Ordering the Cytochrome c-initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9-dependent Manner methods
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In Vitro Association Assays
The ability of caspases-1, -2, -3, -6, -7, -8, -9, and -10 to interact with GST-Apaf-1 fusion proteins was assessed as follows. [ 35 S]Methionine-labeled caspases (5-15-µl aliquots of translation reactions) were brought to 200 µl in GST buffer (50 mM Tris, pH 7.6, 120 mM NaCl, 0.1% CHAPS, 100 µM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin). 2-µl aliquots (∼6 µg protein) of glutathione Sepharose-immobilized GST or GST-Apaf-1 fusion proteins were then added, followed by incubation for 2 h at 4°C under constant rotation. Bead complexes were then washed several times in GST buffer and bound caspases were detected by SDS-PAGE/fluorography.
Preparation of Cell-free Extracts
Cell-free extracts were generated from Jurkat T lymphoblastoid cells or MCF-7 cells as previously described (, ), with the following modifications. Cells (2-5 × 10 8 ) were pelleted and washed twice with PBS, pH 7.2, followed by a single wash with 5 ml of ice-cold cell extract buffer (CEB; 1 20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 µM PMSF, 10 µg/ml leupeptin, 2 µg/ml aprotinin). Cells were then transferred to a 2-ml Dounce-type homogenizer, were pelleted, and two volumes of ice-cold CEB was added to the volume of the packed cell pellet. Cells were allowed to swell under the hypotonic conditions for 15 min on ice. Cells were then disrupted with 20 strokes of a B-type pestle. Lysis was confirmed by examination of a small aliquot of the suspension under a light microscope. Lysates were then transferred...
Cell-free Reactions
Cell-free reactions were typically set up in 10- or 100-µl reaction volumes. For 100-µl scale reactions, 50 µl of cell extract (∼5 mg/ml) and 10 µl of rat liver nuclei were brought to a final volume of 100 µl in CEB, with or without peptides or proteins solubilized in the same buffer. Apoptosis was typically induced by addition of bovine heart cytochrome c to extracts at a final concentration of 50 µg/ml. Where necessary, dATP was also to a final concentration of 1 mM, although many extracts did not require addition of this nucleotide triphosphate. To initiate apoptosis, extracts were incubated at 37°C for periods of up to 3 h. At time points indicated in the text, 2-µl aliquots were removed for determination of percentages of apoptotic nuclei using Hoechst 33342 staining, as previously described (, ). Samples of extract (10-20 _...
YVAD-pNA and DEVD-pNA Cleavage Assay
At times indicated in the text, 10-µl aliquots of cell-free reactions were removed and were diluted to 100 µl by the addition of ice-cold protease reaction buffer (PRB; 50 mM Hepes, pH 7.4, 75 mM NaCl, 0.1% CHAPS, 2 mM dithiothreitol). Samples were held on ice until completion of the experiment and were then divided into two separate 50-µl portions for the separate assessment of YVAD- p -nitroanalide (YVAD-pNA) and DEVD-pNA cleavage activity, respectively. To each 50-µl aliquot, 5.5 µl of a 10 × stock of each peptide (500 µM) was added such that the final concentration of either peptide in the reaction was 50 µM. Reactions were then incubated for 30 min at 37°C, followed by addition of 950 µl ice-cold dH 2 O to stop the reaction. OD 400 readings of each sample were then taken against a blank containing buffer and peptide alone (i.e., no...
SDS-PAGE and Western Blot Analysis
Proteins were subjected to standard SDS-PAGE at 60-70 V and were transferred onto 0.45 µM PVDF membranes (Bio-Rad) for 3 h at 50-75 mA, followed by probing for various proteins using the polyclonal antibodies described under materials. Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies ( Amersham ), followed by detection using the Supersignal chemiluminescence system (Pierce), all as previously described ( ).
Cytochrome c-initiated Apoptosis Is Associated with Proteolytic Processing of Caspase-3, but Not Caspase-1
Previous studies have shown that caspases-3 and -9 are activated in response to cytochrome c (;;;; ). We initially confirmed these observations before assessing the activation of other caspases in this context. Fig. demonstrates that caspase-3 endogenous to Jurkat cell extracts was rapidly converted from the 36-kD proenzyme to the p17/p12 mature form in the presence of cytochrome c. Processing occurred in a two-step manner, with the initial appearance of a p24/p12 intermediate in the extracts, followed by accumulation of the mature p17/ p12 form of the enzyme (Fig., A and C), reminiscent of the mechanism of activation of caspase-3 in response to granzyme B ( ). This was further confirmed by addition of [ 35 S]methionine-labeled caspase-3 to the extracts, which enabled detection of the caspase-3-p12 chain that was not recognized by the anti-caspase-3 polyclonal antibody used...
Cytochrome c Initiates a Cascade of Protease Activation Events Involving Caspases-2, -3, -6, -7, -8, -9, and -10
To explore the range of cytochrome c-inducible caspase activation events in more detail, we monitored the kinetics of activation of all caspases relative to each other in this system. Fig. shows that detectable activation of most caspases, with the exceptions of caspases-8 and -10, appeared to occur contemporaneously, typically within 30 min of addition of cytochrome c to the extracts. In contrast, processing of caspases-8 and -10 were noticeably delayed relative to the other caspases, suggesting that these caspases might be activated late in this pathway.
