A mechanistic view of mitochondrial death decision pores · 1011 Braz J Med Biol Res 40(8) 2007 Mitochondrial pores A mechanistic view of mitochondrial death decision pores 1Departamento

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A mechanistic view of mitochondrialdeath decision pores

1Departamento de Farmacologia, Instituto de Ciências Biomédicas,2Laboratório de Imunopatologia, Instituto de Medicina Tropical,Universidade de São Paulo, São Paulo, SP, Brasil

J.E. Belizário1, J. Alves1,J.M. Occhiucci1,

M. Garay-Malpartida1

and A. Sesso2

Abstract

Mitochondria increase their outer and inner membrane permeability tosolutes, protons and metabolites in response to a variety of extrinsicand intrinsic signaling events. The maintenance of cellular andintraorganelle ionic homeostasis, particularly for Ca2+, can determinecell survival or death. Mitochondrial death decision is centered on twoprocesses: inner membrane permeabilization, such as that promotedby the mitochondrial permeability transition pore, formed across innermembranes when Ca2+ reaches a critical threshold, and mitochondrialouter membrane permeabilization, in which the pro-apoptotic proteinsBID, BAX, and BAK play active roles. Membrane permeabilizationleads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cy-tochrome c initiates the proteolytic activation of caspases, which inturn cleave hundreds of proteins to produce the morphological andbiochemical changes of apoptosis. Voltage-dependent anion channel,cyclophilin D, adenine nucleotide translocase, and the pro-apoptoticproteins BID, BAX, and BAK may be part of the molecular composi-tion of membrane pores leading to mitochondrial permeabilization,but this remains a central question to be resolved. Other transportingpores and channels, including the ceramide channel, the mitochon-drial apoptosis-induced channel, as well as a non-specific outer mem-brane rupture may also be potential release pathways for theseapoptogenic factors. In this review, we discuss the mechanistic mod-els by which reactive oxygen species and caspases, via structural andconformational changes of membrane lipids and proteins, promoteconditions for inner/outer membrane permeabilization, which may befollowed by either opening of pores or a rupture of the outer mitochon-drial membrane.

CorrespondenceJ.E. Belizário

Departamento de Farmacologia

ICB, USP

Av. Lineu Prestes, 1524

05508-900 São Paulo, SP

Brasil

Fax: +55-11-3091-7322

E-mail: [email protected]

Research supported by FAPESP

(No. 01/01000-7), CAPES and CNPq.

Received October 27, 2005

Accepted February 16, 2007

Key words• Mitochondrial outer

membrane permeabilization• Permeability transition pore• Cytochrome c• Reactive oxygen species• Caspases• BCL-2

Introduction

Recent studies have identified, amongthe variety of ways by which cells have beenreported to die, three major forms of celldeath: apoptosis, necrosis and autophagy.

These forms of cell death may follow precisedeath programs that have been characterizedat the morphological and biochemical level(1). Apoptosis is a major mechanism for theremoval and control of superfluous and dam-aged cells during development and normal

Brazilian Journal of Medical and Biological Research (2007) 40: 1011-1024ISSN 0100-879X Review

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adult life (1). Research conducted over thepast few years has provided evidence thatdiverse extracellular and intracellular sig-naling events within apoptosis converge to-ward mitochondria (2-7). In mitochondri-ally mediated apoptosis, inner and/or outermitochondrial membranes undergo a per-meabilization process that causes the releaseand redistribution of small ions, solutes andmetabolites, as well as of cytochrome c, a14-kDa protein that functions as an electroncarrier in the mitochondrial respiratory chain.In mammals, cytochrome c is a necessaryco-factor for activation of caspase-9, a mem-ber of the protease family that coordinatesthe biochemical and morphological eventsof apoptosis (1-7).

It is presumed that cytochrome c andother apoptogenic factors, including Smac/Diablo, HtrA2/Omi and apoptosis-inducingfactor (AIF), are released as a result of theopening of large non-selective pores knownas “permeability transition pores” (PTP) (1,7)or through a proteolipid pore spanning onlythe outer mitochondrial membrane (2,6). Invitro and in vivo studies have demonstratedthat cytoplasmic and mitochondrial proteincomplexes and the membrane lipid environ-ment are involved in the formation and func-tion of these putative pores. Proteins of theBCL-2 family with pro-apoptotic propertiessuch as BID, BAX, and BAK may cooperatein the formation of these pores, together withthe major mitochondrial voltage-dependentanion channel known as VDAC, adenylatenucleotide translocase (ANT) and cyclophi-lin D (Cyclo D). There is also evidence thatthe release of apoptogenic factors could oc-cur through a putative channel named mito-chondrial apoptosis-inducing channel orMAC (8) and through a large channel con-taining ceramide lipids, i.e., the ceramidechannel (9). A rupture of the outer mito-chondrial membrane could also work as apathway for the release of apoptogenic pro-teins (10).

