Caspase signaling in animal development

The caspases are a family of cysteine proteases that function as central regulators of cell death. Recent investigations in Caenorhabditis elegans, Drosophila, and mice indicate that caspases are essential not only in controlling the number of cells involved in sculpting or deleting structures in developing animals, but also in dynamic cell processes such as cell‐fate determination, compensatory proliferation of neighboring cells, and actin cytoskeleton reorganization, in a non‐apoptotic context during development. This review focuses primarily on caspase functions involving their enzymatic activity.     

Caspases - an update

Caspases belong to a family of highly conserved aspartate-specific cysteine proteases and are members of the interleukin-1beta-converting enzyme family, present in multicellular organisms. The caspase gene family consists of 15 mammalian members that are grouped into two major sub-families, namely inflammatory caspases and apoptotic caspases. The apoptotic caspases are further subdivided into two sub-groups, initiator caspases and executioner caspases. The caspases form a caspase-cascade system that plays the central role in the induction, transduction and amplification of intracellular apoptotic signals for cell fate determination, regulation of immunity, and cellular proliferation and differentiation. The substrates of apoptotic caspases have been associated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. The activation of this delicate caspase-cascade system and its functions are regulated by a variety of regulatory molecules, such as the inhibitor of apoptosis protein (IAP), FLICE, calpain, and Ca(2+). Based on the available literature we have reviewed and discussed the members of the caspase family, caspase-cascade system, caspase-regulating molecules and their apoptotic and non-apoptotic functions in cellular life and death. Also recent progress in the molecular structure and physiological role of non-mammalian caspases such as paracaspases, metacaspases and caspase-like-protease family members are included in relation to that of mammalian species.     

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.     

The insect caspases

Developmental and tissue homeostasis is a delicate balance between cell proliferation and cell death. The activation of caspases, a conserved family of cysteine proteases, is a main event in the initiation and execution of programmed cell death. While caspases have been characterized from many organisms, comparatively little is known about insect caspases. In Drosophila melanogaster, seven caspases have been characterized; three initiators and four effectors. In mosquitoes, several putative caspases have been identified in the genomes of Aedes aegypti and Anopheles gambiae. A small number of caspases have been identified in the Lepidoptera, the flour beetle, Tribolium castaneum, and the pea aphid, Acyrthosiphon pisum. The availability of new insect genome sequences will provide a unique opportunity to examine the caspase family across an evolutionarily diverse phylum and will provide valuable insights into their function and regulation.     

昆虫中分离与鉴定的caspase及其作用

天冬氨酸特异性的半胱氨酸蛋白酶(caspase)家族是执行细胞凋亡的主要酶类,对caspase结构及生物学功能的研究有助于更深入的研究细胞凋亡的分子机制。Caspase具有高度保守性,它们具有相似的氨基酸序列、结构和底物特异性。且具有QACRG的五肽活性位点,该活性位点是caspase家族的典型结构。昆虫caspase在caspase依赖型的细胞凋亡中起关键作用,文章介绍和评述昆虫中已经分离、鉴定的caspase及其功能。     

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.     

Regulation of caspase activation in apoptosis: implications for transformation and drug resistance

Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted. This revised version was published online in August 2006 with corrections to the Cover Date.     

A role for caspases in the differentiation of erythroid cells and macrophages

Several cysteine proteases of the caspase family play a central role in many forms of cell death by apoptosis. Other enzymes of the family are involved in cytokine maturation along inflammatory response. In recent years, several caspases involved in cell death were shown to play a role in other cellular processes such as proliferation and differentiation. In the present review, we summarize the current knowledge of the role of caspases in the differentiation of erythroid cells and macrophages. Based on these two examples, we show that the nature of involved enzymes, the pathways leading to their activation in response to specific growth factors, and the specificity of the target proteins that are cleaved by the activated enzymes strongly differ from one cell type to another. Deregulation of these pathways is thought to play a role in the pathophysiology of low-grade myelodysplastic syndromes, characterized by excessive activation of caspases and erythroid precursor apoptosis, and that of chronic myelomonocytic leukemia, characterized by a defective activation of caspases in monocytes exposed to M-CSF, which blocks their differentiation.     

The inflammatory caspases: key players in the host response to pathogenic invasion and sepsis

Caspases are cysteinyl-aspartate-specific proteinases known for their role in apoptosis (cell death or apoptotic caspases) and proinflammatory cytokine maturation (inflammatory caspases). The inflammatory caspases were among the first to be discovered, but only recently have the mechanisms leading to their activation and inhibition begun to be elucidated. In this review, we examine the biochemistry, substrates, and function of this unique family of inflammatory proteases, highlight the most recent findings regarding their regulatory mechanisms, and discuss what remains to be understood about their roles in health and disease.     

