Caspase-like Enzymatic Activity Research Articles

Apoptosis-related genes induced in response to ketamine during early life stages of zebrafish

Increasing evidence supports that ketamine, a widely used anaesthetic, potentiates apoptosis during development through the mitochondrial pathway of apoptosis. Defects in the apoptotic machinery can cause or contribute to the developmental abnormalities previously described in ketamine-exposed zebrafish. The involvement of the apoptotic machinery in ketamine-induced teratogenicity was addressed by assessing the apoptotic signals at 8 and 24 hpf following 20min exposure to ketamine at three stages of early zebrafish embryo development (256 cell, 50% epiboly and 1–4 somites stages). Exposure at the 256-cell stage to ketamine induced an up-regulation of casp8 and pcna at 8 hpf while changes in pcna at the mRNA level were observed at 24 hpf. After the 50% epiboly stage exposure, the mRNA levels of casp9 were increased at 8 and 24 hpf while aifm1 was affected at 24 hpf. Both tp53 and pcna expressions were increased at 8 hpf. After exposure during the 1–4 somites stage, no meaningful changes on transcript levels were observed. The distribution of apoptotic cells and the caspase-like enzymatic activities of caspase-3 and -9 were not affected by ketamine exposure. It is proposed that ketamine exposure at the 256-cell stage induced a cooperative mechanism between proliferation and cellular death while following exposure at the 50% epiboly, a p53-dependent and -independent caspase activation may occur. Finally, at the 1–4 somites stage, the defence mechanisms are already fully in place to protect against ketamine-insult. Thus, ketamine teratogenicity seems to be dependent on the functional mechanisms present in each developmental stage.

Dunaliella tertiolecta (Chlorophyta) Avoids Cell Death Under Ultraviolet Radiation By Triggering Alternative Photoprotective Mechanisms.

The effect of different ultraviolet radiation (UVR) treatments combining PAR (P), UVA (A) and UVB (B) on the molecular physiology of Dunaliella tertiolecta was studied during 6 days to assess the response to chronic UVR exposure. UVR reduced cell growth but did not cause cell death, as shown by the absence of SYTOX Green labeling and cellular morphology. However, caspase-like enzymatic activities (CLs), (regarded as cell death proteases), were active even though the cells were not dying. Maximal quantum yield of fluorescence (Fv /Fm ) and photosynthetic electron transport rate (ETR) dropped. Decreased nonphotochemical quenching (NPQ) paralleled a drop in xanthophyll cycle de-epoxidation under UVB. Reactive oxygen species (ROS) and D1 protein accumulation were inversely correlated. PAB exhibited elevated ROS production at earlier times. Once ROS decayed, D1 protein recovered two-fold compared with P and PA at later stages. Therefore, PsbA gene was still transcribed, suggesting ROS involvement in D1 recovery by its direct effect on mRNA-translation. We add evidence of an UVB-induced positive effect on the cells when P is present, providing photoprotection and resilience, by means of D1 repair. This allowed cells to survive. The photoprotective mechanisms described here (which are counterintuitive in principle) conform to an important ecophysiological response regarding light stress acclimation.

Translating Findings of Proteasome Inhibitors Effects from the in VivoDrosophila Experimental Model to Humans: The Paradigm of the Molecular-Cellular Responses to Bortezomib and Carfilzomib

