The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation

Elaine Beem,1 L. Shannon Holliday,2 and Mark S. Segal1

1Division of Nephrology, Hypertension and Transplantation, Department of Medicine; and 2College of Dentistry, University of Florida, Gainesville, Florida 32610

Submitted 3 June 2003 ; accepted in final form 27 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we demonstrated that both 150 mM KCl and alkaline pH inhibit cytochrome c-mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition, we analyzed the effect of KCl and alkaline pH on the formation of apoptosomes (a large complex consisting of cytochrome c, Apaf-1, and procaspase-9/caspase-9) in vitro. Our results suggest that an initial ~700-kDa apoptosome matures through a 1.4-MDa intermediate before a ~700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial ~700-kDa apoptosome to the 1.4-MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4-MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggests that the 1.4-MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggest that blocking acidification during the induction of apoptosis inhibits activation of procaspase-3. On the basis of these results, we propose a model of apoptosome maturation.

caspase; pH; potassium; apoptosis


THE PROCESS OF APOPTOSIS, or programmed cell death, distinct from necrosis, is characterized by a series of cellular events, such as cell shrinkage, phosphatidylserine externalization (11), membrane blebbing (25), and DNA fragmentation (30). Some of these changes are a direct result of enzymes, collectively termed caspases, which are constitutively produced, highly conserved, aspartic acid-specific cysteine proteases (10). Caspases, normally present in cells in the form of inactive zymogens, are activated by autoproteolysis or cleavage by other caspases. A number of independent pathways have been implicated in the activation of different caspases with a variety of triggering mechanisms involving an assortment of accessory proteins.

Of the many stimuli that induce apoptosis, the majority of them act by releasing cytochrome c from the inner mitochondrial matrix (5, 6, 14, 16, 19, 20, 28). Cytochrome c mediates the assembly of apoptosis protease activating factor-1 (Apaf-1) and procaspase-9 into an active apoptosome complex that leads to the activation of procaspase-3 and dismantling of the cell. Recently, the structure of cytochrome c, dATP, and Apaf-1 was resolved to 27 Å and yielded an Apaf-1 heptamer in a seven-spoked wheel conformation (1). It is thought that the sequential binding of cytochrome c and dATP/ATP to Apaf-1 mediates Apaf-1 oligomerization and leads to the recruitment of the zymogen procaspase-9 (1). The binding of procaspase-9 to the scaffold results in its activation through autocatalysis and sequential activation of its downstream substrate procaspase-3 to form caspase-3, an effector protease that triggers more widespread proteolysis and cell death.

Apaf-1 and procaspase-9 are both counterparts of proteins in the Caenorhabditis elegans apoptotic pathway (CED4 and CED3, respectively) with no ascribed function other than apoptosis. The protein components of the apoptosome are all constitutively produced, the activating nucleotides dATP/ATP are plentiful in the cytosol, and it has been suggested by some that holocytochrome c accidentally released by mitochondrial turnover may be able to initiate the formation of apoptosomes (22). Therefore, the factors necessary for apoptosis may be present in the cytoplasm, yet apoptosis is prevented. We and others have suggested that both pH (22) and ionic charge (8) may be factors that control the process of apoptosis.

Previously, we determined that pH and KCl profoundly affect the ability of cytochrome c to mediate the activation of procaspase-3 within cell lysate. Both normocellular KCl concentration and alkaline pH inhibited the activation of procaspase-3, but while the inhibition by alkaline pH could be overcome by increasing amounts of cytochrome c, KCl inhibition could not. This suggests that these factors affected apoptosome assembly at different points. In this article, we present studies of the effect of alkaline pH and 150 mM KCl on apoptosome formation in vitro.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue culture and reagents. All tissue culture reagents were obtained from Invitrogen (Carlsbad, CA). The human leukemia cell line Jurkat (American Type Culture Collection, Manassas, VA) was cultured at 37°C in RPMI 1640 supplemented with 10% fetal calf serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2. Recombinant active caspase-9 and caspase-3 were purchased from Chemicon (Temecula, CA). Antibodies and other reagents were purchased from the following vendors: anti-Apaf-1 antibody from Trevigen (Gaithersburg, MD), anti-caspase-3 antibody from BD Biosciences-Pharmingen (San Diego, CA), nigericin from Biomol (Plymouth Meeting, PA), and BCECF-AM ester from Molecular Probes (Eugene, OR). Simvastatin was a kind gift from Merck (Rahway, NJ). All other reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated.

