Pathways to caspase activation

Apoptosis or programmed cell death is an active form of cell death which is essential for tissue homeostasis. Many proteins are involved in the molecular signal transduction of apoptosis. The caspase enzymes, a family of specific cysteine proteases, play a central role in cell death machinery. In this review, we mainly discuss the current understanding of several pathways to activate caspases and some key proteins related to these pathways.     

Regulation of apoptosis by Bcl-2 family proteins

For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis.     

Death substrates come alive

Interleukin 1β-converting enzyme (ICE)-like proteases (caspases) play an important role in programmed cell death (apoptosis), and elucidating the consequences of their proteolytic activity is central to our understanding of the molecular mechanisms of cell death. Diverse structural and regulatory proteins and enzymes, including protein kinase Cδ, the retinoblastoma protein (a protein involved in cell survival), the DNA repair enzyme DNA-dependent protein kinase and the nuclear lamins, undergo specific and limited endoproteolytic cleavage by various caspases during apoptosis. Since individual caspases can cleave multiple substrates, the consequences of cleavage of only a single substrate are still poorly understood. Nevertheless, proteolytic activation of protein kinase Cδ may be an important early step in the cell death pathway, and cleavage of the retinoblastoma protein could suppress its cell survival function, whereas proteolytic inactivation of DNA repair enzymes might compromise the ability of the cell to reverse DNA fragmentation. On the other hand, cleavages of nuclear and cytoplasmic structural proteins (e.g. the lamins and Gas2) appear to be required for or contribute to the dramatic rearrangements in cellular architecture that are necessary for the completion of the cell death process. An emerging theme is that parallel and sequential proteolytic activation and inactivation of key protein substrates occurs during the multiple steps of apoptosis.     

Cathepsin-regulated apoptosis

Apoptosis can be mediated by different mechanisms. There is growing evidence that different proteolytic enzymes are involved in the regulation of apoptosis. Cathepsins are proteases which, under physiologic conditions, are localized intralysosomally. In response to certain signals they are released from the lysosomes into the cytoplasm where they trigger apoptotic cell death via various pathways, including the activation of caspases or the release of proapoptotic factors from the mitochondria. Here, we review different mechanisms that induce the release of lysosomal enzymes, and the functional relevance of defined cathepsins in defined models of apoptosis.     

Bacterial secreted effectors and caspase‐3 interactions

Apoptosis is a critical process that intrinsically links organism survival to its ability to induce controlled death. Thus, functional apoptosis allows organisms to remove perceived threats to their survival by targeting those cells that it determines pose a direct risk. Central to this process are apoptotic caspases, enzymes that form a signalling cascade, converting danger signals via initiator caspases into activation of the executioner caspase, caspase‐3. This enzyme begins disassembly of the cell by activating DNA degrading enzymes and degrading the cellular architecture. Interaction of pathogenic bacteria with caspases, and in particular, caspase‐3, can therefore impact both host cell and bacterial survival. With roles outside cell death such as cell differentiation, control of signalling pathways and immunomodulation also being described for caspase‐3, bacterial interactions with caspase‐3 may be of far more significance in infection than previously recognized. In this review, we highlight the ways in which bacterial pathogens have evolved to subvert caspase‐3 both through effector proteins that directly interact with the enzyme or by modulating pathways that influence its activation and activity.     

Regulation of DNA fragmentation: the role of caspases and phosphorylation

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.     

Inhibitor of apoptosis proteins in Drosophila: gatekeepers of death

Regulation of apoptosis is crucial to ensure cellular viability, and failure to do so is linked to several human pathologies. The apoptotic cell death programme culminates in the activation of caspases, a family of highly specific cysteine proteases essential for the destruction of the cell. Although best known for their role in executing apoptosis, caspases also play important signalling roles in non-apoptotic processes, such as regulation of actin dynamics, innate immunity, cell proliferation, differentiation and survival. Under such conditions, caspases are activated without killing the cell. Caspase activation and activity is subject to complex regulation, and various cellular and viral inhibitors have been identified that control the activity of caspases in their apoptotic and non-apoptotic roles. Members of the Inhibitor of APoptosis (IAP) protein family ensure cell viability in Drosophila by directly binding to caspases and regulating their activities in a ubiquitin-dependent manner. The observation that IAPs are essential for cell survival in Drosophila, and are frequently deregulated in human cancer, contributing to tumourigenesis, chemoresistance, disease progression and poor patient survival, highlights the importance of this family of caspase regulators in health and disease. Here we summarise recent advances from Drosophila that start to elucidate how the cellular response to caspase activation is modulated by IAPs and their regulators.     

Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Caspases: evolutionary aspects of their functions in vertebrates

Caspases (cysteine-dependent aspartyl-specific protease) belong to a family of cysteine proteases that mediate proteolytic events indispensable for biological phenomena such as cell death and inflammation. The first caspase was identified as an executioner of apoptotic cell death in the worm Caenorhabditis elegans . Additionally, a large number of caspases have been identified in various animals from sponges to vertebrates. Caspases are thought to play a pivotal role in apoptosis as an evolutionarily conserved function; however, the number of caspases that can be identified is distinct for each species. This indicates that species-specific functions or diversification of physiological roles has been cultivated through caspase evolution. Furthermore, recent studies suggest that caspases are also involved in inflammation and cellular differentiation in mammals. This review highlights vertebrate caspases in their universal and divergent functions and provides insight into the physiological roles of these molecules in animals.     

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.     

Caspase structure, proteolytic substrates, and function during apoptotic cell death

Caspases play an essential role during apoptotic cell death. These enzymes define a new class of cysteine proteases and comprise a multi-gene family with more than a dozen distinct mammalian family members. The discrete and highly limited subset of cellular polypeptides that are cleaved by these proteases is sufficient to account for the majority of cellular and morphological events that occur during cell death. In some cases, caspases also play a contributory role in escalating the propensity for apoptosis, and in doing so may exacerbate disease pathogenesis.     

Mitochondria-dependent apoptosis and cellular pH regulation

Mitochondria play a critical role in apoptosis induction in response to myriad stimuli. These organelles release proteins into the cytosol which trigger caspase activation or perform other functions relevant to apoptosis, including cytochrome c (cyt-c), caspases, AIF, and SMAC (Diablo). The mechanisms by which these proteins escape from mitochondria remain enigmatic. Moreover, it is unclear whether release of these proteins versus disturbances in core mitochondrial functions represents the cell death commitment mechanism. In this regard, suppression of apoptosis using broad-spectrum caspase inhibitory compounds has been reported in many circumstances to prevent the morphological and biochemical manifestations of apoptosis, and yet not protect cells from death and not preserve clonigenic survival. Thus, while mitochondrial damage can be coupled to caspase activation pathways, cell death commitment often occurs upstream of caspase activation when mitochondria-dependent cell death pathways are invoked. Here, we review evidence implicating dysregulation of cellular pH as a component of the cell death mechanism involving mitochondria. Cell Death and Differentiation (2000) 7, 1155 - 1165     

Role of Bcl-2 family proteins and caspases in the regulation of apoptosis

Apoptosis, or programmed cell death, plays a pivotal role in the elimination of unwanted, damaged, or infected cells in multicellular organisms and also in diverse biological processes, including development, cell differentiation, and proliferation. Apoptosis is a highly regulated form of cell death, and dysregulation of apoptosis results in pathological conditions including cancer, autoimmune and neurodegenerative diseases. The Bcl-2 family proteins are key regulators of apoptosis, which include both anti- and pro-apoptotic proteins, and a slight change in the dynamic balance of these proteins may result either in inhibition or promotion of cell death. Execution of apoptosis by various stimuli is initiated by activating either intrinsic or extrinsic pathways which lead to a series of downstream cascade of events, releasing of various apoptotic mediators from mitochondria and activation of caspases, important for the cell fate. In view of recent research advances about underlying mechanism of apoptosis, this review highlights the basics concept of apoptosis and its regulation by Bcl-2 family of protein. Furthermore, this review discusses the interplay of various apoptotic mediators and caspases to decide the fate of the cell. We expect that this review will add to the pool of basic information necessary to understand the mechanism of apoptosis which may implicate in designing better strategy to develop biomedical therapy to control apoptosis.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。     

P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase-1, -3 and -8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP-induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP-induced cell death.     

Caspases: A Molecular Switch Node in the Crosstalk between Autophagy and Apoptosis

Autophagy and apoptosis are two important catabolic processes contributing to the maintenance of cellular and tissue homeostasis. Autophagy controls the turnover of protein aggregates and damaged organelles within cells, while apoptosis is the principal mechanism by which unwanted cells are dismantled and eliminated from organisms. Despite marked differences between these two pathways, they are highly interconnected in determining the fate of cells. Intriguingly, caspases, the primary drivers of apoptotic cell death, play a critical role in mediating the complex crosstalk between autophagy and apoptosis. Pro-apoptotic signals can converge to activate caspases to execute apoptotic cell death. In addition, activated caspases can degrade autophagy proteins (i.e., Beclin-1, Atg5, and Atg7) to shut down the autophagic response. Moreover, caspases can convert pro-autophagic proteins into pro-apoptotic proteints to trigger apoptotic cell death instead. It is clear that caspases are important in both apoptosis and autophagy, thus a detailed deciphering of the role of caspases in these two processes is still required to clarify the functional relationship between them. In this article, we provide a current overview of caspases in its interplay between autophagy and apoptosis. We emphasized that defining the role of caspases in autophagy-apoptosis crosstalk will provide a framework for more precise manipulation of these two processes during cell death.     

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.     

Smac/DIABLO is required for effector caspase activation during apoptosis in human cells

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.