Characterization of the caspase inhibitor IDN-1965 in a model of apoptosis-associated liver injury.

Previous studies have shown that caspase inhibitors are effective at protecting against anti-Fas antibody (alpha-Fas)-mediated liver injury/lethality. The purpose of these experiments was to characterize more fully the efficacy of a broad-spectrum, irreversible caspase inhibitor, IDN-1965 (N-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid), in this model and the role of caspase inhibition in long-term protection. The ED(50) for IDN-1965 by i.p. administration, based on alanine aminotransferase activities, was 0.14 mg/kg. The caspase inhibitor was also efficacious when administered intravenously and orally (ED(50) values of 0.04 and 1.2 mg/kg, respectively). Histologically, marked reduction in Fas-induced apoptosis with IDN-1965 (1 mg/kg, i.p.) was apparent at 6 h. Also, caspase 3-like activities were decreased in a dose-dependent manner, but the inhibition of caspase activity was transient. Immunohistochemical studies demonstrated that IDN-1965 greatly reduced the activation of caspase 3. In survival studies, a single i.p. treatment of 1 mg/kg IDN-1965 or continuous i.p. infusion via osmotic pumps completely blocked lethality measured up to 7 days after alpha-Fas administration. IDN-1965 was also effective in inhibiting liver injury when administered as long as 3 h after or 1 h before alpha-Fas administration. Lastly, Western blot analysis demonstrated that processing of caspases 3, 6, and 8, as well as Bid (a protein responsible for the release of mitochondrial cytochrome C and amplification of the apoptotic cascade) was inhibited by IDN-1965. In conclusion, the broad-spectrum caspase inhibitor IDN-1965 is markedly effective at inhibiting Fas-mediated apoptosis by multiple routes of administration. The therapeutic potential of caspase inhibitors appears promising for the treatment of apoptosis-mediated liver injury based on potency and postinsult efficacy.

Drug discovery opportunities from apoptosis research.

Cell suicide is a normal process that participates in a wide variety of physiological processes, including tissue homeostasis, immune regulation, and fertility. Physiological cell death typically occurs by apoptosis, as opposed to necrosis. Defects in apoptotic cell-death regulation contribute to many diseases, including disorders associated with cell accumulation (e.g. cancer, autoimmunity, inflammation and restenosis) or where cell loss occurs (e.g. stroke, heart failure, neurodegeneration, AIDS and osteoporosis). At the center of the apoptosis machinery is a family of intracellular proteases, known as 'caspases', that are responsible directly or indirectly for the morphological and biochemical events that characterize apoptosis. Multiple positive and negative regulators of these cell-death proteases have been discovered in the genomes of mammals, amphibians, insects, nematodes, and other animal species, as well as a variety of animal viruses. Inputs from signal-transduction pathways into the core of the cell-death machinery have also been identified, demonstrating ways of linking environmental stimuli to cell-death responses or cell-survival maintenance. Knowledge of the molecular mechanisms of apoptosis has provided important insights into the causes of multiple diseases where aberrant cell-death regulation occurs and has revealed new approaches for identifying small-molecule drugs for more effectively treating these illnesses.

Heavy membrane-associated caspase 3: identification, isolation, and characterization.

Heavy membrane preparations from 697 lymphoblastoid cells contain a tightly bound caspase zymogen. This heavy membrane-bound procaspase can be efficiently liberated from membrane preparations using detergents. Alternatively, the procaspase can be rapidly processed and activated from membrane preparations by caspase-1 without detergents. The activated caspase-3 was purified using affinity chromatography and characterized by amino acid sequencing and inhibitor specificity analysis. The sequence indicates that this heavy membrane bound caspase is caspase-3. The kinetic properties and inhibitor binding specificity also show that this purified caspase is enzymologically indistinguishable from cytoplasmic or recombinant caspase-3. However, the N-termini of activated heavy membrane-bound and cytoplasmic caspase-3 are slightly different; peptide sequencing data indicate that the heavy membrane caspase-3 begins at Lys 14, whereas the cytoplasmic enzyme begins at Ser 10. Implications of this structural difference are discussed.

Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions.

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11-deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

DNA damage and activated caspase-3 expression in neurons and astrocytes: evidence for apoptosis in frontotemporal dementia.

Frontotemporal dementia (FTD) is a neurodegenerative disease which affects mainly the frontal and anterior temporal cortex. It is associated with neuronal loss, gliosis, and microvacuolation of lamina I to III in these brain regions. In previous studies we have described neurons with DNA damage in the absence of tangle formation and suggested this may result in tangle-independent mechanisms of neurodegeneration in the AD brain. In the present study, we sought to examine DNA fragmentation and activated caspase-3 expression in FTD brain where tangle formation is largely absent. The results demonstrate that numerous nuclei were TdT positive in all FTD brains examined. Activated caspase-3 immunoreactivity was detected in both neurons and astrocytes and was elevated in FTD cases as compared to control cases. A subset of activated caspase-3-positive cells were also TdT positive. In addition, the cell bodies of a subset of astrocytes showed enlarged, irregular shapes, and vacuolation and their processes appeared fragmented. These degenerating astrocytes were positive for activated caspase-3 and colocalized with robust TdT-labeled nuclei. These findings suggest that a subset of astrocytes exhibit degeneration and that DNA damage and activated caspase-3 may contribute to neuronal cell death and astrocyte degeneration in the FTD brain. Our results suggest that apoptosis may be a mechanism of neuronal cell death in FTD as well as in AD (228).