Cellular proteostasis (homeostasis of the proteome) is ensured by the efficient maintenance of proteome quality and functionality. Central to the proteostasis process is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Recent findings indicate that the over-activation of the proteostasis ensuring mechanisms represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. In line with this concept, proteasome inhibitors (i.e. bortezomib and carfilzomib) have demonstrated clinical efficacy in the treatment of multiple myeloma (MM) and mantle cell lymphoma and are under evaluation for the treatment of other malignancies.By using the fruit fly Drosophila melanogaster as an in vivo platform for screening the effects of proteasome inhibitors at the whole organism level we recently reported that proteasome functionality in flies’ somatic tissues is sex- and tissue- dependent and that it declines with aging. Administration of bortezomib to young flies caused dose-dependent decrease of proteasome activities in the somatic tissues; induction of the proteasome genes expression, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Our in vivo data also showed that carfilzomib was less toxic compared to bortezomib, including neuromusculatory toxicity and effects on flies’ longevity.To address the question whether these findings can be translated to humans we started characterizing proteasome regulation and functionality in both healthy donors, as well as in MM patients treated with either bortezomib or carfilzomib. Initially, we screened isolated red blood cells (RBCs) and peripheral blood mononucleated cells (PBMCs) from male and female healthy donors of different ages; these two cell types represent either an anucleate relatively “long-lived” proteome (RBCs) or cell lineages with the capacity to mobilize genome responses after proteasome inhibition (PBMCs). Our analyses revealed significant variability of basal proteasome peptidase activities among different donors in both RBCs and PBMCs. PBMCs expressed (independently of sex) higher basal proteasome activities as compared to RBCs. Moreover, RBCs isolated from female donors had elevated (as compared to males’ RBCs) basal chymotrypsin-like activity; whereas, males’ PBMCs exhibited higher trypsin-like and caspase-like enzymatic activities as compared to PBMCs from females of similar age. In line with our observations in flies’ somatic tissues, we also found that the proteasome peptidase activities decrease significantly during aging (in a sex-independent manner) in both RBCs and PBMCs.Our studies in isolated RBCs and PBMCs from MM patients treated with either bortezomib or carfilzomib revealed drug-, donor- and cell type-specific readouts. Specifically, in most cases proteasome activities were suppressed in both RBCs and PBMCs after drug administration. Also, we noted that RBCs were particularly sensitive to both inhibitors and their proteasome activities remained low during the entire course of treatment. On the other hand, PBMCs were characterized by phases of relapsed proteasome activities during the periods of no drug administration. Finally, as in the case of the in vivo Drosophila experimental model, proteasome dysfunction in PBMCs triggered in most patients a significant upregulation of the proteasome 20S and 19S genes expression. Moreover, we noted an induction of genes involved in cellular antioxidant responses; this finding is in line with our observations in flies showing that administration of proteasome inhibitors results in increased cellular oxidative stress that mobilizes genomic antioxidant responses. Data on the clinical outcomes of the treated patients in correlation with the recorded molecular responses will be presented at the meeting.Overall, our findings indicate that the molecular-cellular responses to proteasome inhibitors observed at the in vivo Drosophila Experimental model are largely translatable to humans. Moreover, we anticipate that our employed methodologies will set the basis towards a more personalized clinical therapeutic approach for multiple myeloma patients. DisclosuresNo relevant conflicts of interest to declare.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3)

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant–pathogen interaction.

Endoplasmic reticulum stress-induced PCD and caspase-like activities involved

Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed cell death (PCD). PCD can be triggered by various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the ER could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.

An Antimicrobial Peptidomimetic Induces Mucorales Cell Death through Mitochondria-Mediated Apoptosis

The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections.

Differential effect of ultraviolet exposure (UVR) in the stress response of the Dinophycea Gymnodinium sp. and the Chlorophyta Dunaliella tertiolecta: mortality versus survival

Dunaliella tertiolecta (Chlorophyta) and Gymnodinium sp. (Dinophyceae) cells were exposed to ultraviolet radiation (UVR) (PAR, UVA and UVB: PAB) for 6 days either continuously or during a photoperiod. Both UVR treatments were harmful to Gymnodinium but exposure to continuous PAB had the most dramatic effects. Although a number of lesions/damage could have happened during the first few hours of exposure to UVR, in less than 24 h, Gymnodinium lost its ability to detoxify ROS efficiently, photoinhibition occurred, thymine dimers formed in the DNA, caspase-like enzymatic activities DEVDase sharply increased and cells died as determined by SYTOX-green staining. Superoxide dismutase activity did not significantly change with time, and although the catalase activity augmented in both treatments, cells still suffered from the UVR stress. Clearly, UVR was fatal to the dinoflagellate. For the chlorophyte, however, cell numbers increased regardless of the UVR treatment and mortality remained low (<20 %). F v/F m showed an initial decrease but then remained constant for both light treatments. After 6 days of continuous PAB exposure, however, signs of stress (thymine dimers, oxidative stress) paralleled a drop in catalase activity. Results obtained here demonstrate that the dinoflagellate Gymnodinium was much more sensitive and was harmed more rapidly by UVR exposure than the chlorophyte D. tertiolecta. The increased tolerance to UVR exposure of the chlorophyte may provide advantages over other more sensitive phytoplankton species within the photic zone. We provide strong support in the present study for repair being an important component of UV resistance in this species.

Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation

Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F v/F m and the formation of cyclobutane–pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms.

Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide

Salinity stress causes ionic stress (mainly from high Na+ and Cl- levels) and osmotic stress (as a result of inhibition of water uptake by roots and amplified water loss from plant tissue), resulting in cell death and inhibition of growth and ultimately adversely reducing crop productivity. In this report, changes in root nitric oxide content, shoot and root biomass, root H2O2 content, root lipid peroxidation, root cell death, root caspase-like enzymatic activity, root antioxidant enzymatic activity and root ascorbate and glutathione contents/redox states were investigated in maize (Zea mays L. cv Silverking) after long-term (21 d) salt stress (150 mM NaCl) with or without exogenously applied nitric oxide generated from the nitric oxide donor 2,2′-(Hydroxynitrosohydrazano)bis-ethane. In addition to reduced shoot and root biomass, salt stress increased the nitric oxide and H2O2 contents in the maize roots and resulted in elevated lipid peroxidation, caspase-like activity and cell death in the roots. Altered antioxidant enzymatic activities, along with changes in ascorbate and glutathione contents/redox status were observed in the roots in response to salt stress. The detrimental effects of salt stress in the roots were reversed by exogenously applied nitric oxide. These results demonstrate that exogenously applied nitric oxide confers salt stress tolerance in maize by reducing salt stress-induced oxidative stress and caspase-like activity through a process that limits accumulation of reactive oxygen species via enhanced antioxidant enzymatic activity.

A Novel Non-Competitive Chemical Proteasome Inhibitor Displays Preclinical Activity in Myeloma and Leukemia.

The proteasome is an enzymatic complex that rids cells of excess and misfolded proteins and possesses chymotrypin, trypsin, and caspase-like enzymatic activity. To date, all of the proteasome inhibitors approved for clinical use or in clinical trials inhibit the complex competitively by binding the active sites of the enzymes. Here, we report a novel chemical proteasome inhibitor that binds the alpha subunits of the 20S proteasome and inhibits the complex non-competitively through a dual copper-dependent and independent mechanism.In a screen of a focused chemical library for novel proteasome inhibitors, we identified 5-amino-8-hydroxyquinoline (5AHQ). When added to myeloma or leukemia intact cells or cell extracts, 5AHQ inhibited the enzymatic activity of the proteasome at low micromolar concentrations. In order to obtain further insight into the mechanism of action of 5AHQ, we carried out a kinetic analysis of inhibition of the enzymatic activity of purified T. Acidophilium proteasome. By Lineweaver-Burk plot analysis, 5AHQ inhibited the proteasome non-competitively. Next, we investigated the binding of 5AHQ to the proteasome. By NMR analysis, 5AHQ bound the half-proteasome complex comprised of a pair of α-rings, α7-α7, and clear spectral changes were observed that localized to residues Ile159, Val113, Val87, Val82, Leu112, Val89, Val134, Val24 and Leu136 inside the antechamber. In contrast, the competitive inhibitor MG132 that binds the proteolytic chamber did not produce any changes in spectra of α7-α7, as expected. 5AHQ bound copper in a 2:1 stoichiometry with a logβ′ value of 9.09, and the addition of copper to 5AHQ enhanced 5AHQ-mediated inhibition of the proteasome. However, binding intracellular copper was not sufficient to explain the effects of 5AHQ on the proteasome as analogues of 5AHQ that did not bind copper continued to inhibit the proteasome, copper-binding molecules not structurally related to 5AHQ did not affect the proteasome, and 5AHQ inhibited isolated proteasomes in buffers devoid of copper and other heavy metals.Given the effects of 5AHQ on the proteasome, we examined the effects of this molecule on the viability of leukemia and myeloma cell lines. Leukemia, myeloma and solid tumor cell lines were treated with increasing concentrations of 5AHQ for 72 hours and cell viability was measured by the MTS assay. 5AHQ induced cell death in 9/9 myeloma, 6/10 leukemia, and 3/10 solid tumor cell lines with an LD50 ≤5 uM. Cell death was confirmed by Annexin V staining. Consistent with its mechanism of action as a proteasome inhibitor, the ability of 5AHQ to induce cell death matched its ability to inhibit the proteasome. In addition, 5AHQ-mediated cell death was associated with inhibition of the NF-kappaB signalling pathway.As 5AHQ induced cell death in malignant cells, we evaluated the effects of oral 5AHQ in 3 mouse models of leukemia. Sublethally irradiated NOD-SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or intraperitoneally with MDAY-D2 murine leukemia cells. After tumor implantation, mice were treated with 5AHQ (50 mg/kg/day) or buffer control by oral gavage. Oral 5AHQ decreased tumor weight and volume in all 3 mouse models compared to control without causing weight loss or gross organ toxicity.In summary, we have identified a new strategy for inhibition of the proteasome and a lead for a new therapeutic agent for the treatment of hematologic malignancies.