Preparation of cell lysates. Manipulation of lysates was performed at 4°C. Generally, 5 x 108 to 1 x 109 cells provided sufficient lysates for several column fractionations. Cells were washed twice in phosphate-buffered saline (in mM: 137 NaCl, 2.6 KCl, 10 Na2HPO4, and 1.7 KH2PO4, pH 7.4), and the wet weight was determined. Cells were then resuspended in 2 ml of extraction buffer (in mM: 50 HEPES, 50 NaCl, 2 MgCl2, and 5 EGTA, pH 7.1) supplemented with a cocktail of protease inhibitors (1 µg/ml of leupeptin, pepstatin, and phenylmethylsulfonyl fluoride) per gram wet weight of pellet. After shearing the cells in 20 passages through a 27-gauge needle, we clarified the lysate by performing sequential centrifugation at 650 g for 5 min at 4°C, 20,000 g for 5 min at 4°C twice, and 100,000 g for 1 h in a 70.1 Ti rotor in a Beckman L8-70M ultracentrifuge (Beckman Instruments, Fullerton, CA) before filtering it through a 0.45-µm filter (Fisher Scientific, Atlanta, GA). The lysate, with a final protein concentration of ~10 mg/ml as determined by the bicinchoninic acid method of protein determination (Pierce Chemical, Rockford, IL), was aliquoted and stored at –80°C.

Assay of caspase activity. Components of a routine assay, 2.5 µg of lysate protein, bovine heart cytochrome c (as indicated), 0.4 nM ATP, and Z-DEVD-rhodamine 110 substrate (Z-DEVD-R110; Molecular Probes, Eugene, OR), were combined on ice at a final volume of 12 µl in a buffer consisting of 10 mM PIPES, 10 mM DTT, and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), pH 7.1. Kinetic measurements of caspase activity were recorded using a Bio-Tek FL600 microplate fluorescent reader (Bio-Tek Instruments, Winooski, VT). The cleavage of the rhodamine substrate was determined using a 485-nm excitation filter and a 530-nm emission filter at a temperature of 25°C. The pH of the reaction mixtures was adjusted for individual assays within a range from 7.1 to 7.9 by addition of NaOH and measured with the use of a needle pH electrode (MI408; Microelectrodes, Bedford, NH) attached to an AB150 Accumet pH meter (Fisher Scientific). The pH of each reaction was measured both before and after the incubation period.

Column fractionation. Cell lysates, 8 mg of total protein, were separated in 1.5-ml fractions over a 40 x 0.7-cm Sepharose CL-4B column at 4°C. Column runs differed by length of time after activation (with 4.5 µM bovine cytochrome c and 10 µM ATP) and by components added to the standard running buffer, composed of (in mM) 10 PIPES, 5 DTT, 2 EDTA, and 0.1% CHAPS. The normal pH of the activation/running buffer was 7.1, and the standard activation time was 1 h, but additional columns were run at 0 h with no cytochrome c or ATP and at 5, 10, 20, and 30 min after activation. Cytosol was also activated at pH 7.9 or with the addition of 150 mM KCl, and these cytosols were fractionated in running buffer of identical pH and KCl concentration.

Detection of DEVDase and LEHDase activities in column fractions. The presence of active caspase-3 in column fractions was determined using 7 µl of each column fraction and monitoring cleavage of the fluorescent substrate Z-DEVD-R110 (excitation/emission maxima ~485/520 nm) (Molecular Probes) with a Bio-Tek FL600 microplate fluorescent reader (Bio-Tek Instruments) as previously described (26). Caspase-9 activity was also monitored in some column fractions against the reagent Ac-LEHD-AFC (Calbiochem, San Diego, CA) (excitation/emission maxima 400/485 nm). To reduce background activity of caspase-3 against this substrate, Casputin reagent (Biomol Research Laboratories), a specific caspase-3 inhibitor, was added to each test sample at a final concentration of 0.01 U/µl. In our hands, Casputin inhibited 95% of the caspase-3 activity.

Trichloroacetic acid precipitation of column fractions. Detection of apoptosomal protein components in column fractions was enhanced by precipitation with trichloroacetic acid (TCA). Equal volumes of 20% TCA were added to each column fraction, and samples were incubated for 30 min on ice. The samples were precipitated by centrifugation at 21,000 g for 15 min. The pellets were washed in acetone, and the final pellets were dissolved in Laemmli sample buffer [80 mM Tris, pH 8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 100 mM DTT, and bromphenol blue], and heated to 95°C for 5 min.

Immunoblot analysis of column fractions. TCA-precipitated column fractions were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane as previously described (26). The blots were then probed with antibody to Apaf-1, procaspase-9/caspase-9, or procaspase-3/caspase-3. After being washed in 50 mM Tris-buffered saline-0.2% Tween 20 (pH 7.4), the blot was overlaid with horseradish peroxidase-conjugated secondary antibody, and the immunoreactive polypeptides were detected using enhanced chemiluminescence (Pierce). The chemiluminescence was measured digitally using a Fluorchem imaging system and software (Alpha Innotech, San Leandro, CA).

Induction of Jurkat cell apoptosis with simvastatin. Actively growing Jurkat cells were washed and resuspended in fresh RPMI 1640 plus 10% fetal calf serum at a concentration of 8 x 105 cells/ml. Simvastatin (Merck) was added to the cells as the sodium salt, prepared as described by Kita et al. (18) at a final concentration of 50 µM. Cells were incubated at 37°C in 5% CO2 with simvastatin with and without phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) for 48 h before the cells were lysed in Molecular Probes lysis buffer and caspase-3 activity was determined as described above.

