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.     

Proteolytic cleavage during chemotherapy-induced apoptosis

Treatment with anticancer drugs sets into motion a morphologically and biochemically distinct type of cell death called apoptosis. Recent genetic and biochemical studies have suggested that proteases play a prominent role in the active phase of apoptotic cell death. Ongoing studies are aimed at identifying the proteases involved, the substrates that are cleaved, and the means by which the proteolytic process is regulated in nonapoptotic and apoptotic cells. The possibility that these findings will suggest new approaches to treating cancer and other diseases is discussed.     

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during 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.     

Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa

Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.     

Importance of the role of calcium in programmed cell death: a review

Calcium plays an important role in physiological cell death processes such as terminal differentiation and apoptosis. Cell injury occurs when the intracellular calcium pool is disturbed, which in turn may lead to cell death. Calcium in calcium-dependent enzymes, transglutaminases, various proteases, phosphorylases and kinase, is involved in the process of cell death. Examples of such enzymes involved in cell death, and the role of calcium levels in regulation of these enzymes, are described and discussed.     

Non-apoptotic Functions of Caspase-3 in Nervous Tissue

Some enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not necessarily related to cell death in nervous tissue. The activity of caspase-3, an "apoptotic" enzyme, can be measured in normally functioning neurons. The results reported by several groups point to the possibility that caspases may be involved in nervous tissue function as top enzymes in the regulatory proteolytic cascade. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning. The idea of synaptic plasticity modulation by caspase-3 is in line with data reported recently. For example, caspase-3 is possibly involved in the long-term potentiation (LTP) phenomenon since proteins that are key players of molecular mechanisms of LTP induction and maintenance are caspase-3 substrates. Experimental results on blocking LTP by a caspase-3 inhibitor confirm this concept.     

Conventional calpains and programmed cell death

The evidence on the crucial role of a family of calcium-dependent cysteine proteases called calpains in programmed cell death is rich and still growing. However, understanding of the mechanisms of their functions in apoptosis is not full yet. Calpains have been implicated in both physiological and pathological cell death control, especially in various malignancies, but also in the immune system development and function. There is also growing evidence on calpain involvement in apoptosis execution in certain pathological conditions of the central nervous system, in cardiovascular diseases, etc. Understanding of the clinical significance of calpain activation pathways, after intense studies of the influence of calpain activity on drug-induced apoptosis, seems especially important lately, as calpains have become noticed as potential therapeutic targets. To allow pharmacological targeting of these enzymes, thorough knowledge of their patterns of activation and further interactions with already known apoptotic pathways is necessary. A comprehensive summary of both well established and recently obtained information in the field is an important step that may lead to future advances in the use of calpain-targeted agents in the clinic.     

The kinder side of killer proteases: Caspase activation contributes to neuroprotection and CNS remodeling

Caspases are a family of cysteine proteases that are expressed as inactive zymogens and undergo proteolytic maturation in a sequential manner in which initiator caspases cleave and activate the effector caspases 3, 6 and 7. Effector caspases cleave structural proteins, signaling molecules, DNA repair enzymes and proteins which inhibit apoptosis. Activation of effector, or executioner, caspases has historically been viewed as a terminal event in the process of programmed cell death. Emerging evidence now suggests a broader role for activated caspases in cellular maturation, differentiation and other non-lethal events. The importance of activated caspases in normal cell development and signaling has recently been extended to the CNS where these proteases have been shown to contribute to axon guidance, synaptic plasticity and neuroprotection. This review will focus on the adaptive roles activated caspases in maintaining viability, the mechanisms by which caspases are held in check so as not produce apoptotic cell death and the ramifications of these observations in the treatment of neurological disorders.     

Proteolytic enzymes and apoptosis

Information about basic mechanisms of programmed cell death (apoptosis) development with participation of proteolytic enzymes is given in the review. The basic mechanisms of apoptosis launching are conditionally subdivided into three groups, depending on the "points of application" of apoptosis development initiating factor: membrane (receptor-dependent), mitochondrial and nuclear. Attention is accentuated on ubiquitin-dependent proteasomal proteolysis as the key system regulating apoptosis. The possible disturbances of apoptotic program realization are specified under various pathological processes and diseases.     

Role of ICE-related and other proteases in Fas-mediated apoptosis

Interleukin-1beta-converting enzyme (ICE)-like proteases comprise a novel family of unusual cysteine proteases which have been implicated in programmed cell death in both invertebrates and mammals. Current available evidence indicates a role of ICE proteases as central executioners of apoptosis triggered by the cell surface receptor Fas (APO-l). The presence of multiple mammalian ICE proteases with partially overlapping but distinct activities suggests a complex proteolytic cascade which is induced upon Fas ligation. The precise role of single members of the ICE family in Fas-mediated apoptosis, however, is still unclear. Here, we summarize the present knowledge about the relevance of ICE proteases, their potential targets, and interaction with unrelated proteases in cell death mediated by Fas and other apoptotic stimuli.     

"Apoptotic" mechanisms in normal brain plasticity: caspase-3 and long-term potentiation

The analysis of recent data indicates that a few enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not related to cell death in the brain. For example, it can be demonstrated that caspase-3, an "apoptotic" enzyme that is active in neurons is necessary for normal neuroplasticity. Here we discuss the involvement of caspase-3 in long-term potentiation phenomenon. Proteins that are key players of molecular mechanisms of long-term potentiation induction and maintenance are also caspase-3 substrates. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning.     

New concepts in radiation-induced apoptosis: 'premitotic apoptosis' and 'postmitotic apoptosis'

Formerly, the mechanisms responsible for the killing of cells by ionizing radiation were regarded as being divided into two distinct forms, interphase death and reproductive death. Since they were defined based on the classical radiobiological concepts using a clonogenic cell survival assay, biochemical and molecular biological mechanisms involved in the induction of radiation-induced cell death were not fully understood in relation to the modes of cell death. Recent multidisciplinary approaches to cell death mechanism have revealed that radiation-induced cell death is divided into several distinct pathways by the time course and cell-cycle position, and that apoptotic cell death plays a key role in almost every mode of cell death. This review discusses the mechanisms of radiation-induced apoptosis in relation to cellcycle progression and highlights a new concept of the mode of cell death: 'premitotic apoptosis' and 'postmitotic apoptosis'. The former is a rapid apoptotic cell death associated with a prompt activation of caspase-3, a key enzyme of intracellular signaling of apoptosis. Arapid execution of cell killing in premitotic apoptosis is presumably due to the prompt activation of a set of pre-existed molecules following DNA damages. In contrast, the latter is a delayed apoptotic cell death after cell division, and unlike premitotic apoptosis, it neither requires a rapid activation of caspase-3 nor is inhibited by a specific inhibitor, Ac-DEVD-CHO. A downregulation of anti-apoptotic genes such as MAPK and Bcl-2 may play a key role in this mode of cell death. Characterization of these two types of apoptotic cell death regarding the cell cycle regulation and intrcellular signaling will greatly help to understand the mechanisms of radiation-induced apoptosis.     

Current concepts in apoptosis: The physiological suicide program revisited

Apoptosis, or programmed cell death (PCD), involves a complex network of biochemical pathways that normally ensure a homeostatic balance between cellular proliferation and turnover in nearly all tissues. Apoptosis is essential for the body, as its deregulation can lead to several diseases. It plays a major role in a variety of physiological events, including embryonic development, tissue renewal, hormone-induced tissue atrophy, removal of inflammatory cells, and the evolution of granulation tissue into scar tissue. It also has an essential role in wound repair. The various cellular and biochemical mechanisms involved in apoptosis are not fully understood. However, there are two major pathways, the extrinsic pathway (receptor-mediated apoptotic pathway) and the intrinsic pathway (mitochondria-mediated apoptotic pathway), which are both well established. The key component in both is the activation of the caspase cascade. Caspases belong to the family of proteases that ultimately, by cleaving a set of proteins, cause disassembly of the cell. Although the caspase-mediated proteolytic cascade represents a central point in the apoptotic response, its initiation is tightly regulated by a variety of other factors. Among them, Bcl-2 family proteins, TNF and p53 play pivotal roles in the regulation of caspase activation and in the regulation of apoptosis. This review summarizes the established concepts in apoptosis as a physiological cell suicide program, highlighting the recent and significant advances in its study.     

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.     

Programmed cell death and the proteasome

A characteristic feature of apoptotic cell death is the activation of a cascade of cytoplasmic proteases that results in the cleavage of a limited number of target proteins. A central role in these proteolytic events has been assigned to members of the capase family. However, the use of low molecular weight proteasomal inhibitors has also demonstrated that protein degradation or processing by the ubiquitin-proteasome system of the cell has a decisive impact on cell survival and death as well, depending on the cell type and/or the proliferative status of the cells studied. Treatment of proliferating cells with proteasome inhibitors leads to cell death, potentially involving an internal signalling conflict between accumulating levels of the cdk inhibitor p27Kip1 and c-myc. In contrast, in terminally differentiated cells the same compounds have the opposite effect of blocking apoptosis, possibly by preventing proteasome-mediated degradation of a capase inhibitor. In this review the role of proteasome-mediated proteolysis in the dying cell is discussed and apparently conflicting results are integrated into a working hypothesis which functionally locates the proteasome upstream of capase3-like enzymes.     

Apoptosis: alive and kicking in 1997

The various cellular signalling pathways and biochemical activities involved in apoptotic death are now under intense study in many different laboratories. Recent studies using both molecular cloning approaches and in vitro systems have identified a class of highly specific cellular proteases, termed caspases, that appear to have important roles in apoptotic execution. One of these enzymes may lie near the head of the death pathway in certain cells, whereas others may be involved in the final stages of cellular disassembly. Other recent studies using both live cell and in vitro systems have suggested that mitochondria have an essential role in apoptosis. Mitochondria apparently release at least two factors - a protease and cytochrome C - that are capable of triggering apoptotic changes in isolated cell nuclei. The release of the apoptogenic protease appears to be under the control of the Bcl-2 gene product.     

Neurons bearing presenilins: weapons for defense or suicide?

Apoptotic machinery designed for cell's organized self-destruction involve different systems of proteases which cleave vital proteins and disassemble nuclear and cytoplasmic structures, committing the cell to death. The most studied apoptotic proteolytic system is the caspase family, but calpains and the proteasome could play important roles as well. Alzheimer's disease associated presenilins showed to be a substrate for such proteolytic systems, being processed early in several apoptotic models, and recent data suggest that alternative presenilin fragments could regulate cell survival. Mutations in genes encoding presenilins proved to sensitize neurons to apoptosis by different mechanisms e.g. increased caspase-3 activation, oxyradicals production and calcium signaling dysregulation. Here we review the data involving presenilins in apoptosis and discuss a possible role of presenilins in the regulation of apoptotic biochemical machinery.