InteractiveFly: GeneBrief

Cytochrome c proximal: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. Only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis (Arama, 2006).

Apoptosis is a morphologically distinct form of active cellular suicide that serves to eliminate unwanted and potentially dangerous cells. The key enzymes responsible for the execution of apoptosis are an evolutionarily conserved family of cysteine proteases known as caspases. Caspases are present in an inactive or weakly active state in virtually all cells of higher metazoans, and their activity is carefully regulated by both activators and inhibitors. In vertebrates, the mitochondria play an important role in the control of apoptosis: they release cytochrome C and other pro-apoptotic proteins in response to various death signals. In the cytosol, cytochrome C binds to Apaf-1 (Zou, 1997) which in turn promotes the assembly of a multiprotein complex, termed the 'apoptosome', and caspase-9 activation (Rodriguez, 1999; Adams, 2002; Cain, 2002; Salvesen, 2002). In the ensuing 'caspase cascade', many intracellular substrates are cleaved and apoptosis is executed. However, the exact physiological role of cytochrome C for caspase activation remains to be determined, and a recent report on a mutant cytochrome c that fails to activate Apaf-1 in the mouse (Hao, 2005) suggests that cytochrome C is required for caspase activation in only some mammalian cell types (Arama, 2006).

In invertebrates, any role of cytochrome C for the activation of caspases has remained highly controversial. Whereas RNAi experiments in Drosophila S2 cells have failed to reveal a role for cytochrome C in apoptosis, other reports suggest that cytochrome C may promote caspase activation (Dorstyn, 2002, 2004; Zimmermann, 2002). Drosophila contains two Apaf-1 isoforms: one with a WD40 repeat domain, the target for cytochrome C binding, and another lacking this domain, similar to Caenorhabditis elegans Ced-4. The large isoform can directly bind cytochrome C in vitro and promote cytochrome C-dependent caspase activation in lysates from developing embryos (Kanuka, 1999). Furthermore, an overt alteration in the cytochrome C immuno-staining can be detected in doomed cells in some Drosophila tissues, and the mitochondria from apoptotic cells can activate cytosolic caspases (Varkey, 1999). Finally, disruption of one of the two Drosophila cytochrome c genes, cyt-c-d, is associated with a failure to activate caspases in an apoptosis-like process during sperm terminal differentiation in Drosophila (Arama, 2003). In this process, also known as spermatid individualization, the majority of cytoplasm and cellular organelles are eliminated from the developing spermatids in an apoptosis-like process that requires caspase activity (Arama, 2003). However, it was suggested that the mutants used in s previous study (Huh, 2004) may also affect other genes located in the vicinity of the cyt-c-d locus (Arama, 2006).

In order to rigorously address this issue, a series of genetic and transgenic rescue experiments were conducted that unequivocally establish a role of cytochrome C for caspase activation during Drosophila spermatogenesis. First, a point mutation was isolated in cyt-c-d that is defective in caspase activation. Next, it was demonstrated that transgenic expression of cyt-c-d restores effector caspase activation and rescues all the sterility phenotypes associated with various cyt-c-d mutant alleles. The possibility that cyt-c-p functions specifically in respiration was investigated, whereas cyt-c-d plays a role in caspase regulation. Surprisingly, it was found that expression of either cyt-c-d or cyt-c-p can restore caspase activation in cyt-c-d-deficient spermatids, demonstrating that both proteins are functionally equivalent. Other apoptosome proteins in Drosophila, Ark (Apaf-1) and Dronc (caspase-9) are also required for spermatid individualization, and their mutant phenotypes are similar to spermatids with a block in caspase activity. Surprisingly, however, some active caspase-3 staining can still be detected in these mutant testes, suggesting that cytochrome-C-d may function in yet other unknown pathways to promote caspase-3 activation. Finally, several mutants affecting spermatid mitochondria were identifed that provide a strong link between mitochondrial organization and caspase activation during sperm development (Arama, 2006).

In mammals, mitochondria are important for the regulation of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome C, an essential component of the respiratory chain. Cytosolic cytochrome C can bind to and activate Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. The elimination of cytoplasm during terminal differentiation of spermatids in Drosophila involves an apoptosis-like process that requires caspase activity; a P-element insertion (bln¹) in one of the two Drosophila cytochrome c genes, cyt-c-d, has been shown to be associated with male-sterility and loss of effector caspase activation during spermatid individualization. This study demonstrates that the defects in caspase activation and spermatid individualization of bln¹ mutant males can be rescued by transgenic expression of the ORF of cyt-c-d. Furthermore, from screening more than a thousand male-sterile lines with defects in sperm individualization for defects in active-caspase (CM1) staining, a nonsense point mutation was identified in cyt-c-d, that recapitulates all the phenotypes observed for bln¹. Taken together, these results unequivocally demonstrate that cyt-c-d is necessary for effector caspase activation and sperm terminal differentiation in Drosophila (Arama, 2006).