Determination of intracellular pH. Intracellular pH (pHi) was measured with the cell-permeable pH-sensitive fluorescent probe BCECF-AM (Molecular Probes) using the fluorescent ratio technique described by Perez-Sala et al. (23) with some modifications. Cells (4 x 105) were harvested at the conclusion of the experiment and washed in phosphate-buffered saline before being resuspended in 50 µl of a K+-HEPES buffer at pH 7.3 (in mM: 25 HEPES, 145 KCl, 0.8 MgCl2, 1.8 CaCl2, and 5.5 glucose) containing 1 µM BCECF-AM and incubated at 37°C in the dark. After 1 h, the cells were pelleted, washed, and resuspended in the same buffer without BCECF-AM. pHi was determined using fluorescent emission on the Bio-Tek FL600 fluorescent plate reader. We used a dual excitation ratio of pH-sensitive {lambda}1 = 485 nm and isosbestic {lambda}2 = 400 nm with a fixed emission of 535 nm. The ratio of the fluorescence intensities generated at both excitation wavelengths was calculated for each sample. To estimate the pHi, a standard curve was derived for each experiment prepared with Jurkat cells for in situ calibration of pHi. Cells of the same age and concentration were prepared alongside the experimental cells as described above, washed, and resuspended in the K+-HEPES buffer containing the K+ ionophore nigericin plus 1 µM BCECF at pH 6.9, 7.1, 7.3, 7.5, and 7.7 to generate a calibration curve. A standard curve was derived with a typical R2 > 0.95. From these curves, the pHi of the experimental cells was calculated.

Statistical analysis. Statistical significance for differences in activation of caspase-3 activity and pHi was determined using Student's t-test (29).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of caspase activation by pH and KCl. Both alkaline pH and normocellular concentrations of KCl inhibit the ability of cytochrome c to mediate the activation of procaspase-3 within cell lysate (26). Additional cytochrome c overcame the inhibition imposed by alkaline pH but not the inhibition of 150 mM KCl (Fig. 1, A and B). In contrast, more acidic pH was able to temper the inhibition of procaspase-3 activation by 150 mM KCl (Fig. 2). Yet, neither 150 mM KCl nor pH 7.9 inhibited the activity of commercially available active caspase-3 (results not shown); therefore, we hypothesized that alkaline pH and KCl each interfered with the apoptosome-mediated activation of procaspase-3 and at unique entry points in the cascade.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Additional cytochrome c can overcome inhibition of procaspase-3 activation by alkalinity but not by KCl. In standard reaction mixtures and cytochrome c (Cyto. c), pH was adjusted (A) or 150 mM KCl was added (B) as indicated. Procaspase-3 activation was monitored as caspase-3 fluorescent substrate cleavage in a Bio-Tek fluorescent microplate reader at an excitation of 485 nm and an emission of 535 nm. The maximum reaction rate, derived by linear regression, is expressed as nmol DEVD-R110 substrate·min–1·mg cytosolic protein–1. Results are reported as means ± SE of duplicate samples and represent at least 3 different experiments. In A, *P < 0.05 vs. pH 7.9, +166 µg/ml Cyto c. In B, *P < 0.05 vs. –KCl, +21 µg/ml Cyto c.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Acidity can overcome inhibition of procaspase-3 activation by KCl. Cytochrome c was added to a final concentration of 7.1 µg/ml (shaded and solid bars) or withheld (open bar) in the presence (shaded bars) or absence (solid bar) of 150 mM KCl, and the pH of standard reaction mixtures was adjusted to the pH indicated. Procaspase-3 activation was monitored as caspase-3 fluorescent substrate cleavage in a Bio-Tek fluorescent microplate reader at an excitation of 485 nm and an emission of 535 nm. The maximum reaction rate, derived by linear regression, is expressed as nmol DEVD-R110 substrate·min–1·mg cytosolic protein–1. Results are reported as means ± SE of duplicate samples and represent at least 3 different experiments. *P < 0.05 vs. open bar. #P = 0.26 vs. solid bar.

 
Cytochrome c-independent activation of procaspase-3 by recombinant caspase-9. To investigate the inhibition further, we added active recombinant caspase-9 to Jurkat cell lysate at pH 7.9 or in the presence of 150 mM KCl. As expected, active recombinant caspase-9 added to cell lysate activated cytosolic procaspase-3 in the absence of exogenous cytochrome c or ATP (Fig. 3). However, this activation was inhibited by 150 mM KCl, despite the fact that 150 mM KCl did not inhibit the activity of caspase-9 enzyme on its own substrate in a lysate-free buffer or even within lysate buffer (results not shown). A similar experiment could not be performed with regard to pH, because pH 7.9 inhibited the activity of recombinant caspase-9 by 75%. This observation further supports the notion that the structure of the apoptosome is crucial to the caspase-9-mediated activation of procaspase-3.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. KCl can inhibit activation of procaspase-3 by recombinant caspase-9. KCl (150 mM), cytochrome c, and recombinant caspase-9 were added to standard reaction mixtures as indicated. Procaspase-3 activation was monitored as caspase-3 fluorescent substrate cleavage in a Bio-Tek fluorescent microplate reader at an excitation of 485 nm and an emission of 535 nm. The maximum reaction rate, derived by linear regression, is expressed as nmol DEVD-R110 substrate·min–1·mg cytosolic protein–1. Values are means ± SE of duplicate samples and represent at least 3 different experiments. *P < 0.05 vs. –KCl, +caspase-9, –Cyto c.

