Protein disulfide isomerase is cleaved by caspase-3 and -7 during apoptosis

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.     

X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.     

Emerging roles of caspase-3 in apoptosis

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.     

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.     

Apoptosome dependent caspase-3 activation pathway is non-redundant and necessary for apoptosis in sympathetic neurons

Although sympathetic neurons are a well-studied model for neuronal apoptosis, the role of the apoptosome in activating caspases in these neurons remains debated. We find that the ability of sympathetic neurons to undergo apoptosis in response to nerve growth factor (NGF) deprivation is completely dependent on having an intact apoptosome pathway. Genetic deletion of Apaf-1, caspase-9, or caspase-3 prevents apoptosis after NGF deprivation, and importantly, allows these neurons to recover and survive long-term following readdition of NGF. The inability of caspase-3 deficient sympathetic neurons to undergo apoptosis is particularly striking, as apoptosis in dermal fibroblasts and cortical neurons proceeds even in the absence of caspase-3. Our results show that in contrast to dermal fibroblasts and cortical neurons, sympathetic neurons express no detectable levels of caspase-7. The strict requirement for an intact apoptosome, coupled with a lack of effector caspase redundancy, provides sympathetic neurons with a markedly increased control over their apoptotic pathway.     

TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress

14‐3‐3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14‐3‐3 gene family members (TFT1–TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild‐type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺‐ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺‐ATPase to release more protons under LP.     

Alternative, non-secretase processing of Alzheimer's beta-amyloid precursor protein during apoptosis by caspase-6 and -8.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Although the pathogenesis of AD is unknown, it is widely accepted that AD is caused by extracellular accumulation of a neurotoxic peptide, known as Abeta. Mutations in the beta-amyloid precursor protein (APP), from which Abeta arises by proteolysis, are associated with some forms of familial AD (FAD) and result in increased Abeta production. Two other FAD genes, presenilin-1 and -2, have also been shown to regulate Abeta production; however, studies examining the biological role of these FAD genes suggest an alternative theory for the pathogenesis of AD. In fact, all three genes have been shown to regulate programmed cell death, hinting at the possibility that dysregulation of apoptosis plays a primary role in causing neuronal loss in AD. In an attempt to reconcile these two hypotheses, we investigated APP processing during apoptosis and found that APP is processed by the cell death proteases caspase-6 and -8. APP is cleaved by caspases in the intracellular portion of the protein, in a site distinct from those processed by secretases. Moreover, it represents a general effect of apoptosis, because it occurs during cell death induced by several stimuli both in T cells and in neuronal cells.     

Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis

The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin–proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.     

The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C_24:0 CerS activity, and while CerS5 and CerS6 each confer ～ 50% of the C_16:0 CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.     

Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.     

Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway

Claspin is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Here, we show that human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide caused activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin was cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress.     

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.     

Expression of caspase-3 and -7 does not correlate with the extent of apoptosis in primary breast carcinomas

Association between the rate of apoptosis and expression of the several relevant molecules (Bcl-2, pro- and active caspase-3, and caspase-7) was studied in 61 primary breast carcinomas. The rate of apoptosis detected both morphologically and by the TUNEL assay appeared to be high in 18 (30%), moderate in 14 (23%), and low in 29 (48%) carcinomas. High apoptotic index was strongly associated with advanced tumor grade and estrogen receptor positive (ER+) status but not with other investigated clinical or morphological parameters. Among the molecules studied, only the Bcl-2 protein expression demonstrated strong (inverse) correlation with the apoptotic index (p = 0.032). The data of this expected correlation was served as internal control in the study. Interestingly, high levels of the anti-apoptotic protein Bcl-2 was frequently co-incident with increased expression of pro-apoptotic molecules, such as active caspase-3 (p = 0.004) and caspase-7 (p = 0.001). However, expression of caspase-3 or caspase-7 did not show correlation with the extent of apoptosis or any clinico-morphological features, except overrepresentation of ER+ status in tumors expressing caspase-3 (p = 0.009). Thus, these findings indicate a general dysregulation of spontaneous apoptosis in primary breast tumors.     

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.     

Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis

The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).     

The anticancer activity of 3- and 10-bromofascaplysins is mediated by caspase-8, -9, -3-dependent apoptosis

3- and 10-Bromofascaplysins was previously found to possess cytotoxic activity. In this study, we investigated their cancer preventive and proapoptotic properties. These effects were tested on mouse skin epidermal JB6 P⁺ Cl41 cell line, its stable transfectants, and human tumor HL-60, THP-1, SNU-C4, SK-MEL-28, DLD-1, MDA-MB-231, and HeLa cells using a variety of assessments, including a cell viability (MTS) assay, flow cytometry, anchorage-independent soft agar assay, luciferase assay, mitochondrial permeability assay, and Western blotting. 3- and 10-Bromofascaplysins were effective at submicromolar concentrations as the anticancer agents, which exerted their action, at least in part, through the induction of caspase-8, -9, -3-dependent apoptosis.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.     

Spatial and temporal regulation of collagenases—3,—4,and stromelysin —3 implicates distinct functions in apoptosis and tissue remodeling during frog metamorphosis

Matrix metalloproteinases (MMPs) are a family of extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix(ECM).They have been implicated to play important roles in a number of developmental and pathological processes,such as tumor metastasis and inflammation.Relatively few studies have been carried out to investigate the function of MMPs during postembryonic organ-development.Using Xenopus laevis development as a model system,we demonstrate here that three MMPs,stromelysin-3(ST3),collagenases-3(Col3),and Col4,have distinct spatial and temporal expression profiles during metamorphosis as the tadpole transforms into a frog.In situ hybridizations reveal a tight,but distinct,association of individual MMPs with tissue remodeling in the tail and intestine during metamorphosis.In particular,ST3 expression is strongly correlated with apoptosis in both organs as demonstrated by analyses of serial sections with in situ hybridization for ST3 mRNA and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling)for apoptosis,respectively.On the other hand,Col3 and Col4 are present in regions where extensive connective tissue remodeling take place.These results indicate that ST3 is likely to play a role in ECM-remodeling that facilitate apoptotic tissue remodeling or resorption while Col3 and Col4 appear to participate in connective tissue degradation during development.