Two decades ago, the mouse cytochrome c gene was used as a probe for screening a Drosophila genomic library and a fragment was isolated that carried two distinct cytochrome c genes. Northern blot analyses indicated high levels of cyt-c-p expression, while cyt-c-d was reported to be expressed at much lower levels in all stages of development. However, neither the exon/intron organization nor the boundaries of the 5' and 3' UTRs of these genes were determined at the time. As a result, the original Northern analyses were performed with a probe corresponding to the untranscribed genomic region between the two cytochrome c genes that was not suitable to properly assess the size and distribution of cytochrome c transcripts. Unfortunately, this has caused considerable confusion in the field from the start, as even the original report noted that the size of the observed cyt-c-d transcript differed more than two-fold from the predicted size. More recently, relying on the incorrect assumption that cyt-c-d is ubiquitously expressed in the fly, it has been suggested that a loss-of-function mutation in cyt-c-d should lead to severe developmental defects and lethality rather than merely male sterility. However, using a specific cyt-c-d 3' UTR probe reveals a transcript of the predicted size that is absent in cyt-c-d^bln1 mutants. Furthermore, the RT-PCR and immunofluorescence analyses presented in this study indicate that cyt-c-d is mainly expressed in the male germ line and is completely absent during embryonic and larval development, while cyt-c-p is expressed in the soma during all stages of development. In light of these findings, it is not surprising that loss-of-function mutations in cyt-c-d cause male sterility, whereas cyt-c-p mutations lead to embryonic lethality. RT-PCR results suggest that cyt-c-p is also expressed in the testis, although to a much lower extent than cyt-c-d. This expression is attributed primarily to the somatic cells of the testis, since no cytochrome C protein is detected in cyt-c-d^bln1 elongating spermatids, while cyt-c-p RNA is expressed in cyt-c-d^bln1 mutant flies. However, the very low cyt-c-d expression detected in the soma of adult females leaves room for the possibility that cyt-c-d might function in caspase activation in some somatic cells as well (Arama, 2006).

In mammalian cells, release of cytochrome C into the cytosol in response to proapoptotic stimuli can be readily demonstrated. However, previous attempts to detect a similar phenomenon in Drosophila have been unsuccessful. In contrast, apoptotic stimuli can lead to increased cytochrome C immuno-reactivity. A possible limitation is that all these studies were conducted using mammalian antibodies with questionable specificity and sensitivity, and only in a small number of cell types and paradigms. Using an antibody that was raised against Drosophila cytochrome C-d, an increase in a 'grainy signal' was detected upon the onset of individualization, with the highest staining observed in the vicinity of the individualization comple (IC). Since it is highly unlikely that additional cytochrome C-d is being transcribed and imported to the mitochondria at this late stage, the explanation is favored that a conformational change or an exposure of a hidden epitope causes the increase in the intensity of the signal. The activation of Dronc, the Drosophila caspase-9 orthologue, also occurs in association with the IC and depends on the presence of the Drosophila Apaf-1 orthologue, Ark. Moreover, the proapoptotic Hid protein is localized in a similar fashion. What are these structures then, which accumulate apoptotic factors in the vicinity of the IC? One plausible suggestion from the literature is that these structures correspond to 'mitochondrial whorls', which result from the extrusion of material from the minor mitochondrial derivative and constitute the leading component of the IC. These 'whorls' can be labeled using a testes-specific mitochondrial-expressed GFP line. Using this GFP marker, it was found that cytochrome C-d is indeed closely associated with mitochondrial whorls. Therefore, it is possible that an active apoptosome forms in the vicinity of the IC in response to dramatic changes in the mitochondrial architecture that occur at this stage of spermatid differentiation. Similarly, studying the response of Drosophila flight muscle cells to oxygen stress, have recently reported that the cristae within individual mitochondria become locally rearranged in a pattern that they termed a 'swirl'. This process was associated with widespread apoptotic cell death in the flight muscle, which was correlated with a conformational change of cytochrome C manifested by the display of an otherwise hidden epitope. Collectively, these observations suggest that apoptosome-like complexes composed of cytochrome C-d, Ark, and Dronc might be associated with unique mitochondrial swirl-like structures. Consistent with this idea, it was found that the long isoform of Ark that contains the WD40 repeats, the target for cytochrome C binding to mammalian Apaf-1, is the major form detectably expressed in testes (Arama, 2006).

The fact that cytochrome C-d immunoreactivity increases in the vicinity of the IC suggests that the extensive mitochondrial organizations preceding individualization may be partially required for caspase activation. Consistent with this idea, several mutants, such as pln^Z2-0516, which display defects in Nebenkern differentiation and caspase activation. However, not all mitochondrial differentiation events are required for caspase activation. For example, CM1 staining is seen in fuzzy onions, a mutant defective in the mitochondrial fusion event that generates the Nebenkern. In contrast, analysis of the pln mutant indicates that proper elongation of the Nebenkern is essential for caspase activation. Therefore, characterization of other mitochondrial mutants may shed light on the connection between mitochondrial organization and caspase activation during sperm differentiation (Arama, 2006).

What are the mechanisms by which cytochrome C-d activates caspases during late spermatogenesis? In vertebrate cells, following its release into the cytosol, cytochrome C binds to the WD40 domain of the adaptor molecule Apaf-1, which in turn multimerizes and recruits the initiator caspase, caspase-9 via interaction of their CARD domains. This complex, known as the apoptosome, further cleaves and activates effector caspases like caspase-3. Although this model has become the prevailing dogma in the field, the phenotype of mice mutant for a Cyt c with drastically reduced apoptogenic function ('KA allele') suggests that the mechanisms for caspase activation may be more complex than previously thought. In particular, this study suggests that cytochrome C-independent mechanisms for the activation of Apaf-1 and caspase-9 exist, as well as cytochrome C-dependent but Apaf-1-independent mechanisms for apoptosis. These analyses of ark (Apaf-1) and dronc (caspase-9) loss-of-function mutants demonstrate that both genes are required for spermatid individualization, and that their phenotypes, in particular their failure to properly remove the spermatid cytoplasm into the WB, resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes. However, some caspase-3-like activity could still be detected in these mutant testes. This may suggest that either the ark and dronc alleles are not null, or that cytochrome C-d also functions in an apoptosome-independent pathway to promote caspase-3 activation. Therefore, the regulation of caspase activation and apoptosis may be more similar between insects and mammals than has been previously appreciated. Further genetic analysis of this pathway in Drosophila may provide general insights into diverse mechanisms of apoptosis activation (Arama, 2006).

