A Biophysical Model of the Mitochondrial Respiratory System and Oxidative Phosphorylation
This paper reports the purification of Apaf-3, a third protein factor that participates in caspase-3 activation in vitro. Apaf-3 is a member of the caspase family: specifically, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (P. Li, 1997).
The reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9 is reported. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3 (Zhou, 1999).
Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. A novel Apaf-1 cDNA encoding a protein of 1248 amino acids has been identified, containing an insertion of 11 residues between the CARD and ATPase domains and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activates procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. This Apaf-1 was used to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) is unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enables Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibits greater ability to induce apoptosis compared with the wild-type Apaf-1. Procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associates with and activated procaspase-9, but fails to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis (Hu, 1999).
Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. Analysis was performed of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms, and Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, was identified as the major RNA form expressed in all tissues tested. Another expressed isoform, Apaf-1LN, was identified containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP (Benedict, 2000).
In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. This study shows, through fluorescence polarization experiments, that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c.Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c.Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. The cytochrome c.Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins (Purring-Koch, 2000).
Apoptosis in metazoans is executed intracellular caspases. One of the caspase-activating pathways in mammals is initiated by the release of cytochrome c from mitochondria to cytosol, where it binds to Apaf-1 to form a procaspase-9-activating heptameric protein complex named apoptosome. This study reports the reconstitution of this pathway with purified recombinant Apaf-1, procaspase-9, procaspase-3, and cytochrome c from horse heart. Apaf-1 contains a dATP as a cofactor. Cytochrome c binding to Apaf-1 induces hydrolysis of dATP to dADP, which is subsequently replaced by exogenous dATP. The dATP hydrolysis and exchange on Apaf-1 are two required steps for apoptosome formation (Kim, 2005; full text of article).
Apaf-1 generated in insect cells contains dATP as a cofactor. The bound dATP then undergoes one round of hydrolysis to dADP, a process that is stimulated by cytochrome c. This hydrolysis appears to serve two roles: (1) it provides energy for the conformational change need for Apaf-1 to transient from the inactive monomeric state to the oligomeric state, and (2) it allows exogenous dATP (or ATP) to exchange for the dADP that has lower binding affinity for Apaf-1, a critical step for Apaf-1 to form functional apoptosome rather than nonfunctional aggregates. This hydrolysis only happens in one round. Exogenously added dATP will then bind Apaf-1, but remains unhydrolyzed during apoptosome formation (Kim, 2005).
The formation of the nonfunctional Apaf-1 aggregate is also induced by cytochrome c binding to Apaf-1. Cytochrome c was actually reisolated as an inhibitor of Apaf-1 activity when dATP or ATP was not included in the caspase-3-activating reaction. Such aggregates may be identical to the large, inactive Apaf-1 complex first observed in human monocytic tumor cells (Kim, 2005).
Interestingly, the crystal structure of a WD-40-truncated Apaf-1 revealed Apaf-1 in an inactive configuration with an ADP bound to it. Because WD-40 repeats serve as an autoinhibitory role, Apaf-1 without this region should be active, yet may easily become inactive if the exogenous dATP or ATP level is low. This finding is consistent with the observation that WD-40-less Apaf-1 is indeed active in promoting procaspase-9/3 activation but is rather unstable and the only stable configuration might be the dADP or ADP binding form. It is also interesting that this form of Apaf-1 expressed in bacteria contains an ADP but not dADP, whereas full-length Apaf-1 expressed in insect cells contains exclusively dATP. The measured difference between dATP and ATP in binding affinity to Apaf-1 is 10-fold, not enough to explain this exclusive binding of dATP because intracellular ATP level is several orders of magnitude higher than dATP. One possibility is that dATP level in E. coli is very low. Another possibility could be that mammalian and insect cells contain a dATP-specific loading factor for Apaf-1 that is absent in bacteria. Such a factor could potentially function in conjunction with prothymosin- and PHAPI, two proteins that regulate apoptosome activity (Kim, 2005).
The role of cytochrome c in apoptosome formation also becomes clear through the current study. Upon binding to Apaf-1, cytochrome c releases the autoinhibition imposed by the WD-40 repeats and allows Apaf-1 to hydrolyze the bound dATP. This role of cytochrome c also suggests that its simple release from mitochondria and binding to Apaf-1 may not necessarily result in the activation of caspase-9/3. Without exogenous dATP or ATP exchange, cytochrome c binding to Apaf-1 will irreversibly deplete the Apaf-1 protein in cells without activating caspases. Consistently, when intracellular ATP is depleted, cells undergo necrosis in response to stimuli that normally induce apoptosis, and when the cellular ATP levels are restored, the response shifts back to apoptosis. Therefore, it is hypothesized that the nucleotide exchange on Apaf-1 may provide another regulatory step for apoptosis (Kim, 2005).
The question remains whether endogenous Apaf-1 in mammalian cells also exclusively binds dATP. Addressing this question requires improvement on LC-MS method so that the nucleotide bound to endogenous Apaf-1 can be identified by using much less available material (Kim, 2005).
