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.     

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.     

Apoptosis-independent functions of killer caspases

Caspases are well known for their role in the execution of the apoptotic program by cleaving specific target proteins, leading to the dismantling of the cell, as well as for mediating cytokine maturation. Recent work has highlighted novel non-apoptotic activities of apoptotic caspases. These reports indicate that caspases are much more versatile enzymes than we originally expected. In addition to regulating cell survival and cytokine maturation, caspases may be involved in regulating cell differentiation, cell proliferation, spreading and receptor internalization.     

Proteolytic signaling by TNFalpha: caspase activation and IkappaB degradation

Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IkappaBalpha. Caspases are activated using mechanisms of oligomeration and 'self-controlled proteolysis'. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-kappaB is dependent on the degradation of IkappaBalpha. IkappaB kinase (IKK) phosphorylates IkappaBalpha at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-kappaB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-kappaB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-kappaB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die.     

Non-apoptotic functions of apoptosis-regulatory proteins

During the past two decades, apoptotic cell death has been the subject of an intense wave of investigation, leading to the discovery of multiple gene products that govern both its induction and execution. In parallel, it has progressively become evident that most, if not all, proteins that had initially been discovered for their essential role in apoptosis also mediate a wide range of non-apoptotic functions. On the one hand, apoptotic regulators and executioners are involved in non-lethal physiological processes as diverse as cell cycle progression, differentiation, metabolism, autophagy and inflammation. On the other hand, pro-apoptotic proteins can control other modalities of programmed cell death, in particular regulated necrosis. In this review, we summarize the unconventional roles of the apoptotic core machinery from a functional perspective and discuss their pathophysiological implications.     

Caspase-containing complexes in the regulation of cell death and inflammation

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.     

Innate immunity: regulation of caspases by IAP-dependent ubiquitylation

Caspases are widely known as initiators and executioners of cell death. Full activation of caspases leading to cleavage of many cellular substrates was long considered to be a point-of-no-return in the apoptosis pathway. However, it also has been known that activated caspases do not always have the ability to kill, but instead initiate non-apoptotic processes such as cell differentiation or activation of innate immune responses. In this issue of The EMBO Journal, Meinander et al (2012) explore the contribution of polyubiquitination of Dredd, a known initiator caspase, to the activation of innate immunity. The authors show that infection with gram-negative bacteria leads to DIAP2-dependent ubiquitylation of Dredd which in turn is required for processing of Relish (Rel) and expression of antimicrobial peptide (AMP) genes that are indispensable for fighting the infection.     

Non-apoptotic Fas signaling

Fas (Apo-1, CD95) and Fas-Ligand (FasL, CD95L) are typical members of the TNF receptor and TNF ligand family, respectively, with a pivotal role in the regulation of apoptotic processes, including activation-induced cell death, T-cell-induced cytotoxicity, immune privilege and tumor surveillance. Impairment of the FasL/Fas system has been implicated in liver failure, autoimmune diseases and immune deficiency. Thus, the FasL/Fas system was mainly appreciated with respect to its death-inducing capabilities. However, there is increasing evidence that activation of Fas can also result in non-apoptotic responses like cell proliferation or NF-kappaB activation. While the apoptotic features of the FasL/Fas system and the pathways involved are comparably well investigated, the pathways that are utilized by Fas to transduce proliferative and activating signals are poorly understood. This review is focused on the non-apoptotic functions of the FasL/Fas system. In particular, the similarities and differences of the molecular mechanisms of apoptotic and non-apoptotic Fas signaling are addressed.     

The multifaceted activity of insect caspases

Caspases are frequently considered synonymous with apoptotic cell death. Increasing evidence demonstrates that these proteases may exert their activities in non-apoptotic functions. The non-apoptotic roles of caspases may include developmentally regulated autophagy during insect metamorphosis, as well as neuroblast self-renewal and the immune response. Here, we summarize the established knowledge and the recent advances in the multiple roles of insect caspases to highlight their relevance for physiological processes and survival.     

Death by design: apoptosis, necrosis and autophagy

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide.     

Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis

The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via site-specific cleavage. To date, more than 60 proteins have been shown to be substrates of one or more caspases in mammalian cells, and the list is still growing. In this study, to identify human caspase-3 substrates, we digested lysates obtained from a caspase-3-deficient MCF-7 cell line with purified caspase-3 and analyzed eliminated or decreased spots by 2-DE. Proteins degraded by caspase-3, termed as caspase-3 degradome, are involved in a variety of cellular functions, such as stress-responsive proteins, signaling molecules, structural proteins, and unclassified proteins. Interestingly, the cellular level of vinculin, a caspase-3 substrate, was dramatically reduced during the apoptotic process, where the expression level of caspase-3 was increased. This degradomic approach could provide a powerful tool in finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.     

Involvement of protein kinase C-delta in DNA damage-induced apoptosis

Apoptosis is a highly orchestrated cell suicidal program required to maintain a balance between cell proliferation and cell death. A defect in apoptotic machinery can cause cancer. Many anticancer drugs are known to kill tumor cells by inducing apoptosis, and a defect in apoptosis can lead to anticancer drug resistance. Apoptosis is regulated by a complex cellular signaling network. Several members of the protein kinase C (PKC) family serve as substrates for caspases and PKCδ isozyme has been intimately associated with DNA damage-induced apoptosis. It can act both upstream and downstream of caspases. In response to apoptotic stimuli, the full-length and the catalytic fragment of PKCδ may translocate to distinct cellular compartments, including mitochondria and the nucleus, to reach their targets. Both activation and intracellular distribution of PKCδ may have significant impact on apoptosis. This review intends to assimilate recent views regarding the involvement of PKCδ in DNA damage-induced apoptosis.     

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.