Previous observations raised the possibility that the two distinct cytochrome c genes may have evolved to serve distinct functions in respiration and caspase regulation. In order to address this hypothesis, it was asked whether expression of one protein might rescue mutations in the other cytochrome c gene. Surprisingly, it was found that transgenic expression of the cyt-c-p ORF in germ cells rescues caspase activation, spermatid individualization, and sterility of cyt-c-d^-/- flies. Therefore, the ability to activate caspases is not restricted to the cytochrome C-d protein, and it is possible that cytochrome C-p functions in apoptosis in at least some somatic cells (Arama, 2006).

Although cyt-c-d is almost exclusively expressed in the male germ cells, ectopic expression of this protein in the soma can rescue the respiration defect and lethality of cyt-c-p^-/- mutant flies, demonstrating that cytochrome C-d can function in energy metabolism. This raises the question whether the lack of caspase activation could be due to reduced ATP-levels. Although this is a formal possibility, this explanation is considered very unlikely since mutant spermatids complete many other energy-intensive cellular processes. These include the extensive transformation from round spermatids to 1.8 mm long elongated spermatids, a process that involves extensive remodeling and movement of actin filaments, generation of the axonemal tail, mitochondrial reorganization, plasma/axonemal membranes reorganization, and nuclear condensation and elongation. Since all of these processes can occur in the absence of cytochrome C-d, there is no overt shortage of ATP in cyt-c-d mutants. It is therefore considered very unlikely that ATP has become limiting in these mutant cells. Since earlier stage spermatids express cytochrome C-p, sufficient ATP seems to persist to late developmental stages. In mammalian cells, cellular ATP concentration is sufficiently high (around 2 mM) to keep cultured cell alive for several days upon ATP synthase inhibition. Furthermore, cells in which cytochrome c expression is decreased by RNAi still undergo apoptosis in response to various stimuli. Likewise, it appears that cytochrome C is not essential for the function of mature murine sperm, since mice deficient for the testis specific form of cytochrome C, Cyt c_T, are fertile. Taken together, all these observations argue strongly against the possibility that ATP levels in cyt-c-d^-/- mutant spermatids would be insufficient for caspase activation (Arama, 2006).

In conclusion, the results presented in this study definitively demonstrate that cytochrome C-d is essential for caspase activation and spermatid individualization. Both cytochrome C proteins of Drosophila are, at least to some extent, functionally interchangeable. The results also indicate that cytochrome C can promote caspase activation in the absence of a functional apoptosome. Given the powerful genetic techniques available, late spermatogenesis of Drosophila promises to be a powerful system to identify novel pathways for mitochondrial regulation of caspase activation (Arama, 2006).

REGULATION

Regulation of apoptosis and protein interactions

Altered cytochrome c display precedes apoptotic cell death in Drosophila

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. An overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy cells are inactive. These observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases (Varkey, 1999; full text of article).

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. This study demonstrates that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals (Dorstyn, 2002; full text of article).

The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004; full text of article).

The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).

The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).

Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system

Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, it has been demonstrated that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases are inhibited, induce caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid is sufficient to activate caspases in cytosolic extract, and this activity is not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also inducew apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors (Means, 2005; full text of article).

Some activation of caspase-3 is detected in ark and dronc mutant testes, suggests that some of the cytochrome-C-mediated caspase-3 activation is independent of apoptosome components

In vertebrates, mitochondria play an important role in the control of apoptosis by activating the apoptosome, a multiprotein complex that includes caspase-9, Apaf-1, and cytochrome C. Drosophila possesses one Apaf-1 orthologue known either as Hac-1, Dark, or Dapaf-1 which, like its mammalian counterpart, is important in multiple apoptotic pathways. In addition, Drosophila also has a caspase-9 orthologue, Dronc, which, similar to the vertebrate caspase-9, contains a caspase recruitment domain (CARD), and functions in a variety of cell death pathways. Whether Ark and Dronc are required for spermatid individualization was examined. For this purpose, several EMS-derived loss-of-function alleles of both ark and dronc were examined. ark and dronc mutant flies display highly similar phenotypes and most mutant animals die during pupariation. However, some adult 'escapers' emerge that are both male and female sterile. Both ark and dronc mutants displayed severe defects during the spermatid individualization process. In particular, ark and dronc mutant spermatids failed to extrude much of their cytoplasm into a CB, leaving trails of the cytoplasm in what should have been the postindividualized region of the spermatids. Consequently, ark^-/- and dronc^-/- CBs and WBs are highly reduced in size or appear flat, and frequently a large portion of the spermatids' cytoplasm is retained behind in a 'mini' CB structure (white arrowhead in, which often contains part of the IC. The size of the CBs and WBs in ark and dronc mutants is on average only half the size of their wild-type counterparts. These phenotypes are reminiscent of testes that ectopically express the caspase inhibitor gene p35 (Arama, 2003). These results suggest that ark and dronc are required for normal caspase activation and the initiation of an apoptosis-like process essential for spermatid individualization. However, whereas no caspase-3-like activity was detected in cyt-c-d^-/- mutants, some activation of caspase-3 was detected in ark and dronc mutant testes. This suggests that some of the cytochrome-C-mediated caspase-3 activation is independent of apoptosome components. Alternatively, it is possible that the ark and dronc alleles used in this study are not complete nulls and therefore retain some residual function that allows a small amount of cytochrome C-induced caspase activation (Arama, 2006).