 
Effect of KCl and alkaline pH on apoptosome maturation. Lysates were activated by the addition of cytochrome c and ATP for 1 h at pH 7.1, at a K+ concentration of 150 mM KCl, or at pH 7.9. The proteins were separated by size chromatography, caspase activity within the fractions was determined, and the proteins within each fraction were studied by immunoblot analysis. Apaf-1 within lysates activated in the presence of 150 mM KCl eluted with an effective molecular mass of ~700 kDa (Fig. 4A). Immunoblot analysis produced no evidence of autocatalytic activation of procaspase-9 or cleavage of procaspase-3. In addition, LEHDase and DEVDase activity was not detected in column fractions from cytosol activated in the presence of 150 mM KCl (Fig. 5).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 4. Effect of KCl and pH on caspase-9 and caspase-3 association with apoptosomes. Cytosol was activated for 1 h by addition of cytochrome c in the presence of 150 mM KCl (A), at pH 7.1 (B), and at pH 7.9 (C) before being run over a Sepharose CL-4B sizing column. Fractions (1.5 ml) were collected, and the proteins of every other fraction (fraction numbers at top) were separated using SDS-PAGE, transferred to nitrocellulose, and probed with mouse anti-Apaf-1, rabbit anti-caspase-9, or mouse anti-caspase-3 antibody as indicated. Thin arrows indicate the position of procaspase-9; arrowheads indicate the position of p35 caspase-9. Note in A that no caspase-9 immunoreactivity is present; arrowhead in A indicates its expected position. Thick, solid downward arrows indicate the fraction that the 650-kDa protein eluted; the thick, open downward arrow is the predicted fraction in which a 1.4-MDa protein would elute. Results shown represent at least 3 different experiments.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Elution profile of LEHDase (A) and DEVDase (B) activity in column fractions. Cytosol was activated for 1 h by addition of cytochrome c in the presence of 150 mM KCl ({blacktriangledown}), at pH 7.9 ({triangledown}), and at pH 7.1 ({bullet}) before being run over a Sepharose CL-4B sizing column. Fractions (1.5 ml) were collected, and 7 µl of each fraction were assayed for the ability to cleave the fluorescent substrate Ac-LEHD-AFC (A) or Z-DEVD-R110 (B). The maximum reaction rate, derived by linear regression, is expressed as relative fluorescent units (RFU) per minute. Results shown represent at least 3 different experiments.

 
When lysates were activated at pH 7.1, Apaf-1 was found to elute over a broad range of fractions that corresponded to molecular mass 44 kDa to >1.4 MDa (Fig. 4B). All of the procaspase-9 was cleaved into active caspase-9 (Fig. 4B) and eluted in fractions representing aggregates of ~700 kDa. In these same fractions that corresponded to proteins of ~700 kDa, caspase-3 fragments were detected (Fig. 4B). In addition, high levels of LEHDase and DEVDase enzymatic activity were found in these same fractions corresponding to a molecular mass of ~700 kDa and to the presence of the cleaved caspase proteins (Fig. 5).

The results were very different for lysates activated and fractionated at pH 7.9. At this alkaline pH, much of the Apaf-1 was found in the higher molecular mass fractions, corresponding to a predicted molecular mass of 1.4 MDa (Fig. 4C). The Apaf-1 present in these larger molecular mass fractions was associated with caspase-9 protein (Fig. 4C), but minimal LEHDase activity was detected (Fig. 5A). No caspase-3 protein (Fig. 4C) or DEVDase activity (Fig. 5B) was found in the 1.4-MDa fractions associated with caspase-9. In the fractions that corresponded to the ~700-kDa apoptosome, minimal LEHDase and DEVDase activity was detected.

Time course of apoptosome maturation. To better understand the apoptosome maturation process, we analyzed the formation of apoptosomes in lysates activated for various amounts of time at pH 7.1. When lysates were not activated, the majority of the Apaf-1 was found in fractions corresponding to ~700 kDa, and some procaspase-9 and procaspase-3 migrated in these same fractions. Neither caspase-9 nor caspase-3 protein (Fig. 6), nor DEVDase enzymatic activity (Fig. 7), could be detected.



View larger version (63K):
[in this window]
[in a new window]
 
Fig. 6. Time course of apoptosome maturation. Cytosol without cytochrome c added (A) or cytosol activated by addition of cytochrome c for 5 (B), 10 (C), 20 (D), or 30 (E) min before being run over a Sepharose CL-4B sizing column. Fractions (1.5 ml) were collected, and the proteins of every other fraction (fraction number at top) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to the indicated proteins. Thick, solid downward arrow indicates the fraction that the 650-kDa protein eluted; the thick, open downward arrow is the predicted fraction in which a 1.4-MDa protein would elute. Results shown represent at least 2 immunoblot experiments from each column.

