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Originally published In Press as doi:10.1074/jbc.M413269200 on December 7, 2004

J. Biol. Chem., Vol. 280, Issue 8, 6447-6454, February 25, 2005
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Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria*

Brian M. Polster{ddagger}§, Gorka Basañez||, Aitor Etxebarria||**, J. Marie Hardwick§, and David G. Nicholls{ddagger}

From the {ddagger}The Buck Institute for Age Research, Novato, California 94945, ||Unidad de Biofísica (Centro Mixto Consejo Superior de Investigaciones Cientificas), Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, P. O. Box 644, 48080 Bilbao, Spain, and §W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205

Received for publication, November 24, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal cell death in acute and chronic neurodegenerative disorders occurs by a variety of biochemical and morphological alterations that cannot always be classified into discrete pathways such as apoptosis and necrosis. Nevertheless, intracellular calcium deregulation and mitochondrial dysfunction are critical and ubiquitous mediators of neuronal demise (1). Calpains comprise a class of intracellular cysteine proteases that are activated by calcium (see Ref. 2 for review). There are two ubiquitous forms, calpain I (µ-calpain) and calpain II (m-calpain) that are activated by micromolar and millimolar concentrations of Ca2+ in vitro, respectively, as well as a number of tissue-specific isoforms. Several members of the Bcl-2 family of cell death regulators are processed by calpain (3), and calpain cleavage of Bid has been implicated in mitochondrial permeabilization and cell death following ischemia/reperfusion in the heart (4, 5). Although the sequence specificity of calpain proteolysis is incompletely understood and may depend in large part on the tertiary structure of the target protein, some calpain substrates are distinguished by a PEST sequence rich in proline (P), glutamate (E) (or aspartate), and serine (S)/threonine (T) residues (6, 7). PEST sequences are suggested to promote susceptibility to digestion by calpain, and actual proteolysis often occurs elsewhere in the protein (e.g. as in cleavage of I{kappa}B{alpha}) (8).

Apoptosis-inducing factor (AIF) 1 resides primarily in the mitochondrial intermembrane space in healthy cells and contains a predicted PEST sequence (amino acids 529–562)2 near the carboxyl terminus of the protein (9). AIF was suggested to play an essential role in calcium-mediated excitotoxicity initiated by N-methyl-D-aspartate stimulation of cultured neurons (10), an injury model involving calpain I activation (11, 12). AIF is thought to promote cell death by binding to DNA and inducing large scale (~50-kb pair) fragmentation in combination with cyclophilin A or other factors (13–15) and by increasing mitochondrial permeability in a feedback loop following its release (14, 16). Although AIF is nearly 4-fold larger in size than cytochrome c, translocation of AIF to the nucleus has been observed prior to the mitochondrial release of cytochrome c in several model systems of neuronal injury (17). Additionally, smaller forms of released AIF (50–65 kDa compared with ~67 kDa migration behavior of the full-length mature protein) have been detected in vivo following transient focal ischemia (18) and traumatic brain injury (19), as well as in a cell culture model of nitric oxide toxicity (20). Paradoxically, AIF mediates caspase-independent forms of cell death despite the finding that in HeLa cells and mouse embryonic fibroblasts, AIF release from mitochondria during apoptosis appears to require active caspases (21, 22).

Treatment of isolated mitochondria with calcium, agents that promote oxidative stress, or various recombinant proteins is an approach frequently employed to examine the mechanistic underpinnings of the release of toxic proteins normally confined to the intermembrane space (23). In this system, mitochondrial permeabilization by the pro-apoptotic Bcl-2 family members Bid and Bax failed to reconstitute the AIF redistribution observed in cell and tissue models, although apoptogenic cytochrome c, Smac/DIABLO, and Htra2/Omi were faithfully released (21). Multiple studies demonstrated that AIF is anchored to the outer face of the mitochondrial inner membrane, precluding its release following loss of outer membrane integrity (21, 22, 24). Because release of AIF therefore requires detachment or removal of a domain required for this inner membrane association, we tested the hypothesis that proteolysis by calpain I induces AIF release from Bid-permeabilized liver or brain mitochondria. Pure lipid vesicles of mitochondrial inner membrane composition were employed to directly assess the effect of calpain on the ability of AIF to associate with phospholipids.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Recombinant human Bid was purchased from R&D Systems (Minneapolis, MN). Active caspase-2, -3, and -8 were purchased from Gene Therapy Systems (San Diego, CA). Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC) fluorogenic caspase-3 substrate was obtained from Alexis Biochemicals (San Diego, CA). Calpeptin was from Calbiochem. PercollTM was purchased from Amersham Biosciences. Active calpain I purified from human erythrocytes was obtained from Calbiochem and dialyzed into calpain storage buffer (125 mM KCl, 2 mM K2HPO4, 1 mM EGTA, 1 mM EDTA, 5 mM {beta}-mercaptoethanol, 20 mM HEPES, pH 7.0) with 10,000 molecular weight cut-off Slide-A-Lyzer® mini-dialysis units (Pierce). Calpain I-specific activity was recalculated following dialysis by assessing the rate of cleavage of fluorogenic calpain-1 substrate (Calbiochem), and enzyme was stored in aliquots at –80 °C. AIF E-1 primary mouse monoclonal, Bid C-20 primary goat polyclonal, and His H-15 primary rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Cytochrome c 7H8.2C12 primary mouse monoclonal antibody was from BD Pharmingen. AIF primary rabbit polyclonal antibody (AB16501) was from Chemicon (Temecula, CA). Liver phosphatidylcholine, liver phosphatidylethanolamine, and heart cardiolipin were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). All other reagents were from Sigma.

