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Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

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Caspase-3 does not destroy the capacity of complex III to reduce cytochrome c. Isolated mitochondria (500 μg) were incubated in the presence of tBid (25 μg/ml), caspase-3 (25 μg/ml), and/or antimycin A (1 μM) as indicated for 60 min at 37°C. As a control, 500 μg of mitochondria were incubated in water for 20 min at 4°C then incubated ± caspase-3. Reduction of exogenous cytochrome c by complex III was measured as described in Materials and methods.
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fig4: Caspase-3 does not destroy the capacity of complex III to reduce cytochrome c. Isolated mitochondria (500 μg) were incubated in the presence of tBid (25 μg/ml), caspase-3 (25 μg/ml), and/or antimycin A (1 μM) as indicated for 60 min at 37°C. As a control, 500 μg of mitochondria were incubated in water for 20 min at 4°C then incubated ± caspase-3. Reduction of exogenous cytochrome c by complex III was measured as described in Materials and methods.

Mentions: Although we observed intact complex IV activity after caspase treatment of mitochondria, the reduction in oxygen consumption in response to substrates for complex I or II might nevertheless be due to a loss of function of complex III (Fig. 3 A). Therefore, to assess complex III function we examined the ability of accessible complex III to reduce cytochrome c (Kluck et al., 1999). As shown in Fig. 4, intact mitochondria did not reduce exogenously added cytochrome c, whereas tBid-treated mitochondria did (Kluck et al., 1999). This effect was dependent on complex III activity, since the inhibitor antimycin A blocked cytochrome c reduction in this system. Permeabilized mitochondria (by tBid or by hypotonic lysis) treated with caspase-3 displayed full complex III activity in this assay, and thus the function of complex III (at least that of cytochrome c reduction) was not damaged by caspase-3. These results support the idea that caspases damage the function of complexes I and II without affecting those of complex III or complex IV.


Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

Caspase-3 does not destroy the capacity of complex III to reduce cytochrome c. Isolated mitochondria (500 μg) were incubated in the presence of tBid (25 μg/ml), caspase-3 (25 μg/ml), and/or antimycin A (1 μM) as indicated for 60 min at 37°C. As a control, 500 μg of mitochondria were incubated in water for 20 min at 4°C then incubated ± caspase-3. Reduction of exogenous cytochrome c by complex III was measured as described in Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172744&req=5

fig4: Caspase-3 does not destroy the capacity of complex III to reduce cytochrome c. Isolated mitochondria (500 μg) were incubated in the presence of tBid (25 μg/ml), caspase-3 (25 μg/ml), and/or antimycin A (1 μM) as indicated for 60 min at 37°C. As a control, 500 μg of mitochondria were incubated in water for 20 min at 4°C then incubated ± caspase-3. Reduction of exogenous cytochrome c by complex III was measured as described in Materials and methods.
Mentions: Although we observed intact complex IV activity after caspase treatment of mitochondria, the reduction in oxygen consumption in response to substrates for complex I or II might nevertheless be due to a loss of function of complex III (Fig. 3 A). Therefore, to assess complex III function we examined the ability of accessible complex III to reduce cytochrome c (Kluck et al., 1999). As shown in Fig. 4, intact mitochondria did not reduce exogenously added cytochrome c, whereas tBid-treated mitochondria did (Kluck et al., 1999). This effect was dependent on complex III activity, since the inhibitor antimycin A blocked cytochrome c reduction in this system. Permeabilized mitochondria (by tBid or by hypotonic lysis) treated with caspase-3 displayed full complex III activity in this assay, and thus the function of complex III (at least that of cytochrome c reduction) was not damaged by caspase-3. These results support the idea that caspases damage the function of complexes I and II without affecting those of complex III or complex IV.

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

Show MeSH
Related in: MedlinePlus