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7. Elution profile of DEVDase activity in column fractions. Cytosol was activated for 5, 10, 20, 30, or 60 min by addition of cytochrome c before being run over a Sepharose CL-4B sizing column. Fractions (1.5 ml) were collected, and 7 µl of each fraction were assayed for the ability to cleave the fluorescent substrate Z-DEVD-R110. The maximum reaction rate, derived by linear regression, is expressed as RFU per minute.

 
After 5 min of activation, there was a slight shift in the peak of elution of Apaf-1 to the higher molecular mass fractions (Fig. 6B). In addition, active caspase-9 protein was clearly detected in fractions corresponding to the 1.4-MDa apoptosome. There was a slight shift in procaspase-3 protein to the higher molecular mass fractions, but no active caspase-3 subunits or caspase-3 enzymatic activity was detected in these higher molecular mass fractions. After 10 min, Apaf-1, the 35-kDa active subunit of caspase-9, and procaspase-3 were found in the higher molecular mass fractions (Fig. 6C). After 20 min of activation, Apaf-1 and caspase-9 eluted in fractions corresponding to proteins of 1.4 MDa (Fig. 6D). At 30 min, procaspase-9 was completely processed (Fig. 6E). Apaf-1 was still broadly detected across the fractions, but the majority of the detected active subunit of caspase-9 was in the fractions corresponding to the ~700-kDa apoptosome. Also, at this time point, both active caspase-3 fragments and DEVDase activity (Fig. 7) were detected in the fractions that corresponded to the ~700-kDa apoptosome. As discussed above, after 60 min, caspase-3 fragments (Fig. 4B) and enzymatic activity (Fig. 5B) were detected in fractions that corresponded to the ~700-kDa apoptosome.

Unlike the shifting proteins seen over time in immunoblots of lysates activated at pH 7.1, immunoblots of lysates activated in the presence of KCl are identical, regardless of length of activation (data not shown). Earlier time points of lysates activated under alkaline conditions were similar to the early time points of the lysate activated at pH 7.1 (data not shown).

Alkaline pH blocks apoptosis in vivo. The results of in vitro assays may not reflect the actual mechanism in vivo. However, K+-inhibited caspase activation has been demonstrated in vivo by blocking K+ channels (3, 4) during the induction of apoptosis. Relative to pHi, a number of reports have suggested that apoptosis and acidosis are tightly linked in a variety of cells (9, 12, 13, 15, 17, 21), whereas alkaline conditions have been reported to be favorable for proliferation (2, 27). Previously, the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor lovastatin was shown to induce DNA fragmentation in human promyelocytic leukemic HL-60 cells as a result of internal acidification, and both DNA fragmentation and acidification could be blocked by preventing acidification with the addition of phorbol esters (23). To determine whether blocking the activation of procaspase-3 could be the mechanism responsible for inhibition, we studied procaspase-3 activation in Jurkat cells in response to simvastatin exposure. As shown in Fig. 8, Jurkat cells exposed to simvastatin for 48 h demonstrated a marked increase in caspase-3 activity that was accompanied by a decrease in pHi from a baseline of 7.6 to 7.4. Furthermore, when this drop in pHi was blocked by the addition of PMA, caspase-3 activity was inhibited (Fig. 8).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8. Alkaline pH blocks Jurkat cell apoptosis. Jurkat cells were incubated for 48 h in the presence or absence of 10 µM simvastatin (Simv) with or without 5 µM PMA inhibitor as indicated. At 0 and 48 h, cells were collected and intracellular pH (pHi) was determined. In addition, a portion of the cells were lysed, and caspase-3 activity was determined. Results are reported as means ± SE of duplicate samples from 2–3 different experiments. *P < 0.05 compared with caspase-3 activity at 0 h. **P < 0.05 compared with caspase-3 activity in 48 h Simv sample. #P < 0.05 compared with pHi at 0 h. ##P < 0.05 compared with pHi of 48 h Simv sample.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In a previous report, we demonstrated that KCl and alkaline pH inhibited the activation of caspase-3 but not its activity once activated. The ability of alkalinity and K+ to inhibit cytochrome c-mediated activation of caspase-3 is not specific for cytosol derived from Jurkat cells. When the assay was performed with cytosol derived from human umbilical vein endothelial cells, insect cell cytosol derived from SF9 cells, and human embryonic kidney cell cytosol, similar results were obtained (data not shown). From these observations, we hypothesized that the inhibition occurred at the level of the apoptosome, which is one proposed source of procaspase-3 activation. In addition, we showed that inhibition by alkaline pH could be countered by excess cytochrome c, whereas inhibition by KCl could be countered by decreased pH. Realizing that ubiquitous cellular constituents could be the modulators of apoptosis, we compared apoptosomal protein assembly in the presence of these two inhibitors.

In a set of preliminary experiments, we looked at the effects of KCl on the ability of active recombinant caspase-9 to activate procaspase-3 within cell lysate. As expected, the addition of active caspase-9 in the absence of KCl and alkaline pH led to the activation of procaspase-3 without the addition of cytochrome c or ATP. However, in the presence of KCl, caspase-9 did not activate procaspase-3, despite the fact that caspase-9 cleaved its fluorogenic substrate in the presence of KCl and thus was active. This surprising result suggests that caspase-9 does not cleave procaspase-3-free in solution; rather, caspase-9, procaspase-3, or both must be in the proper conformation for cleavage to occur. Alternatively, the conformation of procaspase-3 may be affected by KCl in such a way that caspase-9 cleavage is prevented.