The 14-3-3 proteins are highly conserved molecules that function as intracellular adaptors in a variety of biological processes, such as signal transduction, cell cycle control, and apoptosis. This study shows that a 14-3-3 protein is a heat-shock protein (Hsp) that protects cells against physiological stress as its new cellular function. In Drosophila cells, the 14-3-3zeta is up-regulated under heat stress conditions, a process mediated by a heat shock transcription factor. As the biological action linked to heat stress, 14-3-3zeta interacted with apocytochrome c, a mitochondrial precursor protein of cytochrome c, in heat-treated cells, and the suppression of 14-3-3zeta expression by RNA interference resulted in the formation of significant amounts of aggregated apocytochrome c in the cytosol. The aggregated apocytochrome c was converted to a soluble form by the addition of 14-3-3zeta protein and ATP in vitro. 14-3-3zeta also resolubilized heat-aggregated citrate synthase and facilitated its reactivation in cooperation with Hsp70/Hsp40 in vitro. These observations provide the first direct evidence that a 14-3-3 protein functions as a stress-induced molecular chaperone that dissolves and renaturalizes thermal-aggregated proteins (Yano, 2006; full text of article).

DEVELOPMENTAL BIOLOGY

Rescue results showing that the two cytochrome genes can functionally rescue each other, raise the question of why cyt-c-d^-/- males are sterile if both cytochrome c genes are functionally equivalent. One possible explanation is distinct expression of the two genes, namely that cyt-c-d is testis-specific, whereas cyt-c-p may be restricted to the soma. To examine this possibility, the distribution of transcripts from both cytochrome c genes were examined in the testis and the soma. For this purpose, comparative RT-PCR experiments were performed using specific primers in the unique 5' and 3' UTR sequences of cyt-c-d and cyt-c-p. While cyt-c-p was highly expressed in the soma, cyt-c-d is only weakly expressed there (represented by adult females that lack testes). In contrast, cyt-c-d expression was much higher in testes than cyt-c-p. The low levels of cyt-c-p in testes is attributed to the somatic cells present in this tissue. Furthermore, although the expression of cyt-c-d in the soma of both males and females is much lower than the levels of cyt-c-p, cyt-c-d levels are much higher in adult males than in females, suggesting that the male germ cells provide the main contribution of cyt-c-d in the adult. These results suggest that the distinct phenotypes of cyt-c-d and cyt-c-p are mainly due to their restricted differential expression in the testis and the soma, respectively (Arama, 2006).

In addition to germ cells, the testis also contains somatic cells, such as the testicular wall, muscles cells, and cyst cells. To determine which testicular cell types express cyt-c-d, comparative RT-PCR analyses were first performed with RNA from reproductive tracts of oskar male mutants that are defective in germline development and lack germ cells in the adults. While both cytochrome c genes were expressed in wild type, only cyt-c-p was detected in the germ-cell-less reproductive tracts of sons of oskar^-/-. This indicates that cyt-c-d expression is restricted to the germ cells of the adult male. Next, the developmental stage at which cyt-c-d is expressed in the male germ line was examined. For this purpose, advantage was taken of the fact that testes of adult flies and third instar larvae differ in their repertoire of germ cells. While adult testes contain germ cells in a variety of developmental stages, the most developmentally advanced germ cells present in third instar larval testes are premeiotic spermatocytes. Interestingly, the patterns of cyt-c-d expression in both adult and larval testes are identical, demonstrating that cyt-c-d mRNA accumulates before the entry of spermatocytes into meiosis (Arama, 2006).

The activation of apoptotic effector caspases, as visualized by CM1-staining, is not restricted to the male germ cells but can also be detected in nurse cells during oogenesis. The possibility is considered that caspase activation in this system is also influenced by cyt-c-d. However, no abnormalities during oogenesis were detected in cyt-c-d^-/- flies and the females are fertile. Consistent with this idea, comparative RT-PCR analysis of adult ovaries revealed expression of cyt-c-p but not cyt-c-d (Arama, 2006).

To study the pattern of cytochrome C-d expression in the testis, polyclonal antibodies were raised against four peptides covering the entire length of the protein. Consistent with the findings that no cyt-c-d RNA is expressed in cyt-c-d^bln1 homozygote flies, almost no signal was detected after staining testes of this mutant with the anti-cytochrome C-d antibody. Staining wild-type testes with this antibody revealed a grainy pattern of cytochrome C-d signal along the entire length of elongating spermatids and elongated spermatids. Once an individualization complex (IC) was assembled in the vicinity of the nuclei, an increase in cytochrome C-d staining was detected with the highest intensity found next to the IC. During the caudal translocation of the IC, a significant portion of cytochrome C-d is depleted from the newly individualized part of the spermatids into the CB. Eventually, the newly formed WBs accumulate high levels of cytochrome C-d (Arama, 2006).