Preparation of Mitochondria—Adult (>6 weeks old) C57/B6 mouse and Wistar rat liver mitochondria were isolated by standard differential centrifugation as described previously (25, 26). Non-synaptosomal adult Wistar rat forebrain mitochondria were isolated and purified on a PercollTM gradient as described by Kristian and Fiskum (26). Mitochondrial protein concentration was determined by the Bio-Rad Bradford method. Mitochondrial coupling was assessed polargraphically using a MIP-730 Dip-type O2 microelectrode (Microelectrodes, Inc., Bedford, NH) in a microcuvette (Hellma Cells, Inc., Plainview, NY) with a custom built airtight lid. Acceptor control ratios (ADP-stimulated state 3 rate to resting rate in the presence of oligomycin) for both liver and brain mitochondria were greater than 5 when measured as described previously (27).

Determination of Cytochrome c and AIF Release—Isolated liver or brain mitochondria (0.5 mg/ml) were incubated at 30 °C for 30–60 min in 100 µl of KCl medium (125 mM KCl, 2 mM K2HPO4, 20 mM HEPES, pH 7.0) supplemented with 5 mM succinate, 2 µM rotenone, 4 mM MgCl2, 3 mM ATP, and 1 mM ADP. Succinate was used as a substrate in the presence of rotenone to maintain pyridine nucleotides in a reduced state. However, similar results were obtained with mitochondria respiring on complex I substrates (malate and glutamate, 5 mM each). Calpain I, CaCl2, ruthenium red, calpeptin, EGTA, cyclosporin A (CsA), reduced glutathione, Bid, and caspase-2, -3, and -8 were present in the incubation mixture as noted in the figure legends at the indicated concentrations. Mitochondrial suspensions were centrifuged at 13,400 x g for 5 min at 4 °C at the conclusion of the incubation period. Supernatants were stored with Halt Protease Inhibitor Mixture® (Pierce) at –80 °C. Pellets also stored at –80 °C were resuspended in 100 µl of KCl medium containing 1% Triton X-100 and Halt Protease Inhibitor Mixture® prior to gel loading. Proteins retained in the mitochondrial pellet or released into the supernatant were separated by SDS-PAGE. AIF (1:500 anti-AIF), cytochrome c (1:2,000 anti-cytochrome c), and full-length Bid (1:500 anti-Bid) were detected by immunoblot as described previously (28).

Generation of Recombinant His-tagged Proteins—PET 30a(+) plasmids (Novagen, San Diego, CA) containing a six-histidine (His) tag fused to either the full-length coding sequence for rat AIF or the coding sequence for the mature rat protein (which lacks the first 100 amino-terminal amino acids) were a generous gift from Drs. G. Cao and J. Chen (University of Pittsburgh School of Medicine, Pittsburgh, PA). Plasmids encoding His-tagged proteins were transformed and amplified in Escherichia coli BL21pLysS. Protein expression was induced in 500 ml of bacterial cultures by the addition of 0.1 mM isopropyl-{beta}-D-thiogalactopyranoside for 4 h at 30 °C. Cells were disrupted by a 20-min incubation on ice in the presence of 10 µg/ml lysozyme (Sigma) and protease inhibitors, followed by sonication. His-tagged proteins were batch purified on nickel-nitrilotriacetic acid agarose resin (Qiagen, Valencia, CA), washed, and eluted according to the manufacturer's instructions. The plasmid used for the purification of His-BimEL lacking the carboxyl-terminal hydrophobic tail was a kind gift from Dr. H.-G. Wang (University of South Florida College of Medicine, Tampa, FL), and protein was purified as described (29). Proteins were dialyzed into 125 mM KCl, 20 mM HEPES, pH 7.0 at 4 °C using Slide-A-Lyzer® 10,000 molecular weight cut-off dialysis cassettes (Pierce) and stored at –80 °C.