The formation of these pores in the inner

and outer membrane depends on a variety ofbioenergetic, membrane transport and redoxconditions that ultimately lead to majorchanges in the structure of mitochondrialproteins and lipids. Reactive oxygen species(ROS) generated inside and outside the mi-tochondria are important promoters of chem-ical modification and conformationalchanges of membrane polypeptides and lip-ids (11,12). Caspases are cysteine proteasesthat play a central role in intracellular pro-teolytic pathways by inducing structural andfunctional changes in various vital proteinsinvolved in apoptosis and various other non-apoptotic processes such as inflammation,cell cycle and differentiation (13,14). Vari-ous procaspases and active caspases, includ-ing -3, -7, -8, and -9, are localized in, ortranslocate to, mitochondria during apopto-sis, perhaps controlling the permeabilizationof this organelle (15). More recently, thecritical involvement of caspases in mito-chondrially mediated apoptosis has beendemonstrated using mice lacking caspase-3and -7 (16). The key concept in this model isthat certain critical regulators located in theouter and inner mitochondrial membranes orwithin the matrix could act as caspase sub-strates. Thus, the point-specific cleavage ofone site of substrate proteins could be acommitment step toward membrane perme-abilization.

Reactive oxygen species-inducedmitochondrial structuralmodifications and permeabilitytransition pores

All mammals use O2 for energy produc-tion (11,12). Oxidation is the loss of anelectron by a substance. Under normal meta-bolic conditions, electron-transporting com-plexes I, II, III, and IV plus a non-redox H+-translocating complex, ATP synthase (alsocalled complex V, FoF1-ATP synthase), to-gether with co-enzyme Q and cytochrome c,carry out oxidative phosphorylation. Com-

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plex II is completely encoded by the nucleus,whereas complexes I, III and IV are encodedby nuclear and mitochondrial DNA. Therespiratory enzyme complexes transfer elec-trons (H-→H+ + 2e) from the reducingequivalents NADH or FADH2 to O2, whiletransporting protons across the inner mito-chondrial membrane. The total proton-mo-tive force across the inner mitochondrialmembrane is the sum of a large force derivedfrom the mitochondrial membrane electricalpotential (ρΨm) and a smaller force derivedfrom the H+ concentration gradient (∆pH).This proton-motive force is used to driveprotons from the intermembrane space intothe matrix through ATP synthase, a trans-membrane protein complex that uses theenergy of H+ flow to synthesize ATP fromADP and Pi. This electrochemical protongradient is also required to import mitochon-drial proteins and to regulate metabolite trans-port across the mitochondrial membrane(11,12).

A small percentage of the total O2 con-sumed by the mitochondrial electron trans-port chain in healthy tissues becomes ROS,such as superoxide (O2

.-), hydrogen perox-ide (H2O2) and hydroxyl radical (OH-)(11,12). This ROS production occurs prima-rily in complex I (NADH dehydrogenase)and complex III (ubiquinone-cytochrome creductase). O2 itself is also a free radicalbecause is has two unpaired electrons in itsouter orbit which make it reactive. The twounpaired electrons in O2 have parallel spins,which means that O2 can only oxidize an-other molecule by accepting a pair of elec-trons that have antiparallel spins or one elec-tron at a time. Superoxide has one electronmore than O2. Since only one electron isunpaired in O2

.-, superoxide is more reactivethan O2. However, O2

.- is still not a veryreactive radical, and, in the presence of H+ orHO2

., can reduce O2.- to H2O2 or be oxidized

to O2 (12).

H2O2 is more stable than O2.- and quickly

diffuses across membranes. In the presence

of iron in the ferrous form (Fe2+), H2O2

can be reduced to the highly reactive OH.

radical through the Fenton reaction. O2.- can

react with reactive nitrogen species, suchas nitric oxide and nitrogen dioxide (NO2)to form peroxynitrite (ONOO-). Both theoxygen- and nitric oxide-based radicals at-tack DNA, proteins, lipids, and carbohy-drates to produce DNA strand breaks, pro-tein oxidation and lipid peroxidation. Theamino acids tyrosine, histidine, arginine,lysine, and proline are particularly vulner-able to ROS modification, which translatesto gain or loss of receptor activity, enzymefunction and signal transduction pathways(12,17,18).

Efficient biochemical and bioenergeticmechanisms aimed at controlling ROS pro-duction and ensuring its removal haveemerged (11,12). The first antioxidant en-zyme described is superoxide dismutase(SOD). This enzyme catalyzes the reactionthat converts two O2

.- and two H+ to H2O2

and O2. Three isoforms of this enzyme havebeen well characterized; SOD1, a copper/zinc (Cu/Zn) isoform present in the cytosol;SOD2, a manganese (Mn) isoform present inmitochondria, and SOD3, a Cu/Zn isoformpresent in the extracellular space. Cysteine,glutathione, ascorbic acid (vitamin C), andα-tocopherol (vitamin E) are other impor-tant antioxidants that limit injuries inducedby ROS (11,12).

At the physiological level, both H2O2 andsuperoxide (O2

.-) can act as second messen-gers in cellular signaling that leads to theactivation and inactivation of ion channels,receptors, enzymes (kinases and phospha-tases), and transcription factors (12,17,18).However, above a threshold level, ROS canrandomly attack macromolecules, interfer-ing with vital pathways to cause cell deatheither by apoptosis or necrosis (19,20). Vari-ous mechanisms have been explored to ex-plain how ROS activate cell death signalingpathways (19,20). Earlier studies have shownthat OH. can oxidize thiol (-SH) groups of

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ways (13,14,22). They are synthesized aspro-enzymes containing a prodomain of vari-able length that is attached to the enzymaticsubunits. Upon proteolytic activation andrelease of the pro-domain, the subunits un-dergo a conformational change to form theactive enzyme. Caspases can be classifiedinto two groups: “initiator caspases” containa large prodomain in contrast to “execu-tioner caspases” which are characterized bya small prodomain. Initiator caspases harborprotein-protein interaction modules: the cas-pase recruitment domain (CARD) in cas-pases-1, -2, -4, -5, -9, -11, -12, and deatheffector domain in caspases-8 and -10. Thesemotifs are characterized by the presence ofsix or seven anti-parallel amphipathic α-helices, which allow the recruitment of othersignaling molecules or adaptor molecules inlarge protein complexes, thereby initiatingapoptotic or inflammatory signaling path-ways (13).

Caspase activation can occur secondarilyto triggering the extrinsic or intrinsic path-ways of apoptosis (1,2). The extrinsic path-way of apoptosis is triggered by cytokines inthe CD95/Fas/APO-1, TNF and TRAIL fami-lies (13) upon their binding to membranecell death receptors. An intracellular adaptorprotein, called Fas-associated protein with adeath domain or TNF receptor associatedwith a death domain then recruits the initia-tor procaspase-8, via its death effector do-main, to a death-inducing signaling com-plex. This complex facilitates the processingand full activation of this enzyme that, inturn, promotes the cleavage of specific sub-strates and executioner caspase-3.

A pivotal event in the intrinsic pathwayof apoptosis is the release of cytochrome cfrom the mitochondrial intermembrane space.Once into the cytosol, holo-cytochrome c(that is formed within mitochondria) readilyassociates with the C-terminal region of apop-totic protease-activating factor (Apaf-1) thatcontains 12-13 WD40 repeats. This interac-tion facilitates the binding of dATP with

sensor proteins, activating the PTP (21). ThePTP is considered to function as the point ofno return for both apoptosis and necrosis(20,21). Under a variety of experimentalconditions, Ca2+ is a powerful co-activatorof PTP in response to oxidative stress (19,21).The opening and operation of PTP can beprevented by cyclosporine A, a cyclic pep-tide that binds to Cyclo D, its mitochondrialmatrix molecular receptor. However, thereare situations in which the inhibitory effectis partial, transient or null. In addition, sev-eral studies have shown that cyclosporinesare non-selective inhibitors of seven trans-membrane helix G protein-coupled recep-tors, plasma membrane ion channels andABC transporters (7,21).

Despite extensive research in many labo-ratories, it has been difficult to isolate andidentify the components of PTP, as well asthe extent of contribution of ROS to itsformation and operation (7,20,21). Previousstudies have suggested that PTP is a su-pramolecular complex which may containor be regulated by ANT, VDAC, Cyclo D,and the peripheral benzodiazepine receptor(7,21). While in some pathways the transientopening of PTP is viewed as the first step toapoptosis, in many others, apoptosis can beindependent of this process (2,4-6). Someinvestigators have also considered the possi-bility that the onset of PTP is associated withthe transition from apoptosis to necrosis(20,21).

Caspase activation

Recent findings have provided a newframework for understanding the upstreamand downstream events of mitochondrialdysfunction and the critical roles of proteinsof the BCL-2 and caspase family in apopto-sis (2,4-6).

Caspases constitute a family of evolu-tionarily conserved aspartate-specific cys-teine-dependent proteases that have majorroles in apoptotic and inflammatory path-

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Apaf-1 and exposes its N-terminal CARD,which can now oligomerize and form aprocaspase-9-activating platform. The result-ing oligomeric Apaf-1 complex is able torecruit several inactive procaspase-9 mol-ecules through heterotypic CARD-CARDinteractions to form the so-called apopto-some. The apoptosome then activates initia-tor caspases (13,23).

