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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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Differential inhibition of cytochrome c–initiated processing of caspases-2, -3, -6, -7, -8, and -10 by DEVD-CHO. The  indicated [35S]methionine-labeled caspases were incubated in Jurkat cell extracts in the presence or absence of 10 μg/ml bovine  heart cytochrome c and the indicated concentrations of peptides  or recombinant proteins. Peptides and recombinant proteins  were added to the extracts 30 min before the addition of cytochrome c. After incubation at 37°C for 3 h, reactions were  stopped and caspase processing was subsequently assessed by  SDS-PAGE/fluorography.
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Figure 8: Differential inhibition of cytochrome c–initiated processing of caspases-2, -3, -6, -7, -8, and -10 by DEVD-CHO. The indicated [35S]methionine-labeled caspases were incubated in Jurkat cell extracts in the presence or absence of 10 μg/ml bovine heart cytochrome c and the indicated concentrations of peptides or recombinant proteins. Peptides and recombinant proteins were added to the extracts 30 min before the addition of cytochrome c. After incubation at 37°C for 3 h, reactions were stopped and caspase processing was subsequently assessed by SDS-PAGE/fluorography.

Mentions: As a preliminary approach to ordering the sequence of caspase activation events triggered by cytochrome c, we investigated the effects of the tetrapeptide caspase inhibitors YVAD-CHO and DEVD-CHO, as well as the cowpox virus–derived caspase inhibitor CrmA, on the processing of all caspases downstream of caspase-9. The caspase-1–selective inhibitor YVAD-CHO had similar effects on all caspases, exhibiting little inhibition of caspase activation except at the highest concentration tested (Fig. 8). These data are consistent with YVAD-CHO directly inhibiting caspase-9 at high concentrations and terminating activation of all downstream caspases, in agreement with the results obtained by depletion of caspase-9 from the extracts (Fig. 7). Broadly similar effects on all caspases was also observed using GST-CrmA, with the exception that activation of caspases-8 and -10 were blocked at all concentrations of this inhibitor, whereas processing of all of the other caspases was seen at the lowest concentration tested (0.2 μM). This suggests that caspase-8 processing is downstream of the other caspases in the context of cytochrome c.


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)

Differential inhibition of cytochrome c–initiated processing of caspases-2, -3, -6, -7, -8, and -10 by DEVD-CHO. The  indicated [35S]methionine-labeled caspases were incubated in Jurkat cell extracts in the presence or absence of 10 μg/ml bovine  heart cytochrome c and the indicated concentrations of peptides  or recombinant proteins. Peptides and recombinant proteins  were added to the extracts 30 min before the addition of cytochrome c. After incubation at 37°C for 3 h, reactions were  stopped and caspase processing was subsequently assessed by  SDS-PAGE/fluorography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132895&req=5

Figure 8: Differential inhibition of cytochrome c–initiated processing of caspases-2, -3, -6, -7, -8, and -10 by DEVD-CHO. The indicated [35S]methionine-labeled caspases were incubated in Jurkat cell extracts in the presence or absence of 10 μg/ml bovine heart cytochrome c and the indicated concentrations of peptides or recombinant proteins. Peptides and recombinant proteins were added to the extracts 30 min before the addition of cytochrome c. After incubation at 37°C for 3 h, reactions were stopped and caspase processing was subsequently assessed by SDS-PAGE/fluorography.
Mentions: As a preliminary approach to ordering the sequence of caspase activation events triggered by cytochrome c, we investigated the effects of the tetrapeptide caspase inhibitors YVAD-CHO and DEVD-CHO, as well as the cowpox virus–derived caspase inhibitor CrmA, on the processing of all caspases downstream of caspase-9. The caspase-1–selective inhibitor YVAD-CHO had similar effects on all caspases, exhibiting little inhibition of caspase activation except at the highest concentration tested (Fig. 8). These data are consistent with YVAD-CHO directly inhibiting caspase-9 at high concentrations and terminating activation of all downstream caspases, in agreement with the results obtained by depletion of caspase-9 from the extracts (Fig. 7). Broadly similar effects on all caspases was also observed using GST-CrmA, with the exception that activation of caspases-8 and -10 were blocked at all concentrations of this inhibitor, whereas processing of all of the other caspases was seen at the lowest concentration tested (0.2 μM). This suggests that caspase-8 processing is downstream of the other caspases in the context of cytochrome c.

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