As components of the apoptosome, a caspase-activating complex, cytochrome c (Cyt c) and Apaf-1 are thought to play critical roles during apoptosis. Due to the obligate function of Cyt c in electron transport, its requirement for apoptosis in animals has been difficult to establish. 'Knockin' mice were generated expressing a mutant Cyt c (KA allele), which retains normal electron transfer function but fails to activate Apaf-1. Most KA/KA mice displayed embryonic or perinatal lethality caused by defects in the central nervous system, and surviving mice exhibited impaired lymphocyte homeostasis. Although fibroblasts from the KA/KA mice were resistant to apoptosis, their thymocytes were markedly more sensitive to death stimuli than Apaf-1(-/-) thymocytes. Upon treatment with gamma irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed. These studies indicate the existence of a Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation (Hao, 2005).
The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).
Caspase 9 (Casp9)/Apaf3, a 45 kDa protein (also known as ICE-LAP-6 or Mch6) forms a multiprotein complex containing Apaf1 and cytochrome c. It has been proposed that cytochrome c initiates apoptosis by inducing the formation of the Casp9/Apaf1 complex. Physical association of Casp9 and Apaf1 is mediated by the interaction of their respective caspase recruitment domains (CARDs). CARDs are also found in other caspases with large prodomains, such as Casp4 and Casp8, that can associate with Apaf1 in mammalian cells. The antiapoptotic protein Bcl-XL has also been shown to interact with Casp9 and Apaf1, resulting in the inhibition of Casp9 activation. The association of Casp9 with antiapoptotic as well as proapoptotic proteins suggests a major role for Casp9 in the control of apoptosis in vivo. Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. The absence of Casp9 leads to a dramatic disturbance of telencephalic development that apparently results from decreased apoptosis in this region. The gross morphological features observed in Casp9-/- mice are remarkably similar to those observed in mice lacking Casp3. Both mutants exhibit a profound disturbance of cortical morphology, an expanded germinal zone, and hydrocephaly, suggesting that these mutations affect a common cellular apoptotic pathway dependent on both Casp9 and Casp3. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. The Casp9-dependent, Casp3-independent apoptotic pathway is preferentially triggered in thymocytes in response to dexamethasone. However, the fact that Casp3 is still processed in dexamethasone-treated Casp9-/- thymocytes suggests that dexamethasone also activates a Casp9-independent and Casp3-dependent apoptotic pathway in these cells. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of multiple apoptotic pathways in mammalian cells (Hakem, 1998).
Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. In various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. These results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death (Chandra, 2006).
During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, it was found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c (Waterhouse, 2001).
The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. This study identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. It is shown that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria (Martin, 2005; full text of article).
The release of cytochrome c from mitochondria results in the formation of an Apaf-1-caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90-Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade (Pandey, 2000).
The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Using a cell-free system, it has been shown that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome (Beere, 2001).
Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt c(T)) that is distinct from the cytochrome c expressed in somatic cells (Cyt c(S)). To examine the role of Cyt c(T) in germ cells, mice null for Cyt c(T) were generated. Homozygous Cyt c(T)-/- pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt c(T)-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt c(T)-null mice contained a greater number of immotile spermatozoa than did samples from control mice for epididymal sperm. Cyt c(T)-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt c(T)(-/-) testes and controls. These data indicate that the Cyt c(T)-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation (Narisawa, 2002).
Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, it was found that this cell line (CRL 2613) respires at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, is able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, a mouse knockout was developed for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-alpha-induced apoptosis, but this process is still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways (Vempati, 2007).
Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, mRNA for BIMEL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and this is reduced by dominant-negative c-Jun. Overexpression of BIMEL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim-/- knockout mice die more slowly after NGF withdrawal (Whitfield, 2001).
As components of the apoptosome, a caspase-activating complex, cytochrome c (Cyt c) and Apaf-1 are thought to play critical roles during apoptosis. Due to the obligate function of Cyt c in electron transport, its requirement for apoptosis in animals has been difficult to establish. 'Knockin' mice were generated expressing a mutant Cyt c (KA allele), which retains normal electron transfer function but fails to activate Apaf-1. Most KA/KA mice displayed embryonic or perinatal lethality caused by defects in the central nervous system, and surviving mice exhibited impaired lymphocyte homeostasis. Although fibroblasts from the KA/KA mice were resistant to apoptosis, their thymocytes were markedly more sensitive to death stimuli than Apaf-1(-/-) thymocytes. Upon treatment with gamma irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed. These studies indicate the existence of a Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation (Hao, 2005).
Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis (Cipolat, 2006).
Hydrogen peroxide (H2O2) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H2O2 in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H2O2 generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H2O2 instead of to cytochrome c oxidase. Mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H2O2 three times faster than its counterpart in somatic cells (S-Cc), and the T-Cc heme has the greater resistance to being degraded by H2O2. Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H2O2. Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis (Liu, 2006).
Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, 10 ovarian cancer cell lines were examined for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, it was found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation, compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because no dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation was detected. Lack of APAF-1 activity correlates in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers (Wolf, 2001).
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