To test whether this antibody could also crossreact with cytochrome C-p, testes of cyt-c-d mutant lines were tested that were rescued by transgenic cyt-c-p expression in germ cells. Similar to the cytochrome C-d expression in wild type or after ectopic expression in mutant testes, ectopic cytochrome-C-p expression was also detectable as grainy staining along the entire length of elongated spermatids as well as in CBs and WBs. These results demonstrate that the antibody can detect both forms of the Drosophila cytochrome C molecules. The lack of staining found in cyt-c-d^bln1 elongating spermatids is consistent with the idea that only cyt-c-d and not cyt-c-p is expressed in mature spermatids (Arama, 2006).

EFFECTS OF MUTATION

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

Effector caspases, such as drICE, can display Caspase-3-like (DEVDase) cleaving activity. Therefore, it was asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk. Importantly, this activity was highly reduced in cyt-c-d^Z2-1091 mutant testes. These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system (Arama, 2006).

Because of the cytological proximity between cyt-c-d and cyt-c-p (241 bp maximum between the end of the 3' UTR of cyt-c-d and the beginning of the 5' UTR of cyt-c-p) there is a possibility that the bln¹ P-element insertion in cyt-c-d might also interfere with the expression of cyt-c-p (Huh, 2004). In order to determine whether cyt-c-p expression was altered in cyt-c-d^bln1, RT-PCR analyses was carried out with RNA from wild-type (yw) and cyt-c-d^bln1 adult flies using two sets of primers for each gene specific for either the 5' UTRs of cyt-c-d and cyt-c-p. In agreement with Northern results, no cyt-c-d RNA was detected in cyt-c-d^bln1 flies, confirming that bln¹ is a null allele of cyt-c-d. In contrast, cyt-c-p is expressed in both wild-type and cyt-c-d^bln1 flies (Arama, 2006).

Although the sequence of cyt-c-d and cyt-c-p proteins is highly conserved, they are not identical. In addition, mutations in each gene display distinct phenotypes (Arama, 2003). This raises the possibility that both proteins may have distinct functions in respiration (cytochrome C-p) and caspase activation/apoptosis (cytochrome C-d). To test this hypothesis, it was first asked whether expression of cyt-c-p in developing spermatids is able to substitute for the loss of cyt-c-d. In order to drive expression of transgenes in the male germ line, an expression vector was constructed composed of the hsp83 promoter followed by the 5' and 3' UTRs of cyt-c-d, which are important for the proper temporal regulation of cyt-c-d translation in spermatids, and next the coding regions of either cyt-c-d or cyt-c-p were inserted between both UTRs and transgenic flies were generated with these constructs. At least three independent transgenic lines for each of these constructs were crossed to cyt-c-d^bln1 or cyt-c-d^Z2-1091 flies, and the presence of the appropriate transgene was confirmed by genomic PCR. To validate expression of the transgenes, RT-PCR analysis was inserted with testes RNA in a cyt-c-d^bln1-/- background. Finally, the ability of these transgenes to rescue caspase activation, spermatid individualization, and male sterility was examined in cyt-c-d^bln1 and cyt-c-d^Z2-1091 flies. As a control, transgenic flies containing 'empty vector' (including the hsp83-promotor with the 5' and 3' UTRs of cyt-c-d but without a coding region) were also generated. As expected, no caspase activation was detected in testes of these control flies. In contrast, a transgene with the cyt-c-d open reading frame (ORF) fully rescued CM1-staining, spermatid individualization, and male fertility. This firmly establishes that both the caspase and sterility phenotypes seen in cyt-c-d^bln1 and cyt-c-d^Z2-1091 mutant flies are strictly due to the loss of cytochrome c function, with no detectable contribution from adjacent genes (Arama, 2006).

The ability of cyt-c-p to functionally substitute for the loss of cyt-c-d was tested. Surprisingly, transgenic expression of cyt-c-p was equally effective in rescuing all defects in cyt-c-d^bln1 or and cyt-c-d^Z2-1091 males. It is concluded that both proteins have similar biochemical properties to promote caspase activation and spermatid individualization (Arama, 2006).

l(2)k13905 flies contain a P-element insertion in the 5' UTR of cyt-c-p and die as late embryos or early first instar larva (Arama, 2003). Using RT-PCR, it was found that only cyt-c-p expression was detected in early first instar wild-type larvae, while a dramatic reduction was observed in the cyt-c-p^k13905 mutants. These results are consistent with the phenotypes of cyt-c-d (viable but male sterile) and cyt-c-p (early lethal) mutants (Arama, 2006).

Lethality of cyt-c-p^k13905 homozygotes as well as trans-heterozygotes to Df(2L)Exel6039, a deletion in the region that includes both cyt-c-p and cyt-c-d, is consistent with the idea that cyt-c-p encodes the major cytochrome C responsible for respiration. Whether cyt-c-d could also function in respiration and rescue the early lethality of cyt-c-p^-/- flies was examined. Both cytochrome C proteins were ectopically expressed in cyt-c-p^k13905 mutants using the GAL4-UAS system. The Tub-Gal4 driver line was used to drive cyt-c-p and cyt-c-d expression throughout the lifespan of the fly. Notably, one copy of either the UAS-cyt-c-p or the UAS-cyt-c-d transgenes together with one copy of the driver completely rescued the lethality of cyt-c-p^k13905/Df(2L)Exel6039 flies. It is concluded that both cytochrome C proteins of Drosophila can function in electron transfer/respiration. The complete absence of cyt-c-p from the rescued adult flies is consistent with the idea that cyt-c-p^k13905 is a null allele of cyt-c-p. The faint expression of cyt-c-p in cyt-c-p^k13905 homozygote and cyt-c-p^k13905/Df(2L)Exel6039 trans-heterozygote mutants detected in early first instar larvae only after 30 PCR cycles is attributed to remnants of maternal contribution. This also explains how cyt-c-p^-/- mutant embryos can reach the early first instar larval stage without any zygotic contribution. Finally, it was not possible to rescue the lethality of flies homozygous for the cyt-c-p^k13905 allele, suggesting that the k13905 chromosome carries an additional unrelated lethal mutation (Arama, 2006).