To distinguish between these possibilities, we analyzed the formation of apoptosomes in the presence of KCl and alkaline pH. We fractionated lysates activated with cytochrome c and ATP at pH 7.1 (positive control), at pH 7.9, or in the presence of 150 mM KCl. Distinctly different elution profiles were encountered for the three proteins (Apaf-1, procaspase-9, and procaspase-3) traditionally implicated in cytochrome c-mediated apoptosis. After 1 h of activation at pH 7.1, Apaf-1, caspase-9, and procaspase-3/caspase-3 all coeluted in the region of a ~700-kDa apoptosome or as freed processed enzymes. Procaspase-9 was completely processed to its predominant cleavage products, and LEHDase and DEVDase activities peaked at the ~700-kDa apoptosome. These findings are consistent with previously published studies that suggested that the majority of caspase activity is present in the ~700-kDa apoptosome (8).

In contrast, in lysates activated in the presence of 150 mM KCl, Apaf-1 was present in the fractions corresponding to proteins of ~700 kDa. However, only a portion of procaspase-9 migrated with Apaf-1, and no 35-kDa caspase-9 subunit or LEHDase enzymatic activity was detected. No procaspase-9 or procaspase-3 was detected in higher molecular mass fractions. These data suggest that KCl does not interfere with the association of procaspase-9 with Apaf-1 but certainly inhibits the processing of procaspase-9. This is consistent with previous studies with regard to the ability of KCl to inhibit caspase activity (8). Previously, it was reported that KCl inhibition of apoptosome formation could be overcome by increasing concentrations of cytochrome c (8); however, in our experimental system, additional cytochrome c could not overcome the inhibitory effect of KCl (Fig. 1B). These two conflicting results may be reconciled by the effect of pH. In our system, even after the addition of excess cytochrome c, the pH of the reaction was fixed at 7.1. In experiments performed at pH 6.8, however, the inhibition of KCl could be overcome (Fig. 2).

An antipodal elution pattern emerged with the lysate activated at an alkaline pH. Apaf-1 was detected in fractions corresponding predominately to proteins migrating of 1.4 MDa. Fully processed caspase-9 was associated with the 1.4-MDa apoptosomes but had minimal LEHDase activity. No processing of procaspase-3 protein and only minimal DEVDase activity were detected.

Initially, on the basis of previously published reports (7), we had no reason to suspect that the 1.4-MDa apoptosome was important in the processing of procaspase-9 or procaspase-3. Indeed, the results of alkaline pH fractionation further supported the theory that the 1.4-MDa apoptosome was an aberrant divergence in the process of activation. However, the results of a time course of activation experiment suggested that the ~700-kDa apoptosome is formed very early in the process and may even be constitutively assembled in a benign state. The 1.4-MDa apoptosome was the dynamic manifestation of apoptotic induction, leading directly to the activation of caspase-9.

We detected Apaf-1, procaspase-9, and procaspase-3 eluting together in the region of the ~700-kDa apoptosome even in inactive lysates. We were unable to detect Apaf-1 and procaspase-9 in fractions representing monomeric species. It is possible that this assembly is artifactual. Holocytochrome c was detected eluting with the ~700-kDa apoptosome in inactive lysates (data not shown), even though no exogenous cytochrome c had been added, suggesting that mitochondrial cytochrome c was released during lysate preparation. It has been suggested that intact cells may have holocytochrome c present in the cytosol, albeit at low levels, as a result of mitochondrial turnover (22). Because ATP is readily available in the cytosol, all of the components required for apoptosome assembly may be present in cytoplasm. With this in mind, we postulated that the cell might have developed other safeguards to prevent maturation of apoptosomes and activation of procaspase-3 in the absence of dramatic levels of cytochrome c release. These safeguards include inhibitors of apoptosis, normal intracellular K+ concentrations, and perhaps pH as a modulator.

The ability of normal intracellular K+ concentrations to inhibit apoptosis was previously demonstrated (4). However, the association between acidosis and procaspase activation has not been established definitively. Although a drop in pHi is associated with caspase activity (22), it has not been demonstrated that blocking this acidification would inhibit procaspase-3 activation. We have demonstrated that simvastatin induces both a drop in pHi and a marked increase in caspase-3 activity, both of which are blocked by PMA (Fig. 8). Thus the ability of PMA to block simvastatin-induced apoptosis may occur at the level of activation of procaspase-3 via prevention of cytosolic acidification. Thus there is in vivo evidence that normal intracellular K+ concentrations and alkaline pH can block apoptosis.