Crucial Mitochondrial Impairment upon CDC48 Mutation in Apoptotic Yeast

Mutation in CDC48 (cdc48(S565G)), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48(S565G) yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48(S565G) cells. Mitochondrial dysfunction in the cdc48(S565G) strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis.

A Caspase-Related Protease Regulates Apoptosis in Yeast

Yeast can undergo cell death accompanied by cellular markers of apoptosis. However, orthologs of classical mammalian apoptosis regulators appeared to be missing from the yeast genome, challenging a common mechanism of yeast and mammalian apoptosis. Here we investigate Yor197w, a yeast protein with structural homology to mammalian caspases, and demonstrate caspase-like processing of the protein. Hydrogen peroxide treatment induces apoptosis together with a caspase-like enzymatic activity in yeast. This response is completely abrogated after disruption and strongly stimulated after overexpression of Yor197w. Yor197w also mediates the death process within chronologically aged cultures, pointing to a physiological role in elimination of overaged cells. We conclude that Yor197w indeed functions as a bona fide caspase in yeast and propose the name Yeast Caspase-1 (YCA1, gene YCA1).

The Novel Synthetic Retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437) Causes Apoptosis in Acute Promyelocytic Leukemia Cells Through Rapid Activation of Caspases

The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-γ agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-γ or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro–caspase-3 and pro–caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-, which can be completely inhibited by z-VAD.

The Novel Synthetic Retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437) Causes Apoptosis in Acute Promyelocytic Leukemia Cells Through Rapid Activation of Caspases

AbstractThe synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-γ agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-γ or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro–caspase-3 and pro–caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-α, which can be completely inhibited by z-VAD.

Mitochondrial release of caspase-2 and -9 during the apoptotic process.

The barrier function of mitochondrial membranes is perturbed early during the apoptotic process. Here we show that the mitochondria contain a caspase-like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in addition to three biological activities previously suggested to participate in the apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AIF) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity. All of these factors, which are biochemically distinct, are released upon opening of the permeability transition (PT) pore in a coordinate, Bcl-2-inhibitable fashion. Caspase inhibitors fully neutralize the Z-VAD.afc-cleaving activity, have a limited effect on the AIF activity, and have no effect at all on the DNase activities. Purification of proteins reacting with the biotinylated caspase substrate Z-VAD, immunodetection, and immunodepletion experiments reveal the presence of procaspase-2 and -9 in mitochondria. Upon induction of PT pore opening, these procaspases are released from purified mitochondria and become activated. Similarly, upon induction of apoptosis, both procaspases redistribute from the mitochondrion to the cytosol and are processed to generate enzymatically active caspases. This redistribution is inhibited by Bcl-2. Recombinant caspase-2 and -9 suffice to provoke full-blown apoptosis upon microinjection into cells. Altogether, these data suggest that caspase-2 and -9 zymogens are essentially localized in mitochondria and that the disruption of the outer mitochondrial membrane occurring early during apoptosis may be critical for their subcellular redistribution and activation.