In Vitro Cleavage Assays—For the calpain cleavage assay, His-tagged precursor or mature AIF (10 ng/µl) was incubated with 1.25–5 units/ml of calpain I, 200 µM CaCl2, and 1.5 mM dithiothreitol for 30 min at 30 °C in 20 µl of 30 mM Tris-HCl, pH 7.5. Calpeptin or EGTA were also present when indicated. For the caspase cleavage assay, His-tagged proteins (10 ng/µl) were incubated with 50 units/ml of caspase-2, -3, or -8 for 4 h at 37 °C in 20 µl of KCl medium (pH 7.0) containing 1 mM EDTA, 1 mM EGTA, and 10 mM dithiothreitol. All reactions were terminated by boiling in SDS-PAGE sample buffer for 10 min, protein was separated by 7.5% SDS-PAGE, and proteolytic processing was assessed by immunoblot.

Fluorogenic Caspase-3 Assay—Active caspase-3 was incubated at a concentration of 1.2 units/ml with 60 µM Ac-DEVD-AFC substrate in KCl medium (pH 7.0) containing 5 mM malate, 5 mM glutamate, 4 mM MgCl2,3mM ATP, 1 mM ADP, and 0.25 mM EGTA at 37 °C. Cleavage of the fluorogenic substrate was measured with a PerkinElmer LS50 B fluorimeter using excitation and emission wavelengths of 400 and 505 nm, respectively.

Binding of AIF to Large Unilamellar Vesicles (LUV)—LUV were prepared as described previously, with slight modifications (30). Briefly, a lipid mixture emulating the phospholipid composition of the mitochondrial inner membrane of mouse liver (31) (44 phosphatidylcholine:33 phosphatidylethanolamine:23 cardiolipin, weight/weight) was co-dissolved in chloroform:methanol (2:1), and organic solvents were removed by evaporation under an argon stream followed by incubation under vacuum for 2 h. After resuspension of dry lipid films in sucrose buffer (170 mM sucrose, 10 mM Hepes, pH 7.0), samples were subjected to freeze/thawing and extrusion through two 0.2-µm pore size filters (Nucleopore, San Diego, CA). Sucrose-loaded LUV were then diluted 10-fold with iso-osmotic buffer (100 mM KCl, 10 mM HEPES, pH 7.0), and the liposome suspension was centrifuged at 185,000 x gmax for 1 h. The washing procedure was repeated two more times, and the final pellet was resuspended in iso-osmotic buffer. Sucrose-loaded LUV were incubated in iso-osmotic buffer with proteins as indicated. LUV were subsequently pelleted by centrifugation at 185,000 x gmax for 1 h, and protein contents of supernatant and pellet fractions were determined by SDS-PAGE (7.5% gels) and silver staining. Under these conditions, more than 85% of LUV precipitated to the bottom of the tube, as determined by phosphorus analysis of pellet and supernatant fractions (32).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AIF Release from Isolated Liver or Brain Mitochondria Is Triggered by Calpain and Bid—To examine the ability of calpain I to cleave and release endogenous AIF from mitochondria, we initially used isolated mouse liver mitochondria as a model system because, in contrast to brain mitochondria, these mitochondria represent a homogenous population that is highly sensitive to mitochondrial outer membrane permeabilization by Bid (Fig. 1). Respiring mitochondria were incubated under physiologically relevant concentrations of KCl, Mg2+, and adenine nucleotides, and the Ca2+ uptake inhibitor ruthenium red was included to enable Ca2+ activation of calpain without accumulation of calcium in the mitochondrial matrix. Although measured calcium concentrations as low as 2–3 µM were capable of activating purified calpain I (not shown) consistent with previous reports (2), 200 µM added Ca2+ was employed in this study to overcome EGTA present in the calpain storage buffer (see "Experimental Procedures") and ensure maximal activation.



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  		FIG. 1.
Experimental scheme. Added CaCl2 (200 µM) activates calpain I in the presence of ruthenium red (1µM), which is present to inhibit uptake of Ca2+ into the mitochondrial matrix (Step 1). Calpain I (2.5 units/ml) cleaves Bid (100–200 nM) into a more active form (calpain-cleaved Bid, Step 2), which then induces Bak oligomerization and mitochondrial outer membrane permeabilization (Step 3), granting calpain access to the intermembrane space (IMS). Calpain I then cleaves AIF (Step 4), releasing truncated AIF (tAIF) from an association with the mitochondrial inner membrane. Cytochrome c (C) and tAIF released across the permeabilized outer membrane are then measured by immunoblot in the supernatant of the centrifuged mitochondrial suspension (Step 5).

 
Treatment of mouse liver mitochondria with calpain I (2.5 units/ml) induced slight release of cytochrome c and AIF that was inhibited by the specific calpain inhibitor calpeptin (Fig. 2A, lanes 2 and 3). Although full-length Bid (100 nM) induced essentially complete cytochrome c release from mitochondria (lane 4), indicative of outer membrane permeabilization, AIF redistribution was not detected. However, when mitochondria were treated with the same amount of active calpain I (2.5 units/ml) in the presence of Bid (100 nM), a dramatic enhancement of AIF release occurred compared with calpain treatment alone (compare lane 6 to lane 2). In contrast, co-incubation of Bid with caspase-8 (10 units/ml) or a combination of caspase-8 and caspase-2 or -3 (10 units/ml) led to little AIF release (lanes 9–11). Decreased immunodetection of full-length Bid (see Fig. 3) and cleavage of the fluorogenic caspase-3 substrate Ac-DEVD-AFC (Fig. 2B) confirmed caspase activity under the experimental conditions employed. AIF that was released into the supernatant migrated faster than AIF retained in the mitochondria (see Fig. 4B), suggesting that proteolytic processing had occurred.



