of DLC with other binding partners.     

Regulation of Bcl-2 proteins during anoikis and amorphosis

Adhesion to extracellular matrix regulates cell survival through both integrin engagement and appropriate cell spreading. Numerous signaling pathways converge to affect the levels and posttranslational modifications of Bcl-2 family proteins. Recent work has defined specific roles for different Bcl-2 proteins in the disruption of mitochondrial function that leads to cell death. Using this understanding of Bcl-2 protein function as a framework, we will consider the molecular mechanisms of apoptosis induced by integrin detachment (anoikis) and cell death stimulated by the loss of cytoskeletal architecture (amorphosis).     

Death by association: BH3 domain-only proteins and liver injury

Apoptosis, a prominent form of cell death, is a prime feature of many acute and chronic liver diseases. Apoptosis requires mitochondrial dysfunction, which is regulated by proteins of the Bcl-2 family. Whether or not a cell should live or die is controlled by the interaction of multidomain Bcl-2 proteins with proapoptotic BH3 domain-only proteins of this family. Current models suggest multidomain, antiapoptotic Bcl-2 proteins prevent mitochondrial dysfunction by sequestering and/or preventing activation of its proapoptotic relatives. BH3-only proteins initiate cell death by neutralizing and or ligating multidomain prosurvival Bcl-2 proteins. Thus BH3 domain-only proteins are paramount in the apoptotic process as exemplified by the role of the BH3 domain-only protein Bid in liver injury. In this concise review, we will focus on how these BH3 domain-only proteins are regulated in the cell, their association with the Bcl-2 family of proteins, and finally, current information regarding their involvement in liver cell apoptosis and injury.     

Functional characterization of the Bcl-2 gene family in the zebrafish

Members of the Bcl-2 protein family control the intrinsic apoptosis pathway. To evaluate the importance of this family in vertebrate development, we investigated it in the zebrafish (Danio rerio). We found that the zebrafish genome encodes structural and functional homologs of most mammalian Bcl-2 family members, including multi-Bcl-2-homology (BH) domain proteins and BH3-only proteins. Apoptosis induction by gamma-irradiation required zBax1 and zPuma, and could be prevented by overexpression of homologs of prosurvival Bcl-2 family members. Surprisingly, zebrafish Bax2 (zBax2) was homologous to mammalian Bax by sequence and synteny, yet demonstrated functional conservation with human Bak. Morpholino knockdown of both zMcl-1a and zMcl-1b revealed their critical role in early embryonic zebrafish development, and in the modulation of apoptosis activation through the extrinsic pathway. These data indicate substantial functional similarity between zebrafish and mammalian Bcl-2 family members, and establish the zebrafish as a relevant model for studying the intrinsic apoptosis pathway.     

Galpha12 stimulates apoptosis in epithelial cells through JNK1-mediated Bcl-2 degradation and up-regulation of IkappaBalpha

Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Galpha12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Galpha12 regulates apoptosis in epithelial cells. Inducible expression of Galpha12 or constitutively active (QL)alpha12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLalpha12, but not Galpha12. Inducing QLalpha12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IkappaBalpha protein levels, a potent stimulator of apoptosis. Furthermore, the QLalpha12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Galpha12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Galpha12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Galpha12 were nearly identical to the mechanisms identified in QLalpha12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IkappaBalpha. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Galpha12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.     

Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death

8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax.