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J Biol Chem, Vol. 275, Issue 9, 6067-6070, March 3, 2000

ACCELERATED PUBLICATION
Apaf-1 Oligomerizes into Biologically Active ~700-kDa and Inactive ~1.4-MDa Apoptosome Complexes^*

Kelvin Cain, Shawn B. Bratton, Claudia Langlais, Gail Walker, David G. Brown, Xiao-Ming Sun, and Gerald M. Cohen^§

From the MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of ~1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M_r values of ~1.4 MDa and ~700 kDa. Kinetic analysis demonstrates that in vitro the ~700-kDa complex is produced more rapidly than the ~1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the ~700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the ~700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptotic cell death is characterized by a commitment and an execution phase, the latter being distinguished by a series of stereotypic morphological and biochemical changes, which require the activation of a family of caspases (1, 2). Caspases are constitutively expressed in cells as inactive proenzymes and require proteolytic cleavage to be active. Presently, fourteen caspases have been identified and these can be divided into two classes. "Initiator" caspases, such as caspases-8 and -9, possess long pro-domains and either directly or indirectly activate "effector" caspases, such as caspase-3, -6, and -7, which contain short pro-domains (3-5). Activation of initiator caspases involves interaction of their long pro-domains with either caspase recruitment domains (CARD)¹ (6) or the death effector domains (DEDs) of adapter molecules and results in the formation of cellular complexes. Thus with receptor-mediated cell death, ligation of the CD95 (Fas/APO-1) receptor results in the formation of a death-inducing signaling complex (DISC), in which the death effector domains of the adapter molecule FADD (MORT1) interact with the N-terminal death effector domains of caspase-8 (7, 8). Many apoptotic stimuli cause cell death by inducing the release of cytochrome c (9), which together with Apaf-1 and ATP/dATP facilitates the processing of caspase-9 and initiates a caspase cascade (10, 11). Apaf-1, the first identified mammalian homologue of CED-4, is a 130-kDa protein. It contains an N-terminal CARD domain, followed by a region homologous to CED-4 and a C-terminal domain containing multiple WD-40 repeats, which are involved in protein-protein interactions (11). Both CED-4 and Apaf-1 undergo self-oligomerization, which is required for the activation of CED-3 and caspase-9, respectively (12, 13). In the presence of dATP and cytochrome c, the CARD domains of caspase-9 and Apaf-1 interact to produce an apoptosome complex in which procaspase-9 is processed to its active form, which in turn activates caspase-3 (10, 14, 24).

The apoptosome has been defined as an Apaf-1-containing complex that catalyzes the activation of caspases (15, 16). Recently, using purified recombinant Apaf-1 in the presence of cytochrome c and dATP, the formation of an ~1.4-MDa apoptosome complex has been described (17, 18). This large complex recruits and activates procaspase-9, which dissociates from the apoptosome before activating caspase-3 (17, 18). In contrast, we have isolated an ~700-kDa Apaf-1 apoptosome (aposome)² complex containing activated caspase-9, -3, and -7 from dATP-activated cell lysates (19). The difference in size of the recombinant (~1.4 MDa) and native (~700 kDa) apoptosome complexes led us to question the relative significance of these two large complexes in processing caspases during dATP-dependent activation and apoptotic cell death. In the present study using Superose 6 gel filtration, we demonstrate that both the ~700-kDa and ~ 1.4-MDa apoptosome complexes are formed, following in vitro dATP activation of cell lysates. However, only the ~700-kDa complex is capable of processing and activating effector caspases. Furthermore we also show that, during apoptotic cell death, the ~700 kDa apoptosome complex predominates, supporting the hypothesis that this is the correctly assembled and biologically active form of the apoptosome.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Western Blot Analysis-- Most reagents, including antibodies to caspase-3, -7 and -9, were obtained from indicated sources (15, 21). Protein samples (~20 µg) were resolved on 10 or 12.5% SDS-polyacrylamide gels (PAGE) and transferred onto nitrocellulose membranes (Hybond-C Extra, Amersham Pharmacia Biotech), and antibody binding was detected as described previously (22). To assess the kinetics of apoptosome assembly, Western blots of Apaf-1 from each time point were probed with identical antibody and enhanced chemiluminescence solutions and simultaneously exposed to the same photographic film. The exposed films were scanned with a Molecular Dynamics Densitometer, and the images were analyzed with a Multi-Analyst Image analysis program (Bio-Rad Laboratories, CA).

Cell Culture and Preparation of Control and Caspase-activated Cell Lysates-- Human monocytic tumor cells (THP.1) were grown in RPMI 1640 media with 10% heat-inactivated fetal bovine serum in 5% CO₂ at 37 °C. Apoptosis was induced with etoposide (10 µM), a DNA topoisomerase II inhibitor, or TPCK (75 µM), a chymotrypsin-like serine protease inhibitor, as described previously (22, 23). Lysates (100,000 × g supernatants) from THP.1 cells were prepared as described previously and activated by incubation (10 mg/ml) for 1 h at 37 °C with 2 mM dATP and 2 mM MgCl₂ (19). These cell lysates contained low but detectable levels of cytochrome c and did not require the addition of exogenous cytochrome c for full caspase activation.

Fluorimetric Assay of Caspase Activity-- DEVDase activity (i.e. primarily caspase-3 and -7) of lysates or column fractions was measured (_ex/_em = 405/510 nm) at 37 °C in 96-well plates in 200 µl of assay buffer (20 µM Z-DEVD.AFC, 0.1% CHAPS, 10 mM dithiothreitol, 100 mM HEPES, and 10% sucrose, pH 7.0) using a Wallac Victor² 1420 Multilabel counter. The 96-well plates were assayed for 10 cycles, and cleavage rates were determined by linear regression. The protease activities were expressed as either pmol/min/mg protein or pmol/min/fraction.

Chromatographic Methods-- Lysates were fractionated by size-exclusion chromatography, using a fast protein liquid chromatography protein purification system on analytical (16/60), preparative (26/60) Hi-Prep S300 Sephacryl high-resolution (19) and Superose 6 HR 10/30 columns (Amersham Pharmacia Biotech, Herts, UK). The Sephacryl columns were eluted at 4 °C with 5% (w/v) sucrose, 0.1% (w/v) CHAPS, 20 mM HEPES/NaOH, 5 mM dithiothreitol, pH 7.0, and fractions collected. The Superose 6 column was eluted with the same buffer supplemented with 50 mM NaCl, at 0.4 ml/min, and 0.5- or 1.0-ml fractions were collected. The columns were calibrated with protein standards (Amersham Pharmacia Biotech), including blue dextran, thyroglobulin, ferritin, catalase, bovine serum albumin, ovalbumin, and bovine heart cytochrome c (Sigma) with some of their sizes (kDa) indicated in Fig. 1. Some differences (see Results) were noted in the elution profiles of the effector caspases on S300 and Superose 6 columns. This was most probably because of addition of 50 mM NaCl in the buffer, which ensured good separation of large protein complexes on Superose 6 columns.

Assay of Apoptosome Effector Caspase Activating Activity-- To assess the biological activities of the ~1.4-MDa and ~700-kDa apoptosome complexes, we developed a simple in vitro assay. Briefly, apoptosome complexes were isolated from apoptotic or dATP-treated lysates by Superose 6 gel filtration as described above. The appropriate column fractions (100 µl aliquots) were then incubated with procaspases (25 µg of protein) for 30 min before assaying for DEVDase activity. The procaspases were obtained following fractionation of control lysates (fractions 18-20) by gel filtration on a preparative (26/60) Hi-Prep S300 Sephacryl high-resolution column. These fractions, which contained procaspase-3, -7, and -9, were concentrated in Vivaspin 4 (Vivascience, Lincoln, UK) concentrators (10 kDa cut-off) to a final concentration of 8-14 mg/ml and stored at 80 °C prior to use. In preliminary experiments, it was determined that the isolated active apoptosome complexes required neither dATP/ATP nor cytochrome c to process added procaspases, presumably because these cofactors are only required for the proper oligomerization of Apaf-1. Thus, this assay measures the ability of an assembled Apaf-1 complex to both process and activate effector caspase-3 and -7.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

dATP Activation of Cell Lysates Produces Both ~700-kDa and ~1.4-MDa Apoptosome Complexes-- In our previous study, a Sephacryl S300 column was used to characterize the apoptosome as an ~700-kDa complex (19). Because this column cannot accurately resolve complexes of 700-1500 kDa, we used a Superose 6 high-resolution analytical gel filtration column, which can resolve complexes up to 4 MDa. In control lysates, both procaspase-9 and -3 eluted as their free unprocessed zymogens (Fig. 1A, fractions 22-28) and Apaf-1 eluted as a single peak (Fig. 1A, fractions 18-24), with an estimated M_r  158,000, indicating that it was most likely a monomer. After dATP activation of the lysate for 1 h, a dramatic change in the elution behavior of Apaf-1 and the caspases was observed. Apaf-1 was now associated predominantly with ~1.4-MDa and ~700-kDa apoptosome complexes (Fig. 1B, fractions 6-8 and fractions 10-15, respectively) with a small amount remaining as monomeric Apaf-1 (Fig. 1B, fractions 19-22). After dATP activation, most of the procaspase-9 was processed to its catalytically active large subunits (p35/p37 subunits), which eluted with the ~1.4-MDa and ~700-kDa apoptosome complexes, and also as the free processed caspase (Fig. 1B). Procaspase-3 was completely processed to its catalytically active large subunits (p17/19) (Fig. 1B), which co-eluted with all the DEVDase activity (Fig. 1C, fractions 21-28, M_r = ~60,000).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Two large ~1.4-MDa and ~700-kDa apoptosome complexes are formed following dATP activation of cell lysates. Lysates (10 mg/ml) from THP.1 cells were incubated with or without dATP (2 mM) for 1 h, and the lysate (2 mg) was separated on a Superose 6 column and immunoblotted. A, Apaf-1 from control lysates eluted as a monomer and caspase-3 and -9 as unprocessed zymogens. B, after dATP activation, Apaf-1 eluted both as an ~1.4-MDa and an ~700-kDa apoptosome complex. Almost, all of the caspase-9 was processed to its p35 and 37 forms, which eluted with the ~1.4-MDa and the ~700-kDa apoptosome complexes, and as free heterotetramers. Caspase-3 was processed to its active large subunit. C, the ~700-kDa but not the ~1.4-MDa apoptosome possessed the ability to process and activate most of the effector caspases. The Apaf-1-containing fractions (fractions 5-21, Fig. 1B) were assayed for caspase processing activity by measuring DEVDase activity () produced when the fractions were incubated with "free caspases" as described under "Experimental Procedures." Thus, the ~700-kDa apoptosome complex (fractions 10-15) initially had very little or no DEVDase activity but had the inherent ability to activate exogenous effector caspases. All the fractions from panel B were also initially assayed for DEVDase activity (). The NaCl in the elution buffer resulted in the DEVDase (i.e. free active effector caspase-3 and -7) activity eluting in fractions 22-28. The elution positions of Mr markers are indicated with arrows.

The ~1.4-MDa and ~700-kDa apoptosome complexes were also assayed for their ability to activate exogenous effector caspases. Only the ~700-kDa apoptosome complex (Fig. 1C, fractions 10-15) actively processed the effector caspases as assayed by the induced DEVDase activity. Thus, following dATP activation, two large apoptosome complexes of ~700 kDa and ~1.4 MDa were formed, but only the ~700-kDa complex appeared to constitute the correctly assembled and functionally active apoptosome.

Kinetics of Formation of the ~700-kDa and ~1.4-MDa Apoptosome Complexes-- To understand the relationship between the ~700-kDa and ~1.4-MDa apoptosome complexes, we studied their time-dependent formation and activities following dATP activation. The ~700-kDa complex was more rapidly formed than the ~1.4-MDa complex (Fig. 2A). Production of the ~700-kDa complex was maximal within 5 min of initiating dATP activation, whereas assembly of the ~1.4-MDa complex did not reach a maximum until after 20 min (Fig. 2A). However, only the ~700-kDa apoptosome complex exhibited significant biological activity as assessed by its ability to process caspases to yield active effector caspase activity (DEVDase, Fig. 2B). The biological activity of the assembled ~700-kDa complex was almost maximal 5 min after activation was initiated, whereas the ~1.4-MDa complex was virtually inactive at all time points measured (Fig. 2B).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The ~700-kDa but not the ~1.4-MDa apoptosome complex possesses caspase processing activity. Lysates were activated with dATP as described in the legend to Fig. 1 and at the indicated times fractionated by Superose 6 chromatography, essentially as described under "Experimental Procedures" except that 1-ml fractions were taken. Each fraction was assayed for its caspase processing activity and then subjected to SDS-PAGE. A, the fractions were then analyzed by Western blotting with an antibody to Apaf-1 and quantified by densitometry as described under "Experimental Procedures." The time-dependent formation of the ~1.4-MDa (--) and ~700-kDa (--) apoptosome complexes is shown. The imaged Apaf-1 data is shown for one experiment (similar results were obtained in a second separate experiment). B, caspase processing activity for the ~700-kDa and ~1.4-MDa complexes is shown. This is the average of two separate time course experiments.

During Apoptosis, the ~700-kDa Apoptosome Complex Predominates-- To determine whether the ~700-kDa and/or ~1.4-MDa apoptosome complexes were formed during apoptosis, lysates were prepared from control and apoptotic cells and fractionated using a Superose 6 column. Apoptosis was induced in THP.1 cells by two different stimuli, i.e. etoposide and TPCK (22, 23), which gave 30 and 38% apoptosis, respectively, as determined by Annexin V binding. All of the Apaf-1 in lysates isolated from control cells eluted as the free ~130-kDa monomeric form (Fig. 3A). In marked contrast, Apaf-1, in lysates from etoposide or TPCK-treated THP.1 cells, eluted mainly as the free monomeric form or as the ~700-kDa complex, and little if any Apaf-1 eluted as an ~1.4-MDa complex (Fig. 3, B and C). Furthermore, only the ~700-kDa apoptosome complex processed and activated effector caspases (Fig. 3, B and C), suggesting that this was the biologically active complex which was formed during etoposide- or TPCK-induced apoptosis. Thus, in THP.1 cells, induction of apoptosis by either etoposide or TPCK resulted in the formation of an ~700-kDa caspase processing complex.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Apoptotic cells contain predominantly the active ~700-kDa complex. THP.1 cells were incubated for 6 h either alone (A), with etoposide (10 µM) (B), or with TPCK (75 µM) (C) and the amount of apoptosis assessed as measured by phosphatidylserine externalization (21). Lysates were then freshly prepared and fractionated by Superose 6 gel filtration as described in the legend to Fig. 1. The fractions were run on SDS-PAGE and immunoblotted for Apaf-1 and assayed for caspase processing activity. In both etoposide (B) and TPCK-treated cells (C), Apaf-1 eluted predominantly either as the monomeric form or as a ~700-kDa apoptosome complex. Almost all the caspase processing activity was associated with the ~700-kDa apoptosome complex. Control cell lysates did not have any detectable caspase processing activity (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reconstitution studies with recombinant proteins have shown that Apaf-1 and caspase-9 in the presence of cytochrome c and dATP forms a very large (~1.4 MDa) apoptosome complex (17, 18). This complex was active as judged by its ability to process ³⁵S- labeled procaspase-9 and -3. However, in the current study we have demonstrated that, during dATP-dependent activation of cell lysates, two large apoptosome complexes with M_r values of ~700 kDa and ~1.4 MDa were formed (Figs. 1 and 2). The rate of formation of the ~700-kDa complex was very rapid (within 5-10 min) compared with the formation of the ~1.4-MDa complex (~20 min). Moreover, the ~700-kDa complex efficiently processed exogenous caspases, whereas the ~1.4-MDa complex was relatively inactive (Figs. 1 and 2). Why the ~1.4-MDa apoptosome complex was incapable of caspase processing is unclear because both complexes contained oligomerized Apaf-1 and processed caspase-9 (Fig. 1). We cannot rule out the possibility that an inhibitor is associated with the larger complex or alternatively that the ~1.4-MDa complex may be inappropriately oligomerized and structurally different from the ~1.4-MDa complex previously described (17, 18). Nevertheless, the ~700-kDa complex does not appear to be formed when reconstitution of the apoptosome is carried out with recombinant Apaf-1, procaspase-9, cytochrome c, and dATP (17, 18).

The biological significance of two large Apaf-1 apoptosome complexes is as yet unclear. However, our experiments with apoptotic lysates provide important evidence which indicates that the ~700-kDa complex is the biologically significant complex that is formed during apoptosis. Thus, in THP.1 cells using two unrelated stimuli, i.e. a DNA topoisomerase II inhibitor (etoposide) and a serine chymotrypsin-like protease inhibitor (TPCK), we have demonstrated that the predominant complex formed is an ~700-kDa apoptosome complex (Fig. 3). Taken together our results suggest that the ~700-kDa complex is the major form of the apoptosome formed in vivo and that the correct assembly of the apoptosome may require other factors present in cell lysates, in addition to Apaf-1, cytochrome c, dATP, and caspase-9. Thus, clear differences exist in the apoptosome complexes formed in vitro from recombinant proteins when compared with those assembled in cell lysates and during apoptosis.

In summary, following dATP activation of lysates, two Apaf-1-containing complexes are formed, a biologically inactive ~1.4-MDa and a biologically active ~700-kDa apoptosome. Kinetic analysis demonstrates a more rapid formation of the smaller ~700-kDa complex, which exhibits significant caspase-activating activity. In apoptotic cells, the ~700-kDa complex predominates, and we therefore propose that the ~700-kDa complex is the functionally active form of the apoptosome in apoptosis.

	ACKNOWLEDGEMENTS

Anti-caspase-3 and -caspase-9 antibodies were kind gifts from Dr. D. W. Nicholson, Merck Frosst, Canada, and Dr. D. Green, La Jolla Institute for Allergy and Immunology, San Diego, CA, respectively. Rabbit anti-Apaf-1 was kindly provided by Dr. X. Wang, University of Texas Southwestern Medical Center, Dallas, TX.

	FOOTNOTES

^* 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.

Supported by a Glaxo Wellcome Post Graduate studentship award.

^§ To whom correspondence should be addressed: MRC Toxicology Unit, Hodgkin Bldg., University of Leicester, P. O. Box 138, Lancaster Rd., Leicester, LE1 9HN UK. Tel.: 0116 252 5601; Fax: 0116 252 5616; E-mail: gmc2@le.ac.uk.

² The aposome term has also been used to describe cytoplasmic protrusions released from cells (20). Therefore to avoid confusion we now refer to the ~700 kDa complex as the apoptosome.

	ABBREVIATIONS

The abbreviations used are: AFC, 7-amino-4-trifluoromethylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CARD, caspase recruitment domain; Z-DEVD.AFC, benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; TPCK, N-tosyl-l-phenylalanyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				J. V. Gerasimenko, O. V. Gerasimenko, A. Palejwala, A. V. Tepikin, O. H. Petersen, and A. J. M. Watson Menadione-induced apoptosis: roles of cytosolic Ca2+ elevations and the mitochondrial permeability transition pore J. Cell Sci., January 2, 2002; 115(3): 485 - 497. [Abstract] [Full Text] [PDF]

				K. Cain, C. Langlais, X.-M. Sun, D. G. Brown, and G. M. Cohen Physiological Concentrations of K+ Inhibit Cytochrome c-dependent Formation of the Apoptosome J. Biol. Chem., November 2, 2001; 276(45): 41985 - 41990. [Abstract] [Full Text] [PDF]