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  		FIG. 2.
Active calpain I induces AIF release from Bid-permeabilized liver mitochondria. A, isolated mouse liver mitochondria (0.5 mg/ml) were incubated with 200 µM Ca2+ and 1 µM ruthenium red for 30 min under the conditions described under "Experimental Procedures". Calpain (2.5 units/ml), caspase-2 (casp-2, 10 units/ml), caspase-3 (casp-3, 10 units/ml), caspase-8 (casp-8, 10 units/ml), calpeptin (calpep, 10 µM), and Bid (100 nM) were present in the incubation mixture where indicated. Reduced glutathione (2 mM) was also present to inhibit oxidation of cysteine proteases. In the lane denoted cBid (calpain-cleaved Bid) + calpain + calpep, Bid (100 nM) was preincubated with calpain I (2.5 units/ml) for 15 min prior to addition to the mitochondria, and the 30-min incubation of this mixture with mitochondria was performed in the presence of calpeptin (10 µM). Cytochrome c and AIF release were measured by immunoblot of supernatants following pelleting of the mitochondrial suspensions. Numbers indicate lane number. B, caspase-3 (1.2 units/ml) was added to medium containing 60 µM Ac-DEVD-AFC substrate under the conditions described under "Experimental Procedures," and cleavage was measured flourometrically.

 



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  		FIG. 3.
Active calpain I also induces AIF release from Bidpermeabilized brain mitochondria. Isolated rat brain mitochondria (0.5 mg/ml) were incubated for 30 min as in Fig. 2. All treatments were identical to those described in Fig. 2 for mouse liver mitochondria, with the exception that 200 nM rather than 100 nM Bid was used. Cytochrome c and AIF release were measured in the supernatants as in Fig. 2, and the relative amount of full-length Bid in these supernatants following protease incubation was also measured. Numbers indicate lane number. The Bid antibody was not sensitive enough to detect truncated Bid in either supernatants or pellets.

 



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  		FIG. 4.
Ca2+-induced permeability transition promotes calpeptin-sensitive AIF release from liver mitochondria. A, isolated mouse liver mitochondria (0.5 mg/ml) were incubated for 1 h at 30 °C in 100 µl of KCl medium (pH 7.0) containing 5 mM succinate, 2 µM rotenone, 3mM ATP, 1 mM ADP, and either 0.25 mM EGTA (control), 50 nmol Ca2+/mg mitochondrial protein, or 500 nmol Ca2+/mg mitochondrial protein. Cyclosporin A (CsA, 1 µM) or calpeptin (calpep, 100 µM) were present where indicated. AIF and cytochrome c release were detected by immunoblot in the supernatant as described in Fig. 2. B, isolated mouse liver mitochondria (0.5 mg/ml) were incubated as in panel A but for 30 min in the presence of 100 nM Bid, 200 µM Ca2+,1 µM ruthenium red, and 2 mM reduced glutathione with either calpain I (2.5 units/ml) or caspase-8 (10 units/ml) (lanes 1–3) or in the presence of 500 nmol Ca2+/mg mitochondrial protein with or without calpeptin (calpep, 10 µM) (lanes 4 and 5). AIF cleavage was assessed in supernatant (S) and pellet (P) fractions by immunoblot of proteins transferred from two separate 4–20% Tris-HEPES gradient SDS-PAGE gels run for 3 h at 150 mV. The left panel depicts immunodetection results using a polyclonal antibody raised against a carboxyl-terminal immunogen (amino acids 517–531, Chemicon AB16501), whereas a monoclonal antibody raised against an amino-terminal immunogen (amino acids 1–300, Santa Cruz sc-13116) was used for the immunoblot in the right panel.

 
Bid is cleaved by calpain, as well as by caspases, into a truncated form with higher activity (4, 23). Therefore, the ability of calpain to release AIF in the presence of Bid could be due to calpain-mediated enhancement of Bid-induced pore size, enabling AIF passage, or to increased access of calpain to the intermembrane space, enabling direct cleavage of AIF by calpain. To help distinguish between these possibilities, Bid was preincubated with calpain I to induce processing and this protein mixture was added to the mitochondria in the presence of calpeptin. In the presence of calpain I, but the absence of proteolytic activity, calpain-cleaved Bid failed to release AIF (Fig. 2A, lane 8), demonstrating that calpain activity downstream of Bid cleavage is required for AIF release.

Because AIF has been implicated in neuronal cell death, we performed a similar experiment using isolated rat brain mitochondria to confirm the relevance of using liver mitochondria as a model system. As in liver mitochondria, calpain I alone (2.5 units/ml) induced slight AIF release (Fig. 3, lane 2). Treatment of brain mitochondria with unprocessed Bid (200 nM) failed to induce AIF release (Fig. 3, lane 5), as was the case for liver mitochondria (100 nM Bid, Fig. 2, lanes 4, 5, and 7). In contrast to mouse liver mitochondria, unprocessed Bid also failed to induce cytochrome c release (Fig. 3, lane 5). Activation of Bid by co-incubation with calpain I (2.5 units/ml, lane 4) or caspase-8 (10 units/ml, lane 7) led to cytochrome c release. AIF release, however, occurred only in the presence of Bid and calpain I (lane 4). As in liver mitochondria, Bid that was precleaved with calpain I (calpain-cleaved Bid) failed to release AIF in the absence of calpain activity (lane 6). An immunoblot for Bid confirmed that full-length Bid was proteolyzed to a similar extent in both cases (compare lanes 4 and 6 to lane 5) and that Bid was also cleaved by caspase-8 (lane 7).

Calcium-induced AIF Release from Mouse Liver Mitochondria Requires Calpain—To determine whether an endogenous mitochondrial calpain-like enzyme can mediate AIF release in response to calcium-induced permeability transition, mouse liver mitochondria were overloaded with calcium in the absence and presence of calpeptin. Calcium alone (50 nmol/mg mitochondrial protein) induced both cytochrome c and AIF release from isolated mouse liver mitochondria that was inhibited by CsA (1 µM), a blocker of the classical inner membrane permeability transition pore (Fig. 4A). Released AIF was consistent in size with AIF released by a combination of Bid and calpain (Fig. 4B), and further proteolytic products were not apparent with antibodies raised against either a carboxyl- or an amino-terminal immunogen. CsA was much less effective at inhibiting permeability transition pore opening in response to 500 nmol/mg Ca2+, as evidenced by a reduction in its ability to prevent cytochrome c and AIF release. In contrast to CsA, calpeptin completely inhibited AIF release. However, calpeptin displayed no ability to block the redistribution of cytochrome c even at concentrations up to 100 µM, suggesting that mitochondrial permeability transition was not prevented.

Calpain Cleaves Recombinant AIF—To confirm that AIF can be directly cleaved by calpain, recombinant His-tagged precursor and mature forms of AIF were purified from E. coli and incubated in vitro with active calpain I. Although a few faster migrating bands likely representing degradation products were present, recombinant mature His-AIF migrated at ~67 kDa, consistent with endogenous mitochondrial AIF. His-AIF precursor, containing mitochondrial targeting sequence normally removed upon import, migrated at ~75 kDa. Mature AIF incubated with calpain I in the presence of calcium migrated as a slightly (~2–3 kDa) faster doublet on an SDS-PAGE gel than the protein treated with calcium alone (Fig. 5A). The calpain inhibitor calpeptin and the calcium chelator EGTA both prevented this change in migration, indicating that Ca2+-dependent calpain proteolysis was responsible. When an antibody to the amino-terminal histidine tag rather than an AIF-specific antibody was used to probe for AIF, AIF could not be detected following calpain treatment (Fig. 5B), suggesting that cleavage had occurred at the amino-terminal end of the protein.



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  		FIG. 5.
Recombinant His-tagged mature and precursor AIF proteins are directly cleaved by calpain I, but not by caspase-2, -3, or -8. A, mature His-AIF (10 ng/µl) was incubated with 200 µM Ca2+ alone, 200 µM Ca2+ plus 5 mM EGTA, or 200 µM Ca2+ and the indicated concentration of calpain I in units/ml (U/ml). EGTA (5 mM) or calpeptin (10 µM) was also present where indicated. Protein was detected using an antibody to AIF. B, the blot in panel A was probed using an antibody to His. C, the identical experiment to panel A was performed using recombinant His-AIF precursor protein rather than the mature protein. D, mature His-AIF was incubated with the indicated concentrations of caspase or calpain I under the conditions described under "Experimental Procedures." Protein was detected using an antibody to AIF. E, His-BimEL was either incubated with 50 units/ml caspase-3 or mock-treated as in panel D, and protein was detected using anti-His antibody.

 
AIF is translated as a precursor protein in cells, and the amino-terminal 101 amino acids containing two consecutive mitochondrial targeting sequences are removed upon mitochondrial import (14, 33). The cleavage products (~64–65 kDa) generated by the incubation of His-tagged AIF precursor protein (~75 kDa) with calpain were similar in size to that generated by proteolysis of mature (~67 kDa) AIF (Fig. 5C), suggesting that calpain cleavage occurs just downstream of the site normally processed to generate the mature protein.

Because AIF release is dependent upon caspase activation in some cell types induced to undergo apoptosis (21, 22), we tested whether caspases could directly cleave AIF. Recombinant mature His-tagged AIF was not cleaved by active caspase-2, -3, or -8 in vitro (Fig. 5D). In contrast, caspase-3 cleaved recombinant His-BimEL under identical incubation conditions (Fig. 5E), confirming that caspases were active.

Calpain Cleavage Triggers AIF Detachment from Pure Lipid Vesicles—Mitochondrial subfractionation studies demonstrated that AIF is associated with mitoplast preparations (21, 22, 29), raising the possibility that AIF is anchored to the lipid bilayer of the mitochondrial inner membrane. To test this possibility, we examined the binding of recombinant mature AIF to LUV bearing the phospholipid composition of the mitochondrial inner membrane (31). When recombinant mature AIF was incubated with sucrose-loaded LUV followed by separation of free AIF and LUV-associated AIF by centrifugation, the majority of the protein was found associated with LUV (Fig. 6A). Furthermore, treatment of LUV containing bound AIF with calpain I, but not caspase-8, substantially reduced this association (Fig. 6B). AIF released from the lipid membrane was processed, and EGTA (5 mM) prevented both processing and release. These results are consistent with the hypothesis that calpain proteolysis of AIF at the mitochondria liberates a domain of AIF responsible for its association with phospholipids of the mitochondrial inner membrane.



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  		FIG. 6.
Calpain I mediates detachment of AIF from LUV of mitochondrial inner membrane phospholipid composition. A, mature His-AIF (0.4 µg) was incubated with or without sucrose-filled LUV (1 mM) in iso-osmotic buffer for 1 h at 37 °C in 30 µl, followed by centrifugation at 185,000 x g for 1 h, and determination of protein contents in LUV-free supernatant (S) and LUV-containing pellet (P) fractions by SDS-PAGE and silver staining. B, His-AIF (0.4 µg) incubated with or without LUV as described in panel A was further treated for 2 h at 37 °C with calpain I (0.8 units/ml) in iso-osmotic buffer supplemented with 500 µM CaCl2, 1 mM dithiothreitol and with 5 mM EGTA where indicated or with caspase-8 (20 units/ml) in iso-osmotic buffer supplemented with 1 mM EDTA and 5 mM dithiothreitol. LUV-free (S) and LUV-associated (P) fractions were separated by centrifugation and protein contents determined by SDS-PAGE and silver staining. Results are representative of four independent experiments obtained with two different liposome populations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism of AIF redistribution from mitochondria to the nucleus has been a subject of much contention (17). In this study, we defined a novel mechanism of AIF release that is mediated by direct proteolysis of the protein by calpain, removing its association with the mitochondrial inner membrane. We assessed whether the BH3-only protein Bid was able to release AIF either alone or by increasing the accessibility of proteases to the intermembrane space. We found that the BH3-only protein Bid induces mitochondrial outer membrane permeability to cytochrome c in both mouse liver (Fig. 2) and rat brain (Fig. 3) mitochondria following cleavage, consistent with previous studies showing that truncated Bid induces pore formation by Bak in the outer membrane (23, 34). AIF was not released from either liver or brain mitochondria by caspase- or calpain-cleaved Bid in the absence of protease activity under the same conditions where cytochrome c release was observed.

One explanation for the release of cytochrome c (12.5 kDa) but not AIF (57-kDa theoretical molecular mass) following Bid permeabilization of mitochondria is that Bid induces the formation of a pore of a discrete size that is not large enough for AIF to permeate. Alternatively, the pore induced by Bid may be of sufficient size to allow AIF passage, but an association of AIF with proteins and/or lipids within the intermembrane space may preclude its release. To distinguish between these possibilities, we examined whether AIF could be cleaved and released by calpain in the absence or presence of Bid. We found that calpain I was able to directly cleave mitochondrial AIF to a slightly smaller form. This smaller form was dissociated from mitochondria and found in the supernatant following centrifugation of mitochondrial suspensions (Figs. 2 and 3). Recombinant AIF associated directly with vesicles mimicking the mitochondrial inner membrane in lipid composition, and calpain proteolysis also mediated AIF dissociation from these vesicles (Fig. 6).

In the absence of Bid, little AIF was cleaved and released by calpain I from either liver or brain mitochondria (Figs. 2 and 3), consistent with the inaccessibility of the intermembrane space to external proteases. This nominal release was most likely due to access of calpain I to AIF in the small percentage of broken mitochondria that contributes to the 5–8% background cytochrome c release typically observed in these experiments (see Ref. 35 and Fig. 3, control). When Bid was added in conjunction with the same concentration of active calpain I, AIF proteolysis and release from both liver and brain mitochondria were enhanced despite the finding that Bid alone did not promote AIF release. This result suggests that Bid induces an outer membrane pore large enough for calpain I (a 112-kDa heterodimer composed of catalytic and regulatory subunits) to enter the mitochondrial intermembrane space and cleave AIF. This also argues against the hypothesis that the inability of full-length or cleaved Bid to release AIF is because of an insufficiently large increase in outer membrane permeability. Release of 2000-kDa dextrans by caspase-8-cleaved Bid has been observed from proteoliposomes composed of mitochondrial outer membranes (36), but to our knowledge this is the first direct demonstration of the passage of a protein >110 kDa across the outer membrane in isolated mitochondria treated with Bid without a requirement for cytosolic factors. Nevertheless, we cannot exclude the possibility that calpain itself contributes to the extent of outer membrane permeabilization by Bid by modifying mitochondrial substrates that have yet to be identified.

