Fire and death: the pyrin domain joins the death-domain superfamily

Apoptosis and inflammation are important cellular processes that are highly regulated through specific protein-protein interactions (PPI). Proteins involved in these signaling cascades often carry PPI domains that belong to the death-domain superfamily. This includes the structurally well-characterized Death Domain (DD), the Death Effector Domain (DED) and the Caspase Recruitment Domain (CARD) subfamilies. Recently, a fourth member of the DD superfamily was identified, the Pyrin Domain (PYD). Based on sequence alignments, homology to other domains occurring in death-signalling pathways, and secondary-structure prediction, the PYD was predicted to have an overall fold similar to other DD superfamily members. Just recently, NMR structures of two PYDs have been determined. The PYD structures not only revealed the DD superfamily fold as previously predicted, but also distinct features that are characteristic exclusively for this subfamily. This review summarizes recent findings and developments regarding structural aspects of the DD superfamily, with a special emphasis on the PPIs of the DD superfamily.     

The PYRIN domain: a member of the death domain-fold superfamily

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

Prolonged lipopolysaccharide exposure induces transient immunosuppression in BV2 microglia

Continuous pre-exposure of immune cells to low level of inflammatory stimuli makes them hyporesponsive to subsequent exposure. This pathophysiological adaptation; known as endotoxin tolerance is a general paradigm behind several disease pathogenesis. Current study deals with this immunosuppression with respect to BV2 microglia. We attempted to investigate their immune response under prolonged endotoxin exposure and monitor the same upon withdrawal of the stimuli. BV2 microglia cells were maintained under continual exposure of lipopolysaccharide (LPS) for weeks with regular passage after 72 hr (prolonged LPS exposed cells [PLECs]). PLECs were found to be immunosuppressed with diminished expression of proinflammatory cytokines (IL6, IL1β, TNF-α, and iNOS) and production of nitric oxide, as compared to once LPS exposed cells. Upon remaintenance of cells in normal media without LPS exposure (LPS withdrawal cells [LWCs]), the induced immunosuppression reversed and cells started responding to inflammatory stimuli; revealed by significant expression of proinflammatory cytokines. LWCs showed functional similarities to never LPS exposed cells (NLECs) in phagocytosis activity and their response to anti-inflammatory agents like dexamethasone. Despite their immunoresponsiveness, PLECs were inflamed and showed higher autophagy rate than NLECs. Additionally, we investigated the role of inhibitor of apoptotic proteins (IAPs) in PLECs to understand whether IAPs aids in the survival of microglial cells under stress conditions. Our results revealed that cIAP1 and cIAP2 are induced in PLECs which might play a role in retaining the viability. Furthermore, antagonism of IAPs has significantly induced cell death in PLECs suggesting the role of IAPs in microglial survival under stress condition. Conclusively, our data suggest that continuous exposure of BV2 microglia cells to LPS results in transient immunosuppression and indicates the involvement of IAPs in retaining their viability under inflammatory stress.     

The role of zinc in caspase activation and apoptotic cell death

In addition to its diverse role in many physiological systems, zinc (Zn) has now been shown to be an important regulator of apoptosis. The purpose of this review is to integrate previously published knowledge on Zn and apoptosis with current attempts to elucidate the mechanisms of action of this biometal. This paper begins with an introduction to apoptosis and then briefly reviews the evidence relating Zn to apoptosis. The major focus of this review is the mechanistic actions of Zn and its candidate intracellular targets. In particular, we examine the cytoprotective functions of Zn which suppress major pathways leading to apoptosis, as well as the more direct influence of Zn on the apoptotic regulators, especially the caspase family of enzymes. These two mechanisms are closely related since a decline in intracellular Zn below a critical threshold level may not only trigger pathways leading to caspase activation but may also facilitate the process by which the caspases are activated. Studies by our laboratory in airway epithelial cells show that Zn is co-localized with the precursor form of caspase-3, mitochondria and microtubules, suggesting this Zn is critically placed to control apoptosis. Further understanding the different pools of Zn and how they interact with apoptotic pathways should have importance in human disease.     

Active participation of cell death in development and organismal homeostasis

The precise, regional execution of programmed cell death is required for the proper patterning and sculpting of the embryonic primordium during animal development. In addition, cell death that is not directly involved in sculpting is also widely observed. The most abundant morphological form of programmed cell death in developing animals is apoptosis, and identification of the apoptotic genetic pathways has enabled the study of apoptosis' regulation and roles during development. Genetic and bio-imaging studies have permitted the study of the active roles of cell death in development and organismal homeostasis.     

The p53 tumor suppressor: Critical regulator of life & death in cancer

p53 is the most commonly mutated or deleted known gene in human cancer. The consequences of its disruption are profound, either in the germlines of patients with Li-Fraumeni Syndrome, or in mice with targeted gene knockouts. Abundant evidence suggests that p53 exerts regulation of cell cycle progression as well as apoptotic cell death, both in response to identical environmental or metabolic stressors. The specific decision of cell cycle arrest vs. death may underlie p53's differential ability to trigger death in cancer cells and arrest with repair in non-cancer cells, thus producing a therapeutic index pertinent to cancer therapy. Indeed, p53 status is likely to correlate with prognosis in many human cancers and in multiple animal tumor models. The mechanistic basis for p53's functions are still emerging, and will hopefully yield new therapeutic strategies applicable to treatment of the many poor-prognosis, p53-deficient human malignancies.     