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. This study shows that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, it was found that cyt c-d-as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, these results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells (Mendes, 2006).

In response to apoptotic stimuli, mammalian cells release cytochrome c (Cyt c) from the mitochondria into the cytoplasm where it binds to apoptotic protease-activating factor 1 (Apaf 1). This leads to the recruitment of the zymogen form of caspase 9 to a catalytically active multi-protein complex called the apoptosome (Jiang, 2004). Once activated, the apoptosome, consisting of Cyt c, dATP, Apaf 1 and caspase 9, can cleave and activate downstream caspases, including caspase 3. Genetically modified mice have demonstrated the in vivo importance of several components of this pathway, including Apaf 1, caspase 9, caspase 3 and Cyt c. However, these studies have also shown a surprising degree of complexity and raised questions about how apoptosis is activated in the absence of canonical apoptosome components (Hao, 2005; Mendes, 2006 and references therein).

In Drosophila, the mechanisms leading to the activation of the Apaf 1 homologue are controversial (Kornbluth, 2005). Similar to its mammalian homologue, Drosophila Ark (also called Hac 1/Dapaf 1/Dark) contains a series of WD40 repeats, which, in vitro, can bind Drosophila Cyt c and form an apoptosome-like complex and induce caspase activation. RNA interference knock-down experiments (Zimmermann, 2002; Dorstyn, 2004), however, failed to support a role for cyt c in the apoptosis of S2 culture cells (Mendes, 2006).

The Drosophila genome contains two closely related but distinct cyt c genes: cyt c-d and cyt c-p (Limbach, 1985). cyt c-p is involved in mitochondrial respiration and viability, whereas cyt c-d is required for caspase activation and sperm differentiation (Arama, 2003; Arama, 2006). In the sperm, caspase activation does not lead to cell death, but to sperm maturation. This study reports on the role of cyt c-d in apoptosis during normal development of the Drosophila retina (Mendes, 2006).

In the developing eye, superfluous interommatidial cells (IOCs) and perimeter ommatidial cells (POCs) are eliminated by apoptosis, allowing the precise rearrangement of ommatidia into a honeycomb-like formation. Antibodies raised against the membrane-bound protein Armadillo (Arm) allow the visualization of each cell in the eye lattice. By 42 h after puparium formation (APF), a fixed number of IOCs form an hexagonal array around each photoreceptor cell cluster, comprising four cone cells, three bristle cells, two primary, six secondary and three tertiary pigment cells (Mendes, 2006 and references therein).

To examine the role of the cyt c locus in cell death during pupal eye development, the number of IOCs were compared between wild-type and several cyt c-d male-sterile and viable, loss-of-function alleles at a stage in which cyt c-d is expressed. This analysis focused on the cyt c-d^Z2-1091 allele, since it bears a point mutation that creates a stop codon in the cyt c-d coding region, which does not affect neighbouring open reading frames. At 42 h APF, a time when IOC death is normally complete, cyt c-d^Z2-1091-/- mutant retinas showed extra cells in the secondary or tertiary position. A more pronounced phenotype was observed in cyt c-d^Z2-1091/Df(2L)H20 retinas, indicating that another neighbouring gene included in Df(2L)H20 contributes to the regulation of IOC death, or that cyt c-d^Z2-1091 might be a hypomorphic allele. In addition, the extra IOC phenotype observed in cyt c-d^Z2-1091-/- was rescued by the ectopic expression of cyt c-d in the developing retina. Mutant retinas for the other cyt c-d alleles also showed extra IOCs (Mendes, 2006).

To further characterize the role of cyt c-d, the number of extra IOCs at different stages of pupal development were counted in the mutant retina: at 22 h APF, cyt c-d^Z2-1091-/- retinas already showed extra IOCs; at 48 h APF, they still occasionally showed extra IOCs compared with wild type. The decrease in the number of extra IOCs between 22 and 48 h APF indicates that IOC death is delayed and not completely suppressed in the cyt c-d mutant. Considering that the IOC death process terminates at 36 h APF, it was estimated that IOC apoptosis in cyt c-d^Z2-1091-/- retinas can be delayed up to 12 h. It is proposed that the cyt c-d gene is required for the 'on-time' apoptosis of IOCs during pupal development (Mendes, 2006).

The results show that cyt c-d regulates IOC apoptosis in pupal retinas. It was then asked whether cyt c-d also regulates POC apoptosis. Ommatidia at the edge of the eye (perimeter ommatidia) contain photoreceptor, cone and pigment cells that die by apoptosis. Between 36 and 44 h APF, 80-100 ommatidia are eliminated, allowing the formation of a normal eye edge. POCs were visualized using an anti-Arm antibody in staged cyt c-d^-/- and wild-type retinas. By 38 h APF, POC elimination has just begun, with numerous small ommatidial clusters along the edge of the retinas. By 40 h APF, many wild-type POCs have been eliminated. By contrast, cyt c-d^Z2-1091-/- retinal edges showed more clusters of malformed ommatidia in a thick layer of IOCs. The same phenotype was also visible in all the other cyt c-d alleles. By 54 h APF, POC elimination is complete both in wild-type and mutant retinas. These results indicate that cyt c-d promotes not only IOC elimination but also POC death (Mendes, 2006).