Cytoplasmic proteins named inhibitorsof apoptosis prevent unintended caspase-9activation (24). These proteins bind to andinhibit the newly generated active N termi-nus of caspase-9. This inhibition is relievedafter the release of inhibitors of apoptosis-antagonizing proteins Smac/Diablo andHtrA2/Omi from mitochondria (25,28).These two proteins work as second levelregulators of the apoptotic process. Mito-chondria also release two proteins that haveDNA endonuclease activity: endonucleaseG (Endo G) and the AIF, a 57-kDa flavopro-tein (28). Because of differences in size andshape as well the kinetics of diffusion, it ispresumed that a distinct pore or even a rup-ture of the outer mitochondrial membraneallows for the release of these two proteins(25,28).

Activated caspases promote proteolyticcleavage of various vital proteins duringapoptosis and non-apoptosis processes,which result in either activation or inactiva-tion of their substrates (14,15,22). Cleav-age-induced activation can lead to differen-tial regulation, stabilization, protein com-plex formation, and special localization forthe protein or its fragments (22). In somecases, a first cut by caspases unleashes addi-tional cleavage sites for other proteases. Inother cases, cleavage allows for structuralchanges and exposure of previously hiddenstructures (15). More than 300 proteins havebeen characterized as caspase substrates (15).Plasma membrane receptors and structural,regulatory cytosolic and nuclear proteins arepreferred targets for executioner caspasessuch as caspase-3, -6, and -7 (15).

Mitochondrial outer membranepermeabilization by BCL-2 familyproteins

BCL-2 family members are of particularinterest among substrates that are activated bycaspases during the two pathways of apopto-sis (4,5). The BCL-2 family consists of multi-domain members like BAX, BOK and BAKand the BH3-only group of pro-apoptotic mem-bers, including BID, BAD, BIK, and BIM.The anti-apoptotic members BCL-2 and BCL-xL have 4 BH domains (BH1-4). All of theseproteins have the ability to bind to membranesand form, predominantly under non-physi-ological conditions, ion-conducting channelsin synthetic membranes (4,5).

Caspase-8 promotes the cleavage of BIDto form its C-terminal truncation, tBID, whichmoves to the outer mitochondrial membraneand induces the permeabilization processnamed mitochondrial outer membrane per-meabilization (MOMP) (2), allowing the for-mation of an outer membrane-spanning porethrough which cytochrome c is released (4,5).Studies using BAX and BAK doubly defi-cient cells and knockout mice have shownthat MOMP by BH3-only molecules such asBID and BIM requires BAX and BAK (2,4-6), each of which can form homo-oligomersand hetero-oligomers in the outer mitochon-drial membrane (2,4-6).

The pro-survival proteins BCL-2 andBCL-xL prevent the release of cytochrome cfrom mitochondria induced by many apop-totic signals (4). It seems that BCL-2 mayadapt or regulate mitochondrial homeostasisthrough a combination of different effects,including modulating the formation of ROS,intracellular acidification and proton fluxesin the mitochondria (3). The ability to inter-act and sequester tBID away from BAX andBAK proteins has also being considered offundamental importance for their pro-sur-vival effects (4). Interestingly, BCL-2 andBCL-xL are also cleaved by caspases duringapoptosis when the N-terminal BH4 domain

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is released, enabling the new fragment topromote apoptosis (4-6).

In healthy cells, several BCL-2 members,including BCL-2 and BCL-xL are inserted notonly into the outer membrane of mitochondriabut also into the endoplasmic reticulum (ER).Overexpression of BCL-2 reduces ER (Ca2+)levels and this exerts a protective effect againstsome apoptotic responses (29). The pro-apop-totic members BAX and BAK promote Ca2+

mobilization from the ER to mitochondria(29). Thus, these proteins may operate as regu-lators of ER Ca2+ concentrations and modulatethe propagation of Ca2+ waves into mitochon-dria (29).

BCL-2 family proteins and voltage-dependent anion channel activity

The most common pathway for the trans-location of metabolites through the outer mem-brane under physiological conditions is theVDAC (30-32). Three isoforms (VDAC1,VDAC2 and VDAC3) of molecular massaround 30 kDa have been identified in multi-cellular organisms (31). The archetypalVDAC1 is a large diameter ß barrel structurecomposed of one α helix and 13 ß strandswhose aqueous channel (2.5-3 nm) adoptsmultiple conductance states with special se-lectivity between cations and anions (30).VDAC seems to serve as an important dock-ing site for cytosolic mitochondrial intermem-brane space and inner membrane proteins suchas ANT, hexokinase, Cyclo D, creatine ki-nase, glycerