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1.
Caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain 总被引:10,自引:0,他引:10
Caspase activation resulting from cytochrome c release from the mitochondria is an essential component of the mechanism of apoptosis initiated by a range of factors. The activation of Bid by caspase-8 in this pathway promotes further cytochrome c release, thereby completing a positive feedback loop of caspase activation. Although the identity of the caspases necessary for caspase-8 activation in this pathway are known, it is still unclear which protease directly cleaves caspase-8. In order to identify the factor responsible we undertook a biochemical purification of caspase-8 cleaving activity in cytosolic extracts to which cytochrome c had been added. Here we report that caspase-6 is the only soluble protease in cytochrome c activated Jurkat cell extracts that has significant caspase-8 cleaving activity. Furthermore the caspase-6 that we purified was sufficient to induce Bid dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. We also show that caspase-6 is inactive until the short prodomain is removed. We suggest that the requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis. 相似文献
2.
Guo Y Srinivasula SM Druilhe A Fernandes-Alnemri T Alnemri ES 《The Journal of biological chemistry》2002,277(16):13430-13437
Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway. 相似文献
3.
Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation. 相似文献
4.
Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c. 总被引:41,自引:0,他引:41
D G Kirsch A Doseff B N Chau D S Lim N C de Souza-Pinto R Hansford M B Kastan Y A Lazebnik J M Hardwick 《The Journal of biological chemistry》1999,274(30):21155-21161
Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell. 相似文献
5.
Cif (Cytochrome c Efflux-Inducing Factor) Activity Is Regulated by Bcl-2 and Caspases and Correlates with the Activation of Bid 总被引:1,自引:0,他引:1
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Zhiyong Han Kapil Bhalla Panayotis Pantazis Eric A. Hendrickson James H. Wyche 《Molecular and cellular biology》1999,19(2):1381-1389
The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals. 相似文献
6.
We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with alpha-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c. 相似文献
7.
Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. 总被引:23,自引:2,他引:21
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Mitochondrial cytochrome c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in the activation of DEVD-specific caspases. The apoptosis inhibitors Bcl-2 or Bcl-xL prevent the efflux of cytochrome c from mitochondria. The mechanism responsible for the release of cytochrome c from mitochondria during apoptosis is unknown. Here, we report that cytochrome c release from mitochondria is an early event in the apoptotic process induced by UVB irradiation or staurosporine treatment in CEM or HeLa cells, preceding or at the time of DEVD-specific caspase activation and substrate cleavage. A reduction in mitochondrial transmembrane potential (Deltapsim) occurred considerably later than cytochrome c translocation and caspase activation, and was not necessary for DNA fragmentation. Although zVAD-fmk substantially blocked caspase activity, a reduction in Deltapsim and cell death, it failed to prevent the passage of cytochrome c from mitochondria to the cytosol. Thus the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization. 相似文献
8.
Werner AB de Vries E Tait SW Bontjer I Borst J 《The Journal of biological chemistry》2002,277(43):40760-40767
The death receptor ligand TRAIL arouses much interest for clinical application. We found that TRAIL receptor could induce cytochrome c (Cyt c) release from mitochondria in cells that failed to respond to CD95. Therefore, we examined whether these two closely related death receptors use different intermediates to convey the apoptotic signal to mitochondria. Dominant negative FADD, FLIP(L), or a Bid mutant lacking cleavage sites for caspase-8/10 completely inhibited Cyt c release in response to either receptor. Depletion of Bid from TRAIL- or CD95-activated cytosols blocked their capacity to mediate Cyt c release from mitochondria in vitro, whereas Bax depletion reduced it. We conclude that FADD, caspase-8/10, and caspase-cleaved Bid are required for TRAIL receptor and CD95 signaling to mitochondria, whereas Bax is a common accessory. In vitro, caspase-8 treatment of cytosol from CD95-resistant cells permitted generation of truncated Bid and its association with mitochondria. However, this cytosol impaired the ability of truncated Bid to liberate Cyt c from exogenous mitochondria. We conclude that the TRAIL receptor can bypass or neutralize the activity of cytosolic factor that blocks truncated Bid function. This may benefit the capacity of TRAIL to break apoptosis resistance in tumor cells. 相似文献
9.
Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia 总被引:7,自引:0,他引:7
It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3. 相似文献
10.
The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment 总被引:12,自引:0,他引:12
Kudla G Montessuit S Eskes R Berrier C Martinou JC Ghazi A Antonsson B 《The Journal of biological chemistry》2000,275(30):22713-22718
Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t(c)Bid) and a 6.5 kDa N-terminal fragment (t(n)Bid). Following the cleavage, t(c)Bid translocates to the mitochondria and promotes the release of cytochrome c into the cytosol by a mechanism that is not understood. Here we report that recombinant t(c)Bid can act as a membrane destabilizing agent. t(c)Bid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t(c)Bid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t(c)Bid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t(c)Bid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t(c)Bid on planar bilayers, liposomes, and mitochondria can be inhibited by t(n)Bid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated. 相似文献
11.
Cytochrome c (cyto c) release from mitochondria is a critical event in apoptosis. By investigating the ordering of molecular events during genotoxic stress-induced apoptosis, we found that ionizing radiation (IR) and etoposide induced the release of cyto c from mitochondria in two distinct stages. The early release of low levels of cyto c into the cytosol preceded the activation of caspase 9 and 3, but had no effect on ATP levels or mitochrondrial transmembrane potential (Deltapsim). In contrast, the late stage cyto c release resulted in a drastic loss of mitochondrial cyto c and was associated with reduction of ATP levels and Deltapsim. Moreover, caspases contributed to the late cyto c release since the caspase inhibitor zVAD prevented only the late but not the early-stage cyto c release. Recombinant caspase 3 induced cyto c release from isolated mitochondria in the absence of cytosolic factors. Bcl-2 but not Bid was cleaved during apoptosis after caspase activation. This suggests that Bcl-2 cleavage might contribute to the late cyto c release, which results in mitochondrial dysfunction manifested by the decrease of ATP and Deltapsim. zVAD prevented the reduction of ATP, Deltapsim, and nuclear condensation when added up to 8 h after IR, at the time the caspases were highly activated but when the majority of cyto c was still maintained in the mitochondria. These findings link the feedback loop control of caspase-induced cyto c release with mitochondrial dysfunction manifested by ATP and Deltapsim decline. 相似文献
12.
Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. 总被引:10,自引:2,他引:8
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R M Kluck S J Martin B M Hoffman J S Zhou D R Green D D Newmeyer 《The EMBO journal》1997,16(15):4639-4649
In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors. 相似文献
13.
Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release. 相似文献
14.
Tong X Lin S Fujii M Hou DX 《Biochemical and biophysical research communications》2004,321(3):539-546
Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 microM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH(2)-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine. 相似文献
15.
Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine 总被引:6,自引:0,他引:6
There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation. 相似文献
16.
Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route 总被引:27,自引:0,他引:27
Stoka V Turk B Schendel SL Kim TH Cirman T Snipas SJ Ellerby LM Bredesen D Freeze H Abrahamson M Bromme D Krajewski S Reed JC Yin XM Turk V Salvesen GS 《The Journal of biological chemistry》2001,276(5):3149-3157
We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis. 相似文献
17.
Degli Esposti M Ferry G Masdehors P Boutin JA Hickman JA Dive C 《The Journal of biological chemistry》2003,278(18):15749-15757
Bid is instrumental in death receptor-mediated apoptosis where it is cleaved by caspase 8 at aspartate 60 and aspartate 75 to generate truncated Bid (tBID) forms that facilitate release of mitochondrial cytochrome c. Bid is also cleaved at these sites by caspase 3 that is activated downstream of cytochrome c release after diverse apoptotic stimuli. In this context, tBid may amplify the apoptotic process. Bid is phosphorylated in vitro by casein kinases that regulate its cleavage by caspase 8 (Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L. Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell 8, 601-611). Using a Bid decapeptide substrate, we observed that phosphorylation at threonine 59 inhibited cleavage by caspase 8. This was also seen when recombinant Bid (rBid) and Bid isolated from murine kidney were incubated with casein kinase II. However, there were differences in the susceptibility of rBid and isolated Bid to cleavage by caspases 3 and 8. Caspase 8 cleaved rBid to generate two C-terminal products, p15 and p13 tBid, but produced only p15 tBid from isolated Bid. Contrary to rBid, isolated Bid was resistant to cleavage by caspase 3, yet was readily cleaved within the cytosolic milieu. Our data suggest that one or more distinct cellular mechanisms regulate Bid cleavage by caspases 8 and 3 in situ. 相似文献
18.
Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2. 相似文献
19.
Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax 总被引:2,自引:0,他引:2
Jia L Patwari Y Kelsey SM Newland AC 《Biochemical and biophysical research communications》2001,283(5):1037-1045
We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells. 相似文献
20.
Wang P Valentijn AJ Gilmore AP Streuli CH 《The Journal of biological chemistry》2003,278(22):19917-19925
Adhesion of many cell types to the extracellular matrix is essential to maintain their survival. In the absence of integrin-mediated signals, normal epithelial cells undergo a form of apoptosis termed anoikis. It has been proposed that the activation of initiator caspases is an early event in anoikis, resulting in Bid cleavage and cytochrome c release from mitochondria. We have previously demonstrated that the loss of integrin signaling in mammary epithelial cells results in apoptosis and that this is dependent upon translocation of Bax from the cytosol to the mitochondria. In this paper, we ask whether caspases are required for Bax activation and the associated changes within mitochondria. We show that Bax activation occurs extremely rapidly, within 15 min after loss of integrin-mediated adhesion to extracellular matrix. The conformational changes associated with Bax activation are independent of caspases including the initiator caspase-8. We also examined downstream events in the apoptosis program and found that cytochrome c release occurs after a delay of at least 1 h, with subsequent activation of the effector caspase-3. This delay is not due to a requirement for new protein synthesis, since cycloheximide has no effect on the kinetics of Bax activation, cytochrome c release, caspase-3 cleavage, or apoptosis. Together, our data indicate that the cellular decision for anoikis in mammary epithelial cells occurs in the absence of caspase activation. Moreover, although the conformational changes in Bax are rapid and synchronous, the subsequent events occur stochastically and with considerable delays. 相似文献