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.     

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。     

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.

The activity of ICE-like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis-inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.     

Caspase的活化及其在细胞凋亡中的作用

Caspase是执行细胞凋亡的主要酶类,目前已鉴定的哺乳动物Caspase有14种。Caspase以酶原的形式合成,催化活性很低,必须激活以后才能发挥作用。活化的Caspase通过特异性的裂解一套底物而导致细胞凋亡。与Caspase有关的细胞凋亡通路至少有三种：线粒体／细胞色素c通路、死亡受体通路和内质网通路。Caspase总是与其抑制剂共存,以防止Caspase酶原意外激活而对正常细胞造成损伤。     

Targeted disruption of caspase genes in mice: what they tell us about the functions of individual caspases in apoptosis

Cysteine proteases of the caspase family are crucial mediators of apoptosis. All mammalian cells contain a large number of caspases. Although many caspases are activated in a cell committed to apoptosis, recent data from caspase gene knockout mice suggest that individual caspases may be involved in the cell and stimulus-specific pathways of cell death. The gene disruption studies also establish the functional hierarchy between two structurally distinct classes of caspases. The present review discusses these recent findings and elaborates on how these mutant mouse models have helped the understanding of the mechanisms that govern programmed cell death in the immune and other systems.     

Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue

Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.     

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.     

Old,new and emerging functions of caspases

Caspases are proteases with a well-defined role in apoptosis. However, increasing evidence indicates multiple functions of caspases outside apoptosis. Caspase-1 and caspase-11 have roles in inflammation and mediating inflammatory cell death by pyroptosis. Similarly, caspase-8 has dual role in cell death, mediating both receptor-mediated apoptosis and in its absence, necroptosis. Caspase-8 also functions in maintenance and homeostasis of the adult T-cell population. Caspase-3 has important roles in tissue differentiation, regeneration and neural development in ways that are distinct and do not involve any apoptotic activity. Several other caspases have demonstrated anti-tumor roles. Notable among them are caspase-2, -8 and -14. However, increased caspase-2 and -8 expression in certain types of tumor has also been linked to promoting tumorigenesis. Increased levels of caspase-3 in tumor cells causes apoptosis and secretion of paracrine factors that promotes compensatory proliferation in surrounding normal tissues, tumor cell repopulation and presents a barrier for effective therapeutic strategies. Besides this caspase-2 has emerged as a unique caspase with potential roles in maintaining genomic stability, metabolism, autophagy and aging. The present review focuses on some of these less studied and emerging functions of mammalian caspases.     

Divinations and surprises: genetic analysis of caspase function in mice

Caspases are critical mediators of apoptosis, the principle mechanism by which extra and harmful cells are eliminated to ensure proper development and maintain cellular homeostasis in all multicellular organisms. While compelling evidence suggests that the activation of these otherwise latent intracellular proteases is required for the execution of most, if not all apoptosis in mammals, the presence of more than a dozen caspases presents a major challenge to our understanding of the precise function of individual caspases in vivo. Using a genetic approach, several groups have generated transgenic mice deficient in various caspases so as to investigate their physiological functions. In this review, we will discuss what these studies have revealed about the role of individual caspase in development, apoptosis, and inflammation, with a particular focus on the predictable phenotypes versus the surprises based on in vitro results, as well as the implications of these findings.     

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.     

A subset of caspase substrates functions as the Jekyll and Hyde of apoptosis

Cleavage of caspase substrates is believed to be the commitment point that will lead a cell towards apoptosis. While the cleavage of some caspase substrates participates directly in the dismantling of the cell, others regulate the extent of caspase activation. In this communication, we discuss some recent findings indicating that two caspase substrates, MEKK1 and RasGAP, change their functions from anti- to pro-apoptotic as caspase activity increases. MEKK1 is a MAPK kinase kinase regulating the JNK MAPK pathway. As a full-length protein, MEKK1 generates protective signals (e.g. in cardiomyocytes), but potentiates apoptosis when cleaved by caspases. This switch is mediated by a translocation of the kinase activity from insoluble to soluble cellular structures. RasGAP is a regulator of Ras GTPase family members. As a full-length protein, RasGAP does not modulate apoptosis. However, low caspase activity readily induces the cleavage of RasGAP into an N-terminal fragment that generates potent anti-apoptotic signals. At higher caspase activity, the N-terminal fragment is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP can, thus, be viewed as an apoptostat because it allows the cells to determine when caspases have been mildly activated to fulfill functions other than apoptosis or when caspases are strongly activated to mediate apoptosis.     

Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology

Caspases are the central component of the apoptotic machinery that irreversibly commits a cell to die. Whereas all caspases are structurally similar, those involved in apoptosis can be categorized functionally as either initiator or effector caspases, which are activated by distinct mechanisms. The activated caspases are subject to inhibition by the inhibitor of apoptosis family of proteins. This inhibition can be removed by Smac/DIABLO during apoptosis. The underlying molecular mechanisms of caspase regulation are discussed in this article.     

Nonapoptotic functions of caspases: caspases as regulatory molecules for immunity and cell-fate determination

Caspases are a family of cysteine proteases that are highly conserved in multicellular organisms and function as central regulators of apoptosis. Recent investigations in Caenorhabditis elegans, Drosophila and mice suggest that caspases also function as regulatory molecules for immunity and cell-fate determination. Here, we review genetic studies of nonapoptotic functions of caspases and discuss the regulatory mechanisms of caspases for executing nonapoptotic functions.     

Mechanisms of B cell receptor induced apoptosis

B cell receptor (BCR)-mediated apoptosis plays a key role in the negative selection (deletion) of autoreactive B cells. Mechanisms of BCR-mediated apoptosis have been widely studied in cell lines representing both immature (bone marrow) and mature (germinal center) B cells. However, there is much inconsistency and controversy concerning the possible mechanisms of BCR-mediated apoptosis, which may reflect differences in the origin or the maturational stage of the cell line used. Based on recent studies, collapse of mitochondrial membrane potential (Delta Psi m) seems to be an essential event for BCR-mediated apoptosis in both mature and immature cells. The collapse of Delta Psi m is dependent on the synthesis of new proteins, which are involved in the permeability change of mitochondrial membranes. Mitochondrial dysfunction induces activation of caspases, cysteine proteases, which play a central role in apoptosis. However, instead of caspases, other effector proteases, such as cathepsins or calpains, may also be responsible for the organized destruction of cell components seen during BCR-mediated apoptosis.     

Caspases and apoptosis in fish

Apoptosis has a vital impact on the development and homeostasis of all multicellular organisms. Hence, all metazoan species seem to possess the necessary components of the apoptotic machinery, but in general, their numbers and complexity have increased during evolution. The key apoptotic factors are a cascade of cysteine proteases known as caspases. The fish homologous of almost all the mammalian caspases have also been identified, but several fish-specific caspases with putative distinct functions have also been reported. Despite these differences, the extrinsic and intrinsic pathways have been remarkably well conserved throughout 500 million years of vertebrate evolution. Here, the authors review what is currently known about fish caspases and apoptosis and demonstrate the huge amount of sequence information available from a range of fish species by screening Atlantic salmon genome databases for apoptotic homologous.     

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.     

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.     

HAX-1 protein: multifunctional factor involved in apoptosis, cell migration, endocytosis and mRNA transport

HAX-1 protein, an anti-apoptotic factor, first identified in 1997, is also involved in cell migration, endocytosis and probably mRNA transport. HAX-1 structure indicates similarity to the proteins form Bcl-2 family, although there is no strong homology. HAX-1 is a substrate for Omi/HtrA2, a protease responsible for degradation of the caspases, and functions as an inhibitor of caspase-9, which points to its role in the regulation of apoptosis. Several HAX-1 interactions with proteins involved in apoptosis and cell motility were demonstrated. Another line of inquiry focus on its ability to bind 3' untranslated regions of the certain mRNAs. Some data indicate that it might be involved in mRNA transport. HAX-1 multifunctionality and its involvement in the processes important for the cell status suggest its possible role in oncogenesis and metastasis. It is also known that HAX-1 deficiency or overexpression leads to hereditary or systemic diseases (Kostmann disease, lesional psoriasis, systemic sclerosis). Therefore, detailed analysis of HAX-1 functions could be medically important.