PLA2 activity is required for nuclear shrinkage in caspase-independent cell death

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage.     

Evolution of the folding landscape of effector caspases

Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases [PCPs]) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor, whereas others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor (CA) into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their CA. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (PCP-7, CA) or dimeric (PCP-6) intermediate. In comparison, the unfolding pathway of PCP-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the CA and was retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase–hemoglobinase fold.     

Baculovirus Regulation of Apoptosis

The baculovirus AcMNPV induces apoptosis in a host-specific manner which involves the activation of host caspases (cysteine-dependent, aspartate-specific proteases). AcMNPV carries a novel gene, p35, which encodes a stoichiometric inhibitor of active caspases, thereby blocking apoptosis. P35 is cleaved by caspases and the cleavage products form a stable complex with the caspase. Baculoviruses also carry genes known as iaps (inhibitors of apoptosis), some of which can actively suppress apoptosis by inhibiting the activation of caspases. Members of the IAP family are found in both viral and animal genomes and interact physically with a variety of proteins associated with apoptotic pathways including Reaper, Doom, TRAF2, and some caspases. The ability of baculoviruses to block apoptosis influences their pathogenicity and host range.     

IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms

Some members of the inhibitor of apoptosis (IAP) family suppress apoptosis by neutralizing caspases. The current model suggests that all caspase-regulatory IAPs function as direct enzyme inhibitors, blocking effector caspases by binding to their catalytically active pockets. Here we show that IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Whereas XIAP binds directly to the active-site pockets of effector caspases, we find that regulation of effector caspases by Drosophila IAP1 (DIAP1) requires an evolutionarily conserved IAP-binding motif (IBM) at the neo-amino terminus of the large caspase subunit. Remarkably, unlike XIAP, DIAP1-sequestered effector caspases remain catalytically active, suggesting that DIAP1 does not function as a bona fide enzyme inhibitor. Moreover, we demonstrate that the mammalian IAP c-IAP1 interacts with caspase-7 in an exclusively IBM-dependent, but active site pocket-independent, manner that is mechanistically similar to DIAP1. The importance of IBM-mediated regulation of effector-caspases in vivo is substantiated by the enhanced apoptotic potency of IBM-mutant versions of drICE, DCP-1 and caspase-7.     

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.     

Peptides in apoptosis research.

Apoptosis is a complex process that plays a central role in physiological and pathological cell death. This fast evolving research area has experienced incredible development in the past few years. Progress in the knowledge of the structure of many of the main molecular actors of the apoptotic signal transduction pathways has driven the design of synthetic peptides that in some cases can function as simplified versions of their parent proteins. These molecules are contributing to a better understanding of the activity and regulation of apoptotic proteins and also are setting the basis for the discovery of effective drugs to combat important diseases related to apoptosis. Most applications of peptides in apoptosis research are so far related to caspases, caspase regulatory proteins, such as LAPs and Smac, and proteins of the Bcl-2 family. Additionally, important perspectives are open to other systems, such as the macromolecular assemblies that are responsible for the activation of initiator caspases.     

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.     

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.     

Caspases - controlling intracellular signals by protease zymogen activation

Animal development and homeostasis is a balance between cell proliferation and cell death. Physiologic, and sometimes pathologic, cell death - apoptosis - is driven by activation of a family of proteases known as the caspases, present in almost all nucleated animal cells. The enzymatic properties of these proteases are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side chain for catalyzing peptide bond cleavage. The primary specificity for Asp turns out to be very rare among proteases, and currently the only other known mammalian proteases with the same primary specificity is the physiological caspase activator granzyme B. Like most other proteases, the caspases are synthesized as inactive zymogens whose activation requires limited proteolysis or binding to co-factors. To transmit the apoptotic execution signal, caspase zymogens are sequentially activated through either an intrinsic or an extrinsic pathway. The activation of caspases at the apex of each pathway, the initiators, occurs by recruitment to specific adapter molecules through homophilic interaction domains, and the activated initiators directly process the executioner caspases to their catalytically active forms. In the present communication we review the different mechanisms underlying the selective activation of the caspases.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Mammalian initiator apoptotic caspases

Caspases are a conserved family of cysteine proteases. They play diverse roles in inflammatory responses and apoptotic pathways. Among the caspases is a subgroup whose primary function is to initiate apoptosis. Within their long prodomains, caspases-2, -9 and -12 contain a caspase activation and recruitment domain while caspases-8 and -10 bear death effector domains. Activation follows the recruitment of the procaspase molecule via the prodomain to a high molecular mass complex. Despite sharing some common features, other aspects of the biochemistry, substrate specificity, regulation and signaling mechanisms differ between initiator apoptotic caspases. Defects in expression or activity of these caspases are related to certain pathological conditions including neurodegenerative disorders, autoimmune diseases and cancer.