Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis

Many pro‐apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen‐activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c‐induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf‐1 phosphorylation by the 90‐kDa ribosomal S6 kinase (Rsk). Recruitment of 14‐3‐3ε to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14‐3‐3ε binding to Apaf‐1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk‐catalysed Apaf‐1 phosphorylation and consequent binding of 14‐3‐3ε, resulting in decreased cellular responsiveness to cytochrome c.     

Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase‐8 is a cysteine directed aspartate‐specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase‐8 depends on adaptor‐induced dimerization of procaspase‐8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase‐8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase‐3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase‐8 is a monomer while procaspase‐3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase‐8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase‐8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase‐8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase‐8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.     

Splitting the Apoptosome

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.     

Splitting the apoptosome

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.     

Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.     

Purification and characterization of active caspase‐14 from human epidermis and development of the cleavage site‐directed antibody

Restricted expression of caspase‐14 in differentiating keratinocytes suggests the involvement of caspase‐14 in terminal differentiation. We purified active caspase‐14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764‐fold with a yield of 9.1%. Purified caspase‐14 revealed the highest activity on WEHD‐methylcoumaryl‐amide (MCA), although YVAD‐MCA, another caspase‐1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N‐terminal and C‐terminal analyses demonstrated that the large subunit consisted of Ser⁶‐Asp¹⁴⁶ and N‐terminal of small subunit was identified as Lys¹⁵³. We successfully developed an antiserum (anti‐h14D146) directed against the Asp¹⁴⁶ cleavage site, which reacted only with active caspase‐14 but not with procaspase‐14. Furthermore we confirmed that anti‐h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti‐h14D146 staining was mostly restricted to the cornified layer and co‐localized with some of the TUNEL positive‐granular cells in the normal human epidermis. UV radiation study demonstrated that caspase‐3 was activated and co‐localized with TUNEL‐positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase‐14 activation in response to UV. Our study revealed tightly regulated action of caspase‐14, in which only the terminal differentiation of keratinocytes controls its activation process. J. Cell. Biochem. 109: 487–497, 2010. © 2009 Wiley‐Liss, Inc.     

Cytochrome c and insect cell apoptosis

Abstract The role of cytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy. In Drosophila, the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis, although there are conflicting reports. Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells. Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates, but remarkably caused caspase activation in extracts from human cells. Knockdown of cytochrome c does not protect cells from apoptosis and over‐expression of cytochrome c also does not promote apoptosis. Structural analysis has revealed that cytochrome c is not required for Dapaf‐1 complex assembly. In Lepidoptera, the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence. Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9, Spodoptera litura Sl‐1 and Lymantria dispar LdFB. Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell‐free extracts results in caspase activation, suggesting the activation of caspase is dependent on cytochrome c. Although Apaf‐1 has not been identified in Lepidoptera, the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation. Cytochrome c may be exclusively required for Lepidoptera apoptosis.     

Alpha-fetoprotein positively regulates cytochrome c-mediated caspase activation and apoptosome complex formation.

Previous results have shown that the oncoembryonic marker alpha-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas-dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c-mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP positively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein-protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex.     

Heat shock inhibits caspase‐1 activity while also preventing its inflammasome‐mediated activation by anthrax lethal toxin

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase‐1. We report that heat‐shocking LT‐sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase‐1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen‐activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen‐activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT‐mediated caspase‐1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome‐mediated caspase‐1 activation in macrophages. We found that heat shock induced pro‐caspase‐1 association with a large cellular complex that could prevent its activation. Additionally, while heat‐shocking recombinant caspase‐1 did not affect its activity in vitro, lysates from heat‐shocked cells completely inhibited recombinant active caspase‐1 activity. Our results suggest that heat shock inhibition of active caspase‐1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat‐shocked cells.     

Targeting procaspase‐3 with WF‐208, a novel PAC‐1 derivative,causes selective cancer cell apoptosis

Caspase‐3 is a critical effector caspase in apoptosis cascade, and is often over‐expressed in many cancer tissues. The first synthesized procaspase‐3 activator, PAC‐1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase‐3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF‐208 stood out by its high cytotoxicity against procaspase‐3 overexpressed HL‐60 cells. Compared with PAC‐1, WF‐208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF‐208 activated procaspase‐3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase‐3‐dependent cell death in tumour cells, which possibly because of the zinc‐chelating properties. WF‐208 also showed greater antitumour activity than PAC‐1 in murine xenograft model. In conclusion, we have discovered WF‐208 as a promising procaspase‐3 activating compound, with higher activity and higher cell selectivity than PAC‐1.     

Caspase‐1 inflammasome activity in patients with Staphylococcus aureus bacteremia

The inflammasome is a multiprotein complex that mediates caspase‐1 activation with subsequent maturation of the proinflammatory cytokines IL‐1β and IL‐18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase‐1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent‐labeled inhibitor of caspase‐1), while IL‐1β and IL‐18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase‐1), and IL1B (pro‐IL‐1β) was analyzed by quantitative PCR. We found induced caspase‐1 activity in innate immune cells with subsequent release of IL‐18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase‐1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Chlamydia trachomatis‐infected host cells resist dsRNA‐induced apoptosis

Human pathogenic Chlamydia trachomatis have evolved sophisticated mechanisms to manipulate host cell signalling pathways in order to prevent apoptosis. We show here that host cells infected with C. trachomatis resist apoptosis induced by polyI:C, a synthetic double‐stranded RNA that mimics viral infections. Infected cells displayed significantly reduced levels of PARP cleavage, caspase‐3 activation and a decrease in the TUNEL positive population in the presence of polyI:C. Interestingly, the chlamydial block of apoptosis was upstream of the initiator caspase‐8. Processing of caspase‐8 was reduced in infected cells and coincided with a decrease in Bid truncation and downstream caspase‐9 cleavage. Moreover, the enzymatic activity of caspase‐8, measured by a luminescent substrate, was significantly reduced in infected cells. Caspase‐8 inhibition by Chlamydia was dependent on cFlip as knock‐down of cFlip decreased the chlamydial block of caspase‐8 activation and consequently reduced apoptosis inhibition. Our data implicate that chlamydial infection interferes with the host cell's response to viral infections and thereby influences the fate of the cell.     

Protective effects of astaxanthin on 6‐hydroxydopamine‐induced apoptosis in human neuroblastoma SH‐SY5Y cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although understanding of the pathogenesis of PD remains incomplete, increasing evidence from human and animal studies has suggested that oxidative stress is an important mediator in its pathogenesis. Astaxanthin (Asx), a potent antioxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress‐related diseases. This study examined the protective effects of Asx on 6‐hydroxydopamine (6‐OHDA)‐induced apoptosis in the human neuroblastoma cell line SH‐SY5Y. Pre‐treatment of SH‐SY5Y cells with Asx suppressed 6‐OHDA‐induced apoptosis in a dose‐dependent manner. In addition, Asx strikingly inhibited 6‐OHDA‐induced mitochondrial dysfunctions, including lowered membrane potential and the cleavage of caspase 9, caspase 3, and poly(ADP‐ribose) polymerase. In western blot analysis, 6‐OHDA activated p38 MAPK, c‐jun NH₂‐terminal kinase 1/2, and extracellular signal‐regulated kinase 1/2, while Asx blocked the phosphorylation of p38 MAPK but not c‐jun NH₂‐terminal kinase 1/2 and extracellular signal‐regulated kinase 1/2. Pharmacological approaches showed that the activation of p38 MAPK has a critical role in 6‐OHDA‐induced mitochondrial dysfunctions and apoptosis. Furthermore, Asx markedly abolished 6‐OHDA‐induced reactive oxygen species generation, which resulted in the blockade of p38 MAPK activation and apoptosis induced by 6‐OHDA treatment. Taken together, the present results indicated that the protective effects of Asx on apoptosis in SH‐SY5Y cells may be, at least in part, attributable to the its potent antioxidative ability.     

The antiapoptotic protein AAC‐11 interacts with and regulates Acinus‐mediated DNA fragmentation

The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after caspase cleavage. However, this role has been challenged by recent observations suggesting a contribution of Acinus in apoptotic internucleosomal DNA cleavage. We report here that AAC‐11, a survival protein whose expression prevents apoptosis that occurs on deprivation of growth factors, physiologically binds to Acinus and prevents Acinus‐mediated DNA fragmentation. AAC‐11 was able to protect Acinus from caspase‐3 cleavage in vivo and in vitro, thus interfering with its biological function. Interestingly, AAC‐11 depletion markedly increased cellular sensitivity to anticancer drugs, whereas its expression interfered with drug‐induced cell death. AAC‐11 possesses a leucine‐zipper domain that dictates, upon oligomerization, its interaction with Acinus as well as the antiapoptotic effect of AAC‐11 on drug‐induced cell death. A cell permeable peptide that mimics the leucine‐zipper subdomain of AAC‐11, thus preventing its oligomerization, inhibited the AAC‐11–Acinus complex formation and potentiated drug‐mediated apoptosis in cancer cells. Our results, therefore, show that targeting AAC‐11 might be a potent strategy for cancer treatment by sensitization of tumour cells to chemotherapeutic drugs.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.     

Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation

The apoptosome is an Apaf-1 cytochrome c complex that activates procaspase-9. The three-dimensional structure of the apoptosome has been determined at 27 A resolution, to reveal a wheel-like particle with 7-fold symmetry. Molecular modeling was used to identify the caspase recruitment and WD40 domains within the apoptosome and to infer likely positions of the CED4 homology motif and cytochrome c. This analysis suggests a plausible role for cytochrome c in apoptosome assembly. In a subsequent structure, a noncleavable mutant of procaspase-9 was localized to the central region of the apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the cleavage of procaspase-9 is not required to form an active cell death complex.