Characterization of the bovine BCL2L1 gene and related pseudogenes

We have amplified and characterized partial regions of exons 2 and 3 of the bovine BCL2L1 gene, one of the anti-apoptotic members of the B-cell lymphoma 2 gene family. Cloning and sequencing of the amplified products revealed the existence of several BCL2L1-related sequences, including the bovine BCL2L1 gene and various processed pseudogenes. The bovine BCL2L1 gene revealed two polymorphic nucleotide sequences that resulted in two protein variants, with amino acid replacements at positions 60 and 69. In addition, we report three bovine BCL2L1-related sequences (BCL2L1psi) that probably correspond to intronless processed pseudogenes. These BCL2L1psi pseudogene sequences have accumulated multiple substitutions, deletions and insertions that translated into stop codons or changed the open reading frame of the functional gene. We provide evidence suggesting that the retro-transposition event that originated these processed pseudogenes took place before the divergence of the Cervidae and Bovidae families.     

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44^MAPK), the p38 kinase (p38^MAPK), the c-Jun N-terminal kinases (p46/p54^JNK), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/p44^MAPK as well as of Akt kinase was partially reduced. For p46/p54^JNK and p38^MAPK, elevated phosphorylation was measured. Inhibition of p46/p54^JNK and p38^MAPK activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.     

Apoptotic effects of bovine apo‐lactoferrin on HeLa tumor cells

Lactoferrin (Lf), a cationic iron‐binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron‐free bovine lactoferrin (apo‐bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron‐free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric‐bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl‐2, Sirt1, Mcl‐1, and PARP‐1 were modulated by 1.25 μM of apo‐bLf. In the same cell line, apo‐bLf induced apoptosis together with poly (ADP‐ribose) polymerase cleavage, caspase activation, and a significant drop of NAD⁺. In addition, apo‐bLf–treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo‐bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.     

Caspase activation contributes to delayed death of heat-stressed striatal neurons

Hyperthermia can contribute to brain damage both during development and post-natally. We used rat embryonic striatal neurons in culture to study mechanisms underlying hyperthermia-induced neuronal death. Heat stress at 43 degrees C for 2 h produced no obvious signs of damage during the first 12 h after the stress, but more than 50% of the neurons died during the next 3 days. More than 40% of the neurons had activated caspases 24 h following the heat stress. Caspase-3 activity increased with a delay of more than 10 h following cessation of the heat stress, reaching a peak at approximately 18 h. Neuronal death measured 1-3 days after the stress was reduced by the general caspase inhibitors qVD-OPH (10-20 microm) and zVAD-fmk (50-100 microm). These inhibitors were protective even when added 9 h after cessation of the heat stress, consistent with the delayed activation of caspases. In contrast, blockers of Na+ channels and ionotropic glutamate receptors did not reduce the heat-induced death, indicating that glutamate excitotoxicity was not required for this neuronal death. These results show that the neuronal death produced by heat stress has characteristics of apoptosis, and that caspase inhibitors can delay this death.     

Characterization of Apoptosis Induced by Fucoxanthin in Human Promyelocytic Leukemia Cells

Apoptosis induced by fucoxanthin in HL-60 cells was associated with a loss of mitochondrial membrane potential at an early stage, but not with an increase in reactive oxygen species. Fucoxanthin treatment caused cleavages of procaspase-3 and poly (ADP-ribose) polymerase without any effect on the protein level of Bcl-2, Bcl-X_L, or Bax. Apoptosis induction by fucoxanthin may be mediated via mitochondrial membrane permeabilization and caspase-3 activation.     

Caspase-dependent Cdk activity is a requisite effector of apoptotic death events

The caspase-dependent activation of cyclin-dependent kinases (Cdks) in varied cell types in response to disparate suicidal stimuli has prompted our examination of the role of Cdks in cell death. We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants (DN-Cdks) and Cdk inhibitory genes. Here we demonstrate that Cdk2 activity is necessary for death-associated chromatin condensation and other manifestations of apoptotic death, including cell shrinkage and the loss of adhesion to substrate. Susceptibility to the induction of the cell death pathway, including the activation of the caspase cascade, is unimpaired in cells in which Cdk2 activity is inhibited. The direct visualization of active caspase activity in these cells confirms that death-associated Cdk2 acts downstream of the caspase cascade. Cdk inhibition also does not prevent the loss of mitochondrial membrane potential and membrane phospholipid asymmetry, which may be direct consequences of caspase activity, and dissociates these events from apoptotic condensation. Our data suggest that caspase activity is necessary, but not sufficient, for the full physiological cell death program and that a requisite function of the proteolytic caspase cascade is the activation of effector Cdks.     

Hypoxia and low glucose differentially augments TRAIL-induced apoptotic death

Tumor microenvironment, which is characterized by hypoxia, low-glucose concentrations, high-lactate concentrations, low-extracellular pH, can alter the therapeutic response in tumors. In this study, we investigated whether hypoxia affects TRAIL-induced apoptotic death. When human prostate adenocarcinoma DU-145 cells were treated with 50 ng/mL TRAIL or hypoxia for 4 h, the survival was 45.7 and 32.5%, respectively. The combination of TRAIL and hypoxia synergistically increased cell death. Similar results were observed in human prostate adenocarcinoma LNCaP cells. Western blot analysis showed that the hypoxia augmented TRAIL-induced PARP cleavage as well as the activation of caspase-8 and caspase-3, but not caspase-9. Unlike hypoxia, low glucose promoted caspase-9 activation during TRAIL treatment. These results suggest that hypoxia or low glucose-augmented TRAIL cytotoxicity is mediated through the mitochondria-independent pathway or -dependent pathway, respectively. (Mol Cell Biochem 270: 89–97, 2005)     

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.