The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis.

BACKGROUND: In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported. RESULTS: We report here the crystal structure of a complex of the activated human caspase-8 (proteolytic domain) with the irreversible peptidic inhibitor Z-Glu-Val-Asp-dichloromethylketone at 2.8 A resolution. This is the first structure of a representative of the long prodomain initiator caspases and of the group III substrate specificity class. The overall protein architecture resembles the caspase-1 and caspase-3 folds, but shows distinct structural differences in regions forming the active site. In particular, differences observed in subsites S(3), S(4) and the loops involved in inhibitor interactions explain the preference of caspase-8 for substrates with the sequence (Leu/Val)-Glu-X-Asp. CONCLUSIONS: The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.

Activation of caspase-3 in developmental models of programmed cell death in neurons of the substantia nigra.

Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase-3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase-3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6-hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase-3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The subcellular distribution of activated caspase-3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.

ATP-dependent steps in apoptotic signal transduction.

Apoptotic changes of the nucleus induced by Fas (Apo1/CD95) stimulation are completely blocked by reducing intracellular ATP level. In this study, we examined the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction using two cell lines. In SKW6.4 (type I) cells characterized by rapid formation of the death-inducing signaling complex on Fas treatment, the activation of caspases 8, 9, and 3, cleavage of DFF45 (ICAD), and release of cytochrome c from the mitochondria to the cytoplasm were not affected by reduction of intracellular ATP, although chromatin condensation and nuclear fragmentation were inhibited. On the other hand, in the Fas-mediated apoptosis of Jurkat (type II) cells, which is characterized by involvement of mitochondria and, thus, shares signal transduction mechanisms with apoptosis induced by other stimuli such as genotoxins, activation of the three caspases, cleavage of DFF45 (ICAD), and nuclear changes were blocked by reduction of intracellular ATP, whereas release of cytochrome c was not affected. These results suggested that the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction in type I cells are only located downstream of caspase 3 activation, whereas the activation of caspase 9 by released cytochrome c is the most upstream ATP-dependent step in type II cells. These observations also confirm the existence of two pathways for Fas-mediated apoptotic signal transduction and suggest that the Apaf-1 (Ced-4 homologue) system for caspase 9 activation operates in an ATP-dependent manner in vivo.

Activation of membrane-associated procaspase-3 is regulated by Bcl-2.

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex.

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.

Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia.

We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells.

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-alpha. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step.

Human IAP-like protein regulates programmed cell death downstream of Bcl-xL and cytochrome c.

The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-xL. In contrast, hILP, but not Bcl-xL, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-xL may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.

In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system.

Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabelling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-xL-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.

Conformationally constrained inhibitors of caspase-1 (interleukin-1 beta converting enzyme) and of the human CED-3 homologue caspase-3 (CPP32, apopain).

A systematic study of interleukin-1 beta converting enzyme (ICE, caspase-1) and caspase-3 (CPP32, apopain) inhibitors incorporating a P2-P3 conformationally constrained dipeptide mimetic is reported. Depending on the nature of the P4 substituent, highly selective inhibitors of both Csp-1 or Csp-3 were obtained.

Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release.

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.

Bovine herpesvirus 4 BORFE2 protein inhibits Fas- and tumor necrosis factor receptor 1-induced apoptosis and contains death effector domains shared with other gamma-2 herpesviruses.

Fas- and tumor necrosis factor receptor 1 (TNFR1)-induced apoptosis is mediated by the interaction of FADD with caspase-8. Here, we report that the bovine herpesvirus 4 (BHV4) BORFE2 gene encodes a protein that inhibits Fas- and TNFR1-induced apoptosis and contains death effector domains (DEDs). Using the yeast two-hybrid system, we found that the BORFE2 protein interacts with the prodomain of caspase-8. Furthermore, we show that BHV4 BORFE2 is a member of a family of DED-containing proteins that includes other gamma-2 herpesviruses, such as Kaposi's sarcoma-associated herpesvirus and herpesvirus saimiri.

FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis.

We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.

Identification of the MDM2 oncoprotein as a substrate for CPP32-like apoptotic proteases.

Programmed cell death is mediated by members of the interleukin 1-beta convertase family of proteases, which are activated in response to diverse cell death stimuli. However, the key substrates of these proteases that are responsible for apoptotic cell death have not been identified. Here we report that the MDM2 oncoprotein is cleaved by members of the CPP32 subfamily of interleukin 1-beta convertase proteases both in vitro and in vivo, resulting in the disappearance of MDM2 from apoptotic cells. Because MDM2 functions as a negative regulator of the p53 tumor suppressor and because p53 induces apoptosis in response to a variety of stimuli, this cleavage of MDM2 by CPP32-like proteases may result in deregulation of p53 and contribute directly to the process of apoptotic cell death.