The detection of cyt c-d expression is challenging, since none of the available antibodies allows distinguishing between the two cyt c species or visualize the release of Cyt c during apoptosis of ommatidial cells. The analysis of cyt c RNA transcripts showed that cyt c-p is the prevailing form expressed throughout development and adulthood. The cyt c-d transcript, however, seems to be mainly restricted to the testis (Arama, 2006). Both cyt c transcripts are present at the time of IOC elimination in the retina. This supports the possibility that the two Cyt c proteins can function in the elimination of superfluous retinal cells during pupation. The fact that physiological amounts of cyt c-p cannot substitute for the loss of cyt c-d suggests that the full apoptogenic function of cyt c requires the expression of both cyt c genes (Mendes, 2006).

Elimination of both cyt c genes in the retina might lead to a more pronounced phenotype than cyt c-d mutation alone. Unfortunately, this hypothesis is extremely difficult to test, given the general requirement of cyt c-p for cell survival. In addition, the possibility that the loss of both cyt c genes would lead to a phenotype as pronounced as the complete inhibition of death observed in retina expressing p35is not favored because in cyt c-d^Z2-1091/Df(2L)H20 flies, in which cyt c-d is lost and only one copy of cyt c-p is functional, IOC death is delayed to a level comparable with that in cyt c-d mutants (Mendes, 2006).

Apoptosis is delayed in the cyt c-d^-/- retina. This could be due to a direct role of cyt c-d in the apoptotic process or an indirect consequence of an impaired respiratory function in the mutant retina. To address the latter possibility, ATP levels were measured in several wild-type strains and cyt c-d mutants. No significant difference was found between wild-type and cyt c-d mutants, ruling out an effect in the bioenergetics levels as the cause of extra cells in cyt c-d^-/- retina. To eliminate any consequence in retinal development, cyt c-d^Z2-1091-/- larval and pupal eyes were stained with antibodies against several specific differentiation markers. cyt c-d^Z2-1091-/- larval eye discs stained against Elav (neuronal marker), Boss (R8-specific marker) and Spalt-major (R3, R4, R7, R8 and cone cell marker) appeared as in the wild-type control. Moreover, tangential plastic sections of cyt c-d^Z2-1091-/- adult eyes presented the normal number and arrangement of photoreceptor cells (Mendes, 2006).

Other retinal cell types, including primary pigment, cone and bristle cells, visualized at pupal stages in cyt c-d mutant, appeared normal in shape and number. cyt c-d^Z2-1091-/- retinas were stained at different stages of pupal development (24, 27, 30 and 42 h APF) with an anti-Homothorax (Hth) antibody, which stains secondary and tertiary pigment cell nuclei. All secondary and tertiary cells expressed hth, suggesting that the extra IOCs differentiate normally. Thus, the only phenotype associated with cyt c-d mutations is the appearance of extra secondary and tertiary cells in the eye lattice, with no disruption of early retinal development. For this reason, the cyt c-d mutation can be classified as lattice-specific. To determine whether cyt c-d is required for development progression, the dynamic IOC rearrangement and maturation was measured in staged cyt c-d^Z2-1091-/- and wild-type retinas. Despite the presence of extra IOCs in cyt c-d^Z2-1091-/-, the process of cell sorting and IOC maturation occurs similarly to wild-type retinas (20-27 h APF). Thus, the dynamic rearrangement and maturation of IOCs are not delayed in cyt c-d^Z2-1091-/- retinas, eliminating any significant effect of cyt c-d mutations on the progression of retinal cell differentiation (Mendes, 2006).

Together, these results demonstrate that cyt c-d is not required for respiration, differentiation or developmental progression in the pupal eye, providing the first genetic evidence for a physiological role of Drosophila cyt c in the regulation of developmental apoptosis (Mendes, 2006).

cyt c-d is required for apoptosis progression during pupal eye development in Drosophila. It was asked whether the other homologues of the apoptosome components, Ark and Dronc, are also required for apoptosis in this model. ark and dronc loss-of-function mutant alleles were used; both ark and dronc alleles are strong loss-of-function or null alleles leading to apoptosis defects at early stages in development and lethality (Mendes, 2006).

Using the flipase (FLP)/FLP recombinase target (FRT) technique, ark and dronc mutant clones were generated in the eye. In these clones, visualized by the absence of green fluorescent protein (GFP), an excess was counted of 5.20 and 4.36 IOCs/ommatidium, respectively. These values are comparable with those observed in pupal retinas in which the caspase inhibitor, p35, is ectopically expressed under control of the ubiquitous eye promoter, GMR, showing 5.06 extra IOCs/ommatidium. These values are higher than the total estimated number of IOCs that are dying between 18 and 36 h APF (about 3.5 IOCs/ommatidium). This is probably due to the fact that, in those mutant situations, unwanted IOCs are also rescued during larval development and early pupal development (<18 h APF) (Mendes, 2006).

The number of extra IOCs obtained in dronc mutant clones is similar to the value observed in the retinas of dronc mutant escapers. In addition, clonal analysis showed that only the mutant tissue for ark or dronc, exhibit extra IOCs, not the surrounding non-mutant tissue. This indicates that ark and dronc are required cell-autonomously for IOC apoptosis. In addition, it was found that the combination of cyt c-d mutations and the expression of Dronc dominant negative in the retina induces synergistic reduction of IOC death, suggesting the proximity of these genes in the same pathway (Mendes, 2006).

