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

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
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. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

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Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc-His6), DC3His6, and DC4His6. Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3His6, DC4His6, and hcytc-His6. White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3His6, DC4 His6, or hcytcHis6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
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fig4: Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc-His6), DC3His6, and DC4His6. Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3His6, DC4His6, and hcytc-His6. White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3His6, DC4 His6, or hcytcHis6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.

Mentions: We set out to test whether DC3 or DC4 can induce caspase activation in a cell-free system. Recombinant DC3 and DC4 were generated by coexpressing these proteins with haem lyase in E. coli. The absorbance spectrum of the purified recombinant DC3 and DC4 proteins showed peaks at 419, 520, and 549 nm, similar to the spectrum shared by human cytochrome c (Fig. 4 A). The identity of these purified proteins was confirmed by immunoblotting with cytochrome c antibody (Fig. 4 B). We added recombinant DC3 or DC4 to S100 extracts from Drosophila BG2 or mammalian 293T cells that do not contain endogenous cytochrome c (Fig. 4 C). We found that the addition of DC3 and DC4, in the presence of dATP, did not significantly increase caspase activity in Drosophila BG2 cells (Fig. 4 D). Interestingly, if we used apoptotic extracts from BG2 cells we detected a small but significant increase in caspase activity after the addition of either Drosophila or human cytochrome c (unpublished data). When apoptotic extracts from cells subjected to dc3 and dc4 RNAi were used, the increase in caspase activity was still evident upon addition of recombinant cytochrome c proteins (unpublished data). These results indicate that additional apoptotic factors may augment caspase activation in the presence of cytochrome c. In contrast, we could clearly see that both DC3 and DC4 induced caspase activity in mammalian 293T extracts (Fig. 4 D). This activity is comparable to the increase in caspase activity seen when purified human cytochrome c is added. Interestingly, human cytochrome c was unable to induce caspase activity in BG2 cell extracts. These data indicate that whereas cytochrome c is necessary for caspase activation in mammalian cells, it does not directly activate caspases in Drosophila cells.


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

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc-His6), DC3His6, and DC4His6. Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3His6, DC4His6, and hcytc-His6. White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3His6, DC4 His6, or hcytcHis6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
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Related In: Results  -  Collection

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fig4: Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc-His6), DC3His6, and DC4His6. Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3His6, DC4His6, and hcytc-His6. White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3His6, DC4 His6, or hcytcHis6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
Mentions: We set out to test whether DC3 or DC4 can induce caspase activation in a cell-free system. Recombinant DC3 and DC4 were generated by coexpressing these proteins with haem lyase in E. coli. The absorbance spectrum of the purified recombinant DC3 and DC4 proteins showed peaks at 419, 520, and 549 nm, similar to the spectrum shared by human cytochrome c (Fig. 4 A). The identity of these purified proteins was confirmed by immunoblotting with cytochrome c antibody (Fig. 4 B). We added recombinant DC3 or DC4 to S100 extracts from Drosophila BG2 or mammalian 293T cells that do not contain endogenous cytochrome c (Fig. 4 C). We found that the addition of DC3 and DC4, in the presence of dATP, did not significantly increase caspase activity in Drosophila BG2 cells (Fig. 4 D). Interestingly, if we used apoptotic extracts from BG2 cells we detected a small but significant increase in caspase activity after the addition of either Drosophila or human cytochrome c (unpublished data). When apoptotic extracts from cells subjected to dc3 and dc4 RNAi were used, the increase in caspase activity was still evident upon addition of recombinant cytochrome c proteins (unpublished data). These results indicate that additional apoptotic factors may augment caspase activation in the presence of cytochrome c. In contrast, we could clearly see that both DC3 and DC4 induced caspase activity in mammalian 293T extracts (Fig. 4 D). This activity is comparable to the increase in caspase activity seen when purified human cytochrome c is added. Interestingly, human cytochrome c was unable to induce caspase activity in BG2 cell extracts. These data indicate that whereas cytochrome c is necessary for caspase activation in mammalian cells, it does not directly activate caspases in Drosophila cells.

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
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. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

Show MeSH
Related in: MedlinePlus