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Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Bottom Line: Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions.Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.

ABSTRACT
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 homologue, 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. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated 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 enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal 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.

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Titration of cytochrome c–initiated apoptosis in Jurkat  cell extracts. (A) Rat liver nuclei were incubated in postnuclear  extracts from Jurkat cells (75 μg protein in 20 μl) for 2 h at 37°C  in the presence or absence of the indicated amounts of bovine  heart cytochrome c. As a control, nuclei were also incubated in  the highest concentration of cytochrome c in the absence of cell  extract (CEB was substituted). Apoptosis was assessed by scoring nuclei with features of apoptosis, as described in Fig. 1. Results shown are from a representative experiment and were derived from counts performed on 300 nuclei from several fields.  (B) Cell-free reactions, set up identically to those in A, were incubated for 2 h at 37°C followed by analysis by SDS-PAGE and  Western blotting. Blots were then probed with antibodies directed against the indicated caspases and caspase substrates.
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Figure 3: Titration of cytochrome c–initiated apoptosis in Jurkat cell extracts. (A) Rat liver nuclei were incubated in postnuclear extracts from Jurkat cells (75 μg protein in 20 μl) for 2 h at 37°C in the presence or absence of the indicated amounts of bovine heart cytochrome c. As a control, nuclei were also incubated in the highest concentration of cytochrome c in the absence of cell extract (CEB was substituted). Apoptosis was assessed by scoring nuclei with features of apoptosis, as described in Fig. 1. Results shown are from a representative experiment and were derived from counts performed on 300 nuclei from several fields. (B) Cell-free reactions, set up identically to those in A, were incubated for 2 h at 37°C followed by analysis by SDS-PAGE and Western blotting. Blots were then probed with antibodies directed against the indicated caspases and caspase substrates.

Mentions: Cytochrome c induced apoptotic changes in nuclei added to the extracts at concentrations of as little as 1–5 μg/ml, but had no direct effects on nuclei in the absence of cell extract (Fig. 3 A). Interestingly, at cytochrome c concentrations where only partial caspase-3 activation was observed (5 μg/ml), substrates such as fodrin, PARP, and U1snRNP were almost completely cleaved (Fig. 3 B), suggesting that only a small amount of the total caspase-3 pool is required in order to effect complete proteolysis of these substrates. An alternative explanation is that cytochrome c activates other caspases in the extracts that are capable of cleaving these substrates. Cytochrome c failed to trigger caspase-1 processing at any of the concentrations tested (Fig. 3 B).


Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Titration of cytochrome c–initiated apoptosis in Jurkat  cell extracts. (A) Rat liver nuclei were incubated in postnuclear  extracts from Jurkat cells (75 μg protein in 20 μl) for 2 h at 37°C  in the presence or absence of the indicated amounts of bovine  heart cytochrome c. As a control, nuclei were also incubated in  the highest concentration of cytochrome c in the absence of cell  extract (CEB was substituted). Apoptosis was assessed by scoring nuclei with features of apoptosis, as described in Fig. 1. Results shown are from a representative experiment and were derived from counts performed on 300 nuclei from several fields.  (B) Cell-free reactions, set up identically to those in A, were incubated for 2 h at 37°C followed by analysis by SDS-PAGE and  Western blotting. Blots were then probed with antibodies directed against the indicated caspases and caspase substrates.
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Related In: Results  -  Collection

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Figure 3: Titration of cytochrome c–initiated apoptosis in Jurkat cell extracts. (A) Rat liver nuclei were incubated in postnuclear extracts from Jurkat cells (75 μg protein in 20 μl) for 2 h at 37°C in the presence or absence of the indicated amounts of bovine heart cytochrome c. As a control, nuclei were also incubated in the highest concentration of cytochrome c in the absence of cell extract (CEB was substituted). Apoptosis was assessed by scoring nuclei with features of apoptosis, as described in Fig. 1. Results shown are from a representative experiment and were derived from counts performed on 300 nuclei from several fields. (B) Cell-free reactions, set up identically to those in A, were incubated for 2 h at 37°C followed by analysis by SDS-PAGE and Western blotting. Blots were then probed with antibodies directed against the indicated caspases and caspase substrates.
Mentions: Cytochrome c induced apoptotic changes in nuclei added to the extracts at concentrations of as little as 1–5 μg/ml, but had no direct effects on nuclei in the absence of cell extract (Fig. 3 A). Interestingly, at cytochrome c concentrations where only partial caspase-3 activation was observed (5 μg/ml), substrates such as fodrin, PARP, and U1snRNP were almost completely cleaved (Fig. 3 B), suggesting that only a small amount of the total caspase-3 pool is required in order to effect complete proteolysis of these substrates. An alternative explanation is that cytochrome c activates other caspases in the extracts that are capable of cleaving these substrates. Cytochrome c failed to trigger caspase-1 processing at any of the concentrations tested (Fig. 3 B).

Bottom Line: Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions.Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.

ABSTRACT
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 homologue, 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. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated 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 enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal 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.

Show MeSH
Related in: MedlinePlus