We suggest that K+ and alkaline pH block apoptosis at the level of apoptosome maturation. Our studies suggest that the 1.4-MDa apoptosome is not a "dead end" of apoptosome maturation but a prerequisite for normal maturation. Our model for apoptosome maturation, consistent with our findings, is that initially cytochrome c and dATP/ATP seed Apaf-1 and procaspase-9 oligomerization (Fig. 9A). After this initial association, the maturation of the apoptosome requires the formation of a 1.4-MDa intermediate to permit cleavage of procaspase-9 and association of procaspase-3.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9. Model of apoptosome maturation. A: apoptosome maturation. Cytochrome c (dark shaded circles) and ATP/dATP mediates the oligomerization of Apaf-1 (light blue crescents) and procaspase-9 (light shaded rod) into a ~700-kDa complex. Maturation of the apoptosome to a 1.4-MDa dimer complex is blocked by normal intracellular KCl concentrations. Within the 1.4-MDa complex, procaspase-9 is activated and procaspase-3 associates with the complex. The creation of active apoptosomes is blocked by alkaline pH and requires dissociation of dimers to a ~700-kDa complex. B: apoptosome dimerization. Shown is a representation of 2 of 7 Apaf-1 of the heptamer, modeled after Acehan et al. (1). After Apaf-1 (light blue) binds cytochrome c (Cyto c; dark shaded ovals), its card domain (blue ovals) was free to bind caspase-9 through its card domain (light shaded oval). However, in solution, procaspase-9 does not form spontaneous dimers; apoptosome dimerization may mediate procaspase-9 dimerization. During dimerization, procaspase-9 of one apoptosome cleaves the procaspase-9 of the head-to-head apoptosome, and procaspase-3 associates with the apoptosome. After cleavage of procaspase-9 and procaspase-3, the head-to-head dimer dissociates. Alkalinity may prevent dimer dissociation by promoting apoptosome dimerization or by preventing procaspase-3 cleavage.

 
It is interesting to speculate about the nature of the 1.4-MDa apoptosome. It is reasonable to suspect that it may represent dimerization of ~700-kDa apoptosomes (Fig. 9); there is evidence for such a dimer in the literature (1). Although dimerization of procaspase-9 is critical for its activation, dimers form in solution only at very high concentrations of procaspase-9 (24). Previously, it was proposed that procaspase-9 dimerization occurs when free procaspase-9 associates with procaspase-9 bound to Apaf-1 (1). However, this would occur only if, upon binding Apaf-1, a change in conformation were to occur in procaspase-9 that increased its affinity for free procaspase-9, and this has not been reported. We propose that procaspase-9 dimerization is mediated by apoptosome dimerization (Fig. 9). When a procaspase-9 mutant is used that cannot be cleaved, apoptosome dimers are the predominant structure detected (1). Perhaps procaspase-9 and procaspase-3 cleavage is needed to progress from the 1.4-MDa configuration. KCl may directly prevent the dimerization of Apaf-1, indirectly preventing both procaspase-9 dimerization and autocatalysis.

However, even active caspase-9 is prevented from activating procaspase-3 in the presence of KCl. Under normal and alkaline pH conditions, we were able at some time points to capture procaspase-3 association with the 1.4-MDa apoptosome (Fig. 6; data not shown); thus we think that procaspase-3 associates with the 1.4-MDa apoptosome or that its cleavage is required for maturation of the 1.4-MDa apoptosome to the active ~700-kDa apoptosome. KCl, by inhibiting Apaf-1 dimerization, would block both of these processes.

Alkaline pH blocks the apoptosome in the 1.4-MDa conformation, even though procaspase-9 is cleaved. Perhaps cleavage of procaspase-3 is also required for transformation of the 1.4-MDa apoptosome into active ~700-kDa apoptosomes. In our studies, we did not detect caspase-3 fragments within the 1.4-MDa apoptosome at alkaline pH. Because alkaline pH inhibited caspase-9 activity by ~75% (data not shown), it is not surprising that autocatalysis and procaspase-3 activation might be inhibited under these conditions. Caspase-3 activity is unaffected by alkaline conditions (26) and likely functions normally once activated.

Because procaspase-3 is found within the 1.4-MDa apoptosome under certain experimental conditions (data not shown), cleavage of both procaspase-9 and procaspase-3 may be necessary for transformation of the 1.4-MDa apoptosomes into active ~700-kDa apoptosomes. Alternatively, procaspase-3 may be cleaved when 1.4-MDa apoptosomes are transformed into ~700-kDa apoptosomes and alkaline pH blocks this maturation process. Experiments in our laboratory to investigate these possibilities are underway.

Our results suggest that apoptosome maturation is subverted by physiological factors within the cytosol. KCl apparently prevents the formation of 1.4-MDa apoptosomes, thereby interfering with procaspase-9 autoactivation. On the other hand, alkaline pH permits the formation of the 1.4-MDa apoptosome, but the apoptosome is retained in this larger configuration, possibly by interfering with the cleavage of procaspase-3.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02537 and DK-59821 (to M. S. Segal) as well as by Gatorade research funds.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Segal, PO Box 100224, Gainesville, FL 32610 (E-mail: segalms{at}medicine.ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, and Akey CW. Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell 9: 423–432, 2002.[ISI][Medline]

2. Baltz JM. Intracellular pH regulation in the early embryo. Bioessays 15: 523–530, 1993.[ISI][Medline]

3. Bortner CD and Cidlowski JA. Caspase independent/dependent regulation of K+, cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J Biol Chem 274: 21953–21962, 1999.[Abstract/Free Full Text]

