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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 induces loss of ΔΨm in isolated, tBid-treated mitochondria. Mouse liver mitochondria (20 μg) were incubated with FCCP (10 μM), tBid (20 μg/ml), cytochrome c (100 μM), caspase-3 (0.25 μg/ml), and/or zVAD-fmk (100 μM) as indicated, then stained with TMRE, and analyzed by flow cytometry. Low fluorescence indicates a loss of ΔΨm.
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fig2: Caspase-3 induces loss of ΔΨm in isolated, tBid-treated mitochondria. Mouse liver mitochondria (20 μg) were incubated with FCCP (10 μM), tBid (20 μg/ml), cytochrome c (100 μM), caspase-3 (0.25 μg/ml), and/or zVAD-fmk (100 μM) as indicated, then stained with TMRE, and analyzed by flow cytometry. Low fluorescence indicates a loss of ΔΨm.

Mentions: During apoptosis, the mitochondrial outer membrane becomes permeable due to the action of pro-apoptotic Bcl-2 family proteins. Using isolated mitochondria, we asked whether such outer membrane permeabilization, with or without caspase activity, is sufficient to account for the loss of ΔΨm in mitochondria during apoptosis. ΔΨm was examined by uptake of TMRE (Fig. 2). The uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), caused a dissipation of ΔΨm as expected. To induce outer membrane permeabilization as it occurs during apoptosis, we treated the mitochondria with the activated BH3-only protein, truncated Bid (tBid) (caspase-free; see Materials and methods), which caused a rapid release of cytochrome c (Fig. 3 B, inset) but did not disrupt ΔΨm (Fig. 2). This effect required that the mitochondria be maintained at high density (as in the experiment shown), since dilution of the treated organelles caused a loss of ΔΨm as the cytochrome c is diluted (Waterhouse et al., 2001b). Although apoptosis in most cases does not depend on the action of tBid (Yin et al., 1999), we employed this protein as a model, since it is likely that the mitochondrial permeabilization induced by tBid is similar to that occurring during many forms of apoptosis (Korsmeyer et al., 2000). Treatment of the isolated mitochondria with caspase-3 also failed to affect ΔΨm (Fig. 2). However, addition of both tBid and caspase-3 caused a loss of ΔΨm, and this effect was dependent on the caspase activity as zVAD-fmk inhibited the effect. Since tBid induces the permeabilization of the mitochondrial outer membrane (Kuwana et al., 2002), it is therefore likely that it acts to permit caspase-3 access to the intermembrane space, necessary for the protease to affect ΔΨm. Although the mechanism is unknown, tBid can cause the outer mitochondrial membrane to become permeable to dextrans of higher molecular weight than that of active caspase-3 (Kuwana et al., 2002).


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 induces loss of ΔΨm in isolated, tBid-treated mitochondria. Mouse liver mitochondria (20 μg) were incubated with FCCP (10 μM), tBid (20 μg/ml), cytochrome c (100 μM), caspase-3 (0.25 μg/ml), and/or zVAD-fmk (100 μM) as indicated, then stained with TMRE, and analyzed by flow cytometry. Low fluorescence indicates a loss of ΔΨm.
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Related In: Results  -  Collection

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

fig2: Caspase-3 induces loss of ΔΨm in isolated, tBid-treated mitochondria. Mouse liver mitochondria (20 μg) were incubated with FCCP (10 μM), tBid (20 μg/ml), cytochrome c (100 μM), caspase-3 (0.25 μg/ml), and/or zVAD-fmk (100 μM) as indicated, then stained with TMRE, and analyzed by flow cytometry. Low fluorescence indicates a loss of ΔΨm.
Mentions: During apoptosis, the mitochondrial outer membrane becomes permeable due to the action of pro-apoptotic Bcl-2 family proteins. Using isolated mitochondria, we asked whether such outer membrane permeabilization, with or without caspase activity, is sufficient to account for the loss of ΔΨm in mitochondria during apoptosis. ΔΨm was examined by uptake of TMRE (Fig. 2). The uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), caused a dissipation of ΔΨm as expected. To induce outer membrane permeabilization as it occurs during apoptosis, we treated the mitochondria with the activated BH3-only protein, truncated Bid (tBid) (caspase-free; see Materials and methods), which caused a rapid release of cytochrome c (Fig. 3 B, inset) but did not disrupt ΔΨm (Fig. 2). This effect required that the mitochondria be maintained at high density (as in the experiment shown), since dilution of the treated organelles caused a loss of ΔΨm as the cytochrome c is diluted (Waterhouse et al., 2001b). Although apoptosis in most cases does not depend on the action of tBid (Yin et al., 1999), we employed this protein as a model, since it is likely that the mitochondrial permeabilization induced by tBid is similar to that occurring during many forms of apoptosis (Korsmeyer et al., 2000). Treatment of the isolated mitochondria with caspase-3 also failed to affect ΔΨm (Fig. 2). However, addition of both tBid and caspase-3 caused a loss of ΔΨm, and this effect was dependent on the caspase activity as zVAD-fmk inhibited the effect. Since tBid induces the permeabilization of the mitochondrial outer membrane (Kuwana et al., 2002), it is therefore likely that it acts to permit caspase-3 access to the intermembrane space, necessary for the protease to affect ΔΨm. Although the mechanism is unknown, tBid can cause the outer mitochondrial membrane to become permeable to dextrans of higher molecular weight than that of active caspase-3 (Kuwana et al., 2002).

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