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The ability of caspases-1, -2, -3, -6, -7, -8, -9, and -10 to interact with GST-Apaf-1 fusion proteins was assessed as follows. [ 35 S]Methionine-labeled caspases (5-15-µl aliquots of translation reactions) were brought to 200 µl in GST buffer (50 mM Tris, pH 7.6, 120 mM NaCl, 0.1% CHAPS, 100 µM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin). 2-µl aliquots (∼6 µg protein) of glutathione Sepharose-immobilized GST or GST-Apaf-1 fusion proteins were then added, followed by incubation for 2 h at 4°C under constant rotation. Bead complexes were then washed several times in GST buffer and bound caspases were detected by SDS-PAGE/fluorography.
Cell-free extracts were generated from Jurkat T lymphoblastoid cells or MCF-7 cells as previously described (, ), with the following modifications. Cells (2-5 × 10 8 ) were pelleted and washed twice with PBS, pH 7.2, followed by a single wash with 5 ml of ice-cold cell extract buffer (CEB; 1 20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 µM PMSF, 10 µg/ml leupeptin, 2 µg/ml aprotinin). Cells were then transferred to a 2-ml Dounce-type homogenizer, were pelleted, and two volumes of ice-cold CEB was added to the volume of the packed cell pellet. Cells were allowed to swell under the hypotonic conditions for 15 min on ice. Cells were then disrupted with 20 strokes of a B-type pestle. Lysis was confirmed by examination of a small aliquot of the suspension under a light microscope. Lysates were then transferred to Eppendorf tubes and were centrifuged at 15,000 g for 15 min at 4°C (S15 or postnuclear extracts). The supernatant was removed while taking care to avoid the pellet. Supernatants were then frozen in aliquots at -70°C until required.
Cell-free reactions were typically set up in 10- or 100-µl reaction volumes. For 100-µl scale reactions, 50 µl of cell extract (∼5 mg/ml) and 10 µl of rat liver nuclei were brought to a final volume of 100 µl in CEB, with or without peptides or proteins solubilized in the same buffer. Apoptosis was typically induced by addition of bovine heart cytochrome c to extracts at a final concentration of 50 µg/ml. Where necessary, dATP was also to a final concentration of 1 mM, although many extracts did not require addition of this nucleotide triphosphate. To initiate apoptosis, extracts were incubated at 37°C for periods of up to 3 h. At time points indicated in the text, 2-µl aliquots were removed for determination of percentages of apoptotic nuclei using Hoechst 33342 staining, as previously described (, ). Samples of extract (10-20 µl) were also removed at times indicated in the text and frozen at -70°C for subsequent SDS-PAGE/Western blot or fluorographic determination of substrate cleavage profiles or caspase activation.
At times indicated in the text, 10-µl aliquots of cell-free reactions were removed and were diluted to 100 µl by the addition of ice-cold protease reaction buffer (PRB; 50 mM Hepes, pH 7.4, 75 mM NaCl, 0.1% CHAPS, 2 mM dithiothreitol). Samples were held on ice until completion of the experiment and were then divided into two separate 50-µl portions for the separate assessment of YVAD- p -nitroanalide (YVAD-pNA) and DEVD-pNA cleavage activity, respectively. To each 50-µl aliquot, 5.5 µl of a 10 × stock of each peptide (500 µM) was added such that the final concentration of either peptide in the reaction was 50 µM. Reactions were then incubated for 30 min at 37°C, followed by addition of 950 µl ice-cold dH 2 O to stop the reaction. OD 400 readings of each sample were then taken against a blank containing buffer and peptide alone (i.e., no extract).
Proteins were subjected to standard SDS-PAGE at 60-70 V and were transferred onto 0.45 µM PVDF membranes (Bio-Rad) for 3 h at 50-75 mA, followed by probing for various proteins using the polyclonal antibodies described under materials. Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies ( Amersham ), followed by detection using the Supersignal chemiluminescence system (Pierce), all as previously described ( ).
Previous studies have shown that caspases-3 and -9 are activated in response to cytochrome c (;;;; ). We initially confirmed these observations before assessing the activation of other caspases in this context. Fig. demonstrates that caspase-3 endogenous to Jurkat cell extracts was rapidly converted from the 36-kD proenzyme to the p17/p12 mature form in the presence of cytochrome c. Processing occurred in a two-step manner, with the initial appearance of a p24/p12 intermediate in the extracts, followed by accumulation of the mature p17/ p12 form of the enzyme (Fig., A and C), reminiscent of the mechanism of activation of caspase-3 in response to granzyme B ( ). This was further confirmed by addition of [ 35 S]methionine-labeled caspase-3 to the extracts, which enabled detection of the caspase-3-p12 chain that was not recognized by the anti-caspase-3 polyclonal antibody used (Fig. B). In direct contrast, conversion of caspase-1 (ICE) to its mature form was not detected in the same extracts over an identical time course (Fig. A). To further confirm that processed caspases were active, we used synthetic tetrapeptide substrates that are preferentially cleaved by caspase-1...
To explore the range of cytochrome c-inducible caspase activation events in more detail, we monitored the kinetics of activation of all caspases relative to each other in this system. Fig. shows that detectable activation of most caspases, with the exceptions of caspases-8 and -10, appeared to occur contemporaneously, typically within 30 min of addition of cytochrome c to the extracts. In contrast, processing of caspases-8 and -10 were noticeably delayed relative to the other caspases, suggesting that these caspases might be activated late in this pathway.
Machine-readable layer
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