The role of ark and dronc in POC apoptosis was examined. In ark and dronc mutant clones, POCs are rescued and TUNEL is blocked. Moreover, the mutant retinas present extra POCs that are never eliminated, as seen in GMR-p35. Thus, Dronc has a pivotal role as an initiator caspase in the pupal retina-which differs from embryonic tissues-in which Dronc is required for most, but not all, cell death (Mendes, 2006).

To rule out the possibility that developmental defects in ark or dronc mutant retinas indirectly affect cell death, retinal cell differentiation was examined in ark and dronc mutant clones using several larval and pupal eye differentiation markers. It was found that retinal cell differentiation is normal in ark and dronc mutant clones (Mendes, 2006).

Together, these results demonstrate that ark and dronc are required for the initiation and/or execution of IOC and POC apoptosis, placing these genes hierarchically at the top of the apoptotic cascade during pupal eye development (Mendes, 2006).

To further explore the role of cyt c-d in the regulation of caspase activation, the elimination of sensory organs (macrochaetes) was used as a model. A recent study proposed that caspase activation does not lead to apoptosis but inhibits the Wingless pathway to ensure the correct number of sensory organ precursors (SOPs). Consistently, loss-of-function mutations in ark or dronc lead to the appearance of extra bristles on the Drosophila notum. To determine the role of cyt c-d during SOP development, the number of posterior scutellar bristles on the thorax of cyt c-d mutant flies was counted. In all the cyt c-d mutant alleles examined, a significant number of flies was found that had one extra bristle. Using a recently characterized allele of ark (ark^N5), an extra bristle cell phenotype was observed. As for the extra IOCs, ark has a more pronounced phenotype than cyt c-d mutants, suggesting that similar mechanisms lead to caspase activation in the two models. Together, these results provide further support that cyt c-d promotes caspase activation required for accurate developmental progression (Mendes, 2006).

Extra bristles were identified in flies mutant for the executioner caspase dcp 1 (dcp-1^prev1), for which no role in the regulation of bristle cell number has yet been reported. Interestingly, no extra bristle phenotype was found in drICE mutant (drICE¹⁷), suggesting that dcp 1 could be the main executioner caspase in this model (Mendes, 2006).

In most tissues, with the exception of developing testis, SOP and retina, cyt c-d has no apparent role in caspase activation or apoptosis, suggesting that apoptosis can occur in the absence of this protein. The existence of a Cyt-c-independent pathway for apoptosis in Drosophila was previously proposed on the basis of RNA interference studies in Drosophila cell lines or a cell-free system, showing that apoptosis can occur independent of Cyt c function but requires Ark (Zimmermann, 2002; Dorstyn, 2004; Means, 2005). Therefore, at least in some models, Ark-dependent caspase activation might be either constitutive or regulated by other pathways. In support of the latter, ectopic expression of Ark is not sufficient to trigger apoptosis in vivo, suggesting that Ark must be activated to function (Akdemir, 2006). Likewise, analysis of mice devoid of Cyt c apoptogenic function (K72A) indicates that caspase activation in thymocytes can occur independently of Cyt c (Hao, 2005). Mammalian Apaf 1 might either have some constitutive activity or might be regulated by factors other than Cyt c (Mendes, 2006).

Conversely, the results indicate that a Cyt-c-dependent mechanism for apoptosis in the retina might be necessary for the rapid removal of a precise number of cells during development. An even stricter requirement is observed during sperm or SOP development, in which imbalanced caspase activation or loss of cyt c-d function leads to male sterility and extra bristle cells, respectively (Arama, 2006). The results indicate that Cyt c is able to promote the activation of Ark to form an apoptosome that leads to Dronc activation and cell death. In support of this hypothesis, Dronc is recruited into a >700 kDa complex in Drosophila cell extracts supplemented with Cyt c and dATP (Dorstyn, 2002), similar to the mammalian apoptosome. In addition, Ark interacts with Cyt c, an interaction dependent on the WD40 domain of Ark. However, recent structural data suggest that Ark does not require Cyt c to form an apoptosome-like structure (Yu, 2006). Although that study used horse and not Drosophila Cyt c for the apoptosome assembly, Drosophila apoptosome formation might not require Cyt c. If so, it raises a question on the inhibitory function of the WD40 domain of Ark. The WD40 is conserved between vertebrates and Drosophila but not Caenorhabditis elegans, in which it is thought to maintain Apaf 1 in an inactive conformation that is relieved on Cyt c binding. How Ark activation in vivo is dependent on Cyt c awaits further analysis (Mendes, 2006).

EVOLUTIONARY HOMOLOGS

cytochrome c and respiration

Kimball's Biology Pages

A Biophysical Model of the Mitochondrial Respiratory System and Oxidative Phosphorylation

Pathways: Mitochondrial

Electron Transport and Cellular Respiration

Apaf-1 interaction with cytochrome c

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).

Ordering the cytochrome c-initiated caspase cascade downstream of cytochrome c

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).

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome

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).

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

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).

Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c

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).

Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90

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).

Testis-specific cytochrome c-null mice produce functional sperm but undergo early testicular atrophy

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).

Role of cytochrome c in apoptosis: Increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of cytochrome c release

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).

Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial Cytochrome c release

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 BIM_EL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and this is reduced by dominant-negative c-Jun. Overexpression of BI_MEL 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).

Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation

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).

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

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).

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

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).

Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent APAF-1

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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date revised: 22 February 2022

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