胱冬肽酶非依赖性的细胞程序性死亡

在多细胞有机体的组织内稳态维持和正常发育过程中,细胞程序性死亡发挥着重要的作用。细胞程序性死亡有多种形式(如细胞凋亡、类细胞凋亡和类坏死等),其中了解较清楚的是细胞凋亡。一直以来,胱冬肽酶(caspase)被认为是细胞凋亡发生中关键的一种蛋白酶。但是最近的研究表明,包括细胞凋亡在内的一些细胞程序性死亡可以以一种不依赖胱冬肽酶的方式发生。细胞程序性死亡与胱冬肽酶之间存在非依赖性关系。     

AIF-Mediated Programmed Necrosis: A Highly Orchestrated Way to Die

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly regulated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.     

2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens. At least in some instances, teratogens induce cell death in areas of normal PCD, suggesting that there is a link between programmed and teratogen-induced cell death. Although researchers recognized early on that cell death is an integral part of both normal and abnormal development, little was known about the mechanisms of cell death. In 1972, Kerr et al. ('72) showed conclusively that cell deaths, induced in a variety of contexts, followed a reproducible pattern, which they termed apoptosis. The next breakthrough came in the 1980s when Horvitz and his colleagues identified specific cell death genes (ced) that controlled PCD in the roundworm, Caenorhabditis elegans (C. elegans). Identification of ced genes in the roundworm quickly led to the isolation of their mammalian homologues. Subsequent research in the 1990s led to the identification of a cadre of proteins controlling cell death in mammals, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, Bcl-2 family proteins, and IAPs. Two major pathways of apoptosis have now been elucidated, the receptor-mediated and the mitochondrial apoptotic pathways. The latter pathway, induced by a wide variety of toxic agents, is activated by the release of cytochrome c from mitochondria. Cytochrome c then facilitates the activation of a caspase cascade involving caspase-9 and -3. Activation of these caspases results in the cleavage of a variety of cellular proteins leading to the orderly demise of the cell. Work from my laboratory in the last 5 years has shown that teratogens, such as hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine, induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway, i.e., mitochondrial release of cytochrome c, activation of caspase-9 and -3, inactivation of poly (ADP-ribose) polymerase (PARP), and systematic degradation of DNA. Our work, as well as the work of others, has also shown that different tissues within the early post implantation mammalian embryo are differentially sensitive to the cell death inducing potential of teratogens, from exquisite sensitivity of cells in the developing central nervous system to complete resistance of cells in the developing heart. More importantly, we have shown that the resistance of heart cells is directly related to the failure to activate the mitochondrial apoptotic pathway in these cells. Thus, whether a cell dies in response to a teratogen and therefore contributes to the pathogenesis culminating in birth defects, depends, at least in part, by the cell's ability to regulate the mitochondrial apoptotic pathway. Future research aimed at understanding this regulation should provide insight not only into the mechanism of teratogen-induced cell death but also the role of cell death in the genesis of birth defects.     

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway

Accumulation of misfolded proteins and alterations in Ca2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1(-/-) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.     

Mitochondrial involvement in tracheary element programmed cell death

The mitochondria pathway is regarded as a central component of some types of programmed cell death (PCD) in animal cells where specific signals cause the release of cytochrome c from mitochondria to trigger a proteolytic cascade involving caspases. However, plant cells lack canonical caspases, therefore a role for the mitochondria in programmed cell death in plant cells is not obvious. Using plant cells which terminally differentiate, we provide evidence supporting the involvement of mitochondria in PCD, however the release of cytochrome c is insufficient to trigger the PCD. Prior to execution of cellular autolysis initiated by the rupture of the large central vacuole to release sequestered hydrolases, mitochondria adopt a definable morphology, the inner membrane depolarizes prior to death, and cytochrome c is released from mitochondria. However, PCD can be blocked despite translocation of cytochrome c. These results suggest a role for the mitochondria in this PCD but do not support the current animal model for a causative role of cytochrome c in triggering PCD.     

The autophagosomal-lysosomal compartment in programmed cell death

In the last decade a tremendous progress has been achieved in understanding the control of apoptosis by survival and death factors as well as the molecular mechanisms of preparation and execution of the cell's suicide. However, accumulating evidence suggests that programmed cell death (PCD) is not confined to apoptosis but that cells use different pathways for active self-destruction as reflected by different morphology: condensation prominent, type I or apoptosis; autophagy prominent, type II; etc. Autophagic PCD appears to be a phylogenetically old phenomenon, it may occur in physiological and disease states. Recently, distinct biochemical and molecular features have been be assigned to this type of PCD. However, autophagic and apoptotic PCD should not be considered as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in a cell's response to changes of environmental conditions, both physiological or pathological. Furthermore, recent data suggest that diverse or relatively unspecific signals such as photodamage or lysosomotropic agents may be mediated by lysosomal cysteine proteases (cathepsins) to caspases and thus, apoptosis. The present paper reviews morphological, functional and biochemical/molecular data suggesting the participation of the autophagosomal-lysosomal compartment in programmed cell death.     

Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.     

Apoptosis: checkpoint at the mitochondrial frontier.

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.     

The role of Apaf-1 in programmed cell death: from worm to tumor

Apoptosis or programmed cell death is an important process to eliminate unnecessary or hazardous cells. Apaf-1, a mammalian homologue of CED-4 of C. elegans, is the essential adaptor molecule in the mitochondrial pathway of apoptosis. Mice lacking Apaf-1 show accumulation of neurons in the developing central nervous system due to reduced apoptosis. Apaf-1-deficient cells are remarkably resistant to various apoptotic stimuli. Apaf-1-mediated apoptosis plays a role in the prevention of tumorigenesis. However, Apaf-1-independent cell death pathways are also indicated. In this review, we will summarize what has been learned about the role of Apaf-1 by biochemical and genetical approaches.     

Caspases in yeast apoptosis-like death: facts and artefacts

Various findings suggest that programmed cell death (PCD) is induced in yeast as a response to the impact of a deleterious environment and/or an intracellular defect. Moreover, the specifically localized PCD within multicellular colonies seems to be important for the safe degradation of cell subpopulations to simple compounds that can be used as nutrients by healthy survivors occurring in propitious colony areas, being thus important for proper development and survival of the yeast population. In spite of this, the question remains whether yeast dies by real apoptosis, i.e. death involving caspases, or by other kinds of PCD. A large group of mammalian caspases includes those that are responsible for monitoring of the stimulus and initiating the dying process, as well as those involved in the execution of death. Additionally, paracaspases and metacaspases, that share some homology with real caspases, but possibly differ in substrate specificity, have been identified in plants, fungi, Dictyostelium and metazoa. In yeast, one homologue of caspases, metacaspase Mca1p/Yca1p, has been identified so far, although there are several indications of the presence of other caspase-like activities in yeast. In this minireview, we summarize various data on the possible involvement of Mca1p and other caspase-like activities in yeast PCD.     

Programmed cell death in plants

The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.     

Adult Apaf-1-deficient mice exhibit male infertility

Release of cytochrome c from the mitochondria, and subsequent binding to apoptotic protease-activating factor-1 (Apaf-1), is a key trigger of apoptotic events. A complex composed of Apaf-1, dATP, and cytochrome c activates a series of cytoplasmic proteases called caspases, leading to apoptotic cell death. We have disrupted the Apaf-1 gene in the mouse. Like previous reports on this knockout model, we find that most Apaf-1 mutants die perinatally and frequently exhibit exencephaly and cranioschesis. We additionally find that the neural lesions that develop in the knockout are due to an excess of neural progenitor cells that manifests as early as embryonic day 9.5 in development. In contrast to previous reports on the Apaf-1 knockout mice, we find that 5% of the mutants successfully survive to adulthood. In these survivors, the brain develops normally, but in males, there is degeneration of spermatogonia resulting in the virtual absence of sperm. Thus, cytochrome c-mediated apoptosis is not absolutely required for normal neural development, but is essential for spermatogenesis. These findings strongly suggest that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death.     

植物Metacaspase研究进展

过敏性坏死反应是植物的一种重要的抗病机制, 类似于动物细胞凋亡, 它是一种程序性细胞死亡(programmed cell death, PCD)过程。目前, 已经确定半胱天冬蛋白酶(caspase)在动物PCD过程中起核心作用。在植物中, 尚未发现其直系同源蛋白, 但是有一类与其结构相似的蛋白酶, 称为metacaspase。在植物不同的PCD过程中, 有的依赖于metacaspase, 而有的则不依赖于该类蛋白酶。目前对metacaspase的结构和功能已有了初步的研究, 对其深入的研究则进展缓慢, 其具体的生物学功能和在PCD信号路径中的定位有待进一步探索。     

Caspase-mediated programmed cell death pathways as potential therapeutic targets in cancer

The caspase family is well characterized as playing a crucial role in modulation of programmed cell death (PCD), which is a genetically regulated, evolutionarily conserved process with numerous links to many human diseases, most notably cancer. In this review, we focus on summarizing the intricate relationships between some members of the caspase family and their key apoptotic mediators, involving tumour necrosis factor receptors, the Bcl-2 family, cytochrome c, Apaf-1 and IAPs in cancer initiation and progression. We elucidate new emerging types of cross-talk between several caspases and autophagy-related genes (Atgs) in cancer. Moreover, we focus on presenting several PCD-modulating agents that may target caspases-3, -8 and -9, and their substrates PARP-1 and Beclin-1, which may help us harness caspase-modulated PCD pathways for future drug discovery.     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。     

AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.