4. Bortner CD, Hughes FM Jr, and Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 272: 32436–32442, 1997.[Abstract/Free Full Text]

5. Brenner C and Kroemer G. Apoptosis: mitochondria—the death signal integrators. Science 289: 1150–1151, 2000.[Free Full Text]

6. Cai J, Yang J, and Jones DP. Mitochondrial control of apoptosis: the role of cytochrome c. Biochim Biophys Acta 1366: 139–149, 1998.[ISI][Medline]

7. Cain K, Bratton SB, Langlais C, Walker G, Brown DG, Sun XM, and Cohen GM. Apaf-1 oligomerizes into biologically active ~700-kDa and inactive ~1.4-MDa apoptosome complexes. J Biol Chem 275: 6067–6070, 2000.[Abstract/Free Full Text]

8. Cain K, Langlais C, Sun XM, Brown DG, and Cohen GM. Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 276: 41985–41990, 2001.[Abstract/Free Full Text]

9. Chen Q, Benson RS, Whetton AD, Brant SR, Donowitz M, Montrose MH, Dive C, and Watson AJ. Role of acid/base homeostasis in the suppression of apoptosis in haemopoietic cells by v-Abl protein tyrosine kinase. J Cell Sci 110: 379–387, 1997.[Abstract/Free Full Text]

10. Cohen GM. Caspases: the executioners of apoptosis. Biochem J 326: 1–16, 1997.[ISI][Medline]

11. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, and Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148: 2207–2216, 1992.[Abstract/Free Full Text]

12. Furlong IJ, Ascaso R, Lopez Rivas A, and Collins MK. Intracellular acidification induces apoptosis by stimulating ICE-like protease activity. J Cell Sci 110: 653–661, 1997.[Abstract/Free Full Text]

13. Gendron MC, Schrantz N, Métivier D, Kroemer G, Maciorowska Z, Sureau F, Koester S, and Petit PX. Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation. Biochem J 353: 357–367, 2001.[CrossRef][ISI][Medline]

14. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]

15. Hirpara JL, Clément MV, and Pervaiz S. Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells. J Biol Chem 276: 514–521, 2001.[Abstract/Free Full Text]

16. Hiura TS, Li N, Kaplan R, Horwitz M, Seagrave JC, and Nel AE. The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles. J Immunol 165: 2703–2711, 2000.[Abstract/Free Full Text]

17. Iguchi K, Usui S, Ishida R, and Hirano K. Imidazole-induced cell death, associated with intracellular acidification, caspase-3 activation, DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. Apoptosis 7: 519–525, 2002.[CrossRef][ISI][Medline]

18. Kita T, Brown MS, and Goldstein JL. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J Clin Invest 66: 1094–1100, 1980.[ISI][Medline]

19. Kroemer G, Dallaporta B, and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: 619–642, 1998.[CrossRef][ISI][Medline]

20. Li H, Kolluri SK, Gu J, Dawson MI, Cao X, Hobbs PD, Lin B, Chen G, Lu J, Lin F, Xie Z, Fontana JA, Reed JC, and Zhang X. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289: 1159–1164, 2000.[Abstract/Free Full Text]

21. Liu D, Martino G, Thangaraju M, Sharma M, Halwani F, Shen SH, Patel YC, and Srikant CB. Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis. J Biol Chem 275: 9244–9250, 2000.[Abstract/Free Full Text]

22. Matsuyama S, Llopis J, Deveraux QL, Tsien RY, and Reed JC. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2: 318–325, 2000.[CrossRef][ISI][Medline]

23. Pérez-Sala D, Collado-Escobar D, and Mollinedo F. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease. J Biol Chem 270: 6235–6242, 1995.[Abstract/Free Full Text]

24. Renatus M, Stennicke HR, Scott FL, Liddington RC, and Salvesen GS. Dimer formation drives the activation of the cell death protease caspase 9. Proc Natl Acad Sci USA 98: 14250–14255, 2001.[Abstract/Free Full Text]

25. Robertson AM, Bird CC, Waddell AW, and Currie AR. Morphological aspects of glucocorticoid-induced cell death in human lymphoblastoid cells. J Pathol 126: 181–187, 1978.[ISI][Medline]

26. Segal MS and Beem E. Effect of pH, ionic charge, and osmolality on cytochrome c-mediated caspase-3 activity. Am J Physiol Cell Physiol 281: C1196–C1204, 2001.[Abstract/Free Full Text]

27. Siczkowski M and Ng LL. Phorbol ester activation of the rat vascular myocyte Na+-H+ exchanger isoform 1. Hypertension 27: 859–866, 1996.[Abstract/Free Full Text]

28. Von Ahsen O, Renken C, Perkins G, Kluck RM, Bossy-Wetzel E, and Newmeyer DD. Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release. J Cell Biol 150: 1027–1036, 2000.[Abstract/Free Full Text]

29. Winer BJ. Statistical Principles in Experimental Design (2nd ed.). New York: McGraw-Hill, 1971.

30. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556, 1980.[ISI][Medline]