The enhancement of calpain-induced release from brain mitochondria by Bid was less pronounced than that observed from liver mitochondria (compare Figs. 2 and 3). This is likely due to the additional requirement for Bid cleavage to increase outer membrane permeability in brain mitochondria, leading to a kinetic delay in the ability of calpain I to access the intermembrane space. Treatment of liver mitochondria with a lower Bid concentration (20 nM) in the presence of calpain I also led to less enhancement of AIF release compared with background AIF release with calpain I alone.3 Calpain cleavage of Bid was required for cytochrome c release at this concentration of Bid, as was the case for brain mitochondria. Experiments are underway to examine the kinetics of AIF release by calpain in the presence of Bid.

To further demonstrate that AIF is most likely directly cleaved by calpain I rather than another endogenous intermembrane space protease in mitochondria treated with calpain and Bid, we produced recombinant His-tagged precursor and mature forms of AIF and incubated purified protein directly with calpain I. Calpain I cleaved AIF in the absence of other mitochondrial proteins in a Ca2+-dependent fashion (Fig. 5). Truncated AIF could be recognized by an AIF antibody, but not by a His antibody, suggesting that cleavage of AIF occurred at sites very close to the amino terminus, leading to the removal of the 6-histidine epitope. Calpain I also cleaved AIF precursor protein into two closely migrating bands similar in size to those produced upon cleavage of mature recombinant AIF or endogenous mitochondrial AIF (compare Fig. 5C to 5A and 4B). This confirms that AIF is cleaved near the amino rather than the carboxyl terminus, because a carboxyl-terminal cleavage of AIF precursor would produce a larger product than calpain-processed mature AIF due to the additional sequence at the amino terminus. Because calpain cleavage sites are most commonly found in unstructured regions of proteins, we predict that proteolysis occurs within or slightly downstream of amino acids 102–121 of AIF, an unstructured domain at the amino terminus of the mature protein (102 being the first amino acid) that precedes the FAD binding domain (9, 15). Our finding that proteolysis of AIF near the amino terminus induces its release is consistent with a recent study (24) that used antibody epitope mapping combined with proteinase K treatment to demonstrate that the domain mediating attachment of mature AIF to the mitochondrial inner membrane was located within the first 100 amino acids.

It is important to note that the molecular mass of the primary AIF band detected in mitochondria by immunoblot is ~67 kDa. AIF, like many flavin-containing proteins, migrates slower than its predicted molecular mass (57 kDa) (37). Because AIF is translated as a precursor protein before being clipped upon mitochondrial import, this has often created confusion in the identification of AIF by SDS-PAGE, with the 67-kDa band sometimes identified as precursor AIF protein, or mature AIF sometimes identified as 57 kDa without the use of molecular mass markers. We found that recombinant AIF protein corresponding to the mature form and containing a small 6-histidine tag migrated at ~67 kDa, similar to endogenous protein, whereas precursor His-AIF migrated at ~75 kDa. Our finding that AIF can be processed into closely migrating smaller forms (~64–65 kDa) by calpain additionally stresses the importance of showing molecular mass markers when analyzing AIF by SDS-PAGE.

One of the most puzzling conundrums in the cell death field concerns the repeated observation of AIF translocation to the nucleus prior to cytochrome c release from mitochondria (10, 17, 38, 39). Notably, calpain cleavage within or downstream of amino acids 102–121 of AIF precursor protein would remove the mitochondrial targeting peptide. This presents the possibility that intracellular Ca2+ elevations (e.g. in excitotoxic injury paradigms) that activate calpain prevent AIF located in the cytosol or associated with other intracellular organelles from targeting the mitochondria by removing the signal sequence. This would be predicted to lead to the accumulation of AIF in the cytosol and nucleus (based on its two nuclear localization sequences (14)), giving the appearance of AIF translocation from mitochondria prior to mitochondrial permeabilization. Consistent with this hypothesis, subcellular fractionation and immunodetection of AIF has demonstrated an increase of AIF in the nuclear fraction without a readily apparent decrease in the mitochondrial fraction (10).

Mitochondrial Ca2+ overload has been widely hypothesized to play a role in releasing mitochondrial intermembrane space proteins by permeability transition-induced outer membrane rupture (23, 40), and a previous study demonstrated AIF release from isolated mouse liver mitochondria following permeability transition (41). We confirmed previous observations of CsA-suppressible AIF release from isolated mouse liver mitochondria. However, we also extended these findings by showing that the specific calpain inhibitor calpeptin completely inhibited AIF release without impairing permeability transition pore-mediated release of cytochrome c (Fig. 4A). This observation, together with the finding that AIF released from mitochondria following Ca2+ overload was comparable in size with calpain I-cleaved AIF (Fig. 4B), implicates the involvement of an endogenous mitochondrial calpain-like enzyme in permeability transition pore-induced AIF redistribution. Preliminary experiments have detected calpain activity and multiple calpain isoforms in our preparations of isolated liver and brain mitochondria,3 consistent with a role for mitochondrial calpain in AIF release.

In some cell types, AIF release during apoptosis has been shown to be dependent on caspase activation (21, 22), whereas in other death paradigms, including excitotoxic stimulation of cortical neurons (10), caspase inhibition did not impair AIF translocation to the nucleus. Therefore we investigated whether caspases, like calpain I, could promote AIF release. Recombinant His-AIF was not cleaved by caspase-2, -3, or -8 under conditions where processing of another His-tagged protein by caspase-3 occurred (Fig. 5). Caspase-8 was able to induce the release of a small fraction of AIF from isolated liver mitochondria in the presence of Bid (Fig. 2). AIF release was not noticeably enhanced by the co-presence of caspase-2, -3 (Fig. 2) -6, or -73 and did not occur with full-length Bid despite similar amounts of cytochrome c release. We did not investigate the mechanism of minor AIF release by caspases in the present study; however, the size of released AIF was consistent with calpain cleavage (Fig. 4B) and may reflect the ability of caspases to cleave the endogenous calpain inhibitor protein calpastatin (42, 43).

Pathological cell death does not always occur by a discrete mechanism that can be labeled apoptosis or necrosis but can be characterized by a continuous spectrum of events that occur in dying cells, depending on the intracellular and extracellular milieu (40). Thus, in classical apoptosis (e.g. that induced by the protein kinase inhibitor staurosporine), AIF release may occur by a pathway involving caspase activation, consistent with some mammalian cell models (21, 22) and with the requirement for the caspase-3-like enzyme CED-3 for the release of the Caenorhabditis elegans AIF homologue Wah-1 (44). However, in other forms of death that require intracellular Ca2+ deregulation, we propose that AIF redistribution within the cell is mediated by calpain processing. AIF translocation and apoptosis in rat hippocampal neurons induced by hydrogen peroxide or by the pneumococcal toxin pneumolysin were both inhibited by Ca2+ chelation with BAPTA-AM (45), but not by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (46), supporting a caspase-independent role for Ca2+ in AIF redistribution in intact neurons.

Calpains cleave multiple substrates potentially involved in cell death, including cyclin-dependent kinase-5 (47), the plasma membrane Ca2+ ATPase isoform 1 (48), and calcineurin (49). Additionally, calpain inhibition is neuroprotective in vivo (49–52) as well as in cell culture (12, 49). The present study has identified AIF as a novel calpain substrate that has been implicated in neuronal death, demonstrating that its proteolysis regulates detachment from an association with the mitochondrial inner membrane and release from the intermembrane space. The requirement for low micromolar concentrations of calcium for calpain I activation suggests that calpain activation may follow Ca2+ deregulation in cells, because intracellular buffering and Ca2+ extrusion systems maintain intracellular calcium below 0.5 µM in healthy cells (1, 53). However, close spatial coupling of calpain to N-methyl-D-aspartate receptors, putative calpain-activating proteins, or intracellular Ca2+ stores may enable transient calpain activation to occur prior to a catastrophic Ca2+ crisis (11, 54). Future studies will be geared toward defining the time course of calpain activation in relationship to intracellular calcium changes in neurons, as well as assessing whether AIF cleavage by calpain plays a role in calcium-mediated excitotoxicity.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants NS44791 (to B. M. P.), NS73581 (to J. M. H.), and NS41908 (to D. G. N.). This work was also supported in part by funds from the Ministerio de Ciencia y Tecnología, Spain, Grant BMC 2002-00784 (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** A predoctoral student supported by the Basque government. Back

To whom correspondence should be addressed: The Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945. Tel.: 415-209-2209; Fax: 415-209-2232; E-mail: bpolster{at}buckinstitute.org.

1 The abbreviations used are: AIF, apoptosis-inducing factor; Ac-DEVD-AFC, Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; CsA, cyclosporin A; His, 6-histidine; LUV, large unilamellar vesicle. Back

2 A PESTfind score of +11.97 was determined for this region using the PESTfind algorithm available at www.at.embnet.org/embnet/tools/bio/PESTfind/. Back

3 B. M. Polster, A. Etxebarria, G. Basañez, J. M. Hardwick, and D. G. Nicholls, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. J. Chen, G. Cao, and H.-G. Wang for the DNA plasmids used to produce recombinant proteins, Drs. J. Gafni, L. M. Ellerby, and R. Rao for laboratory reagents, and Dr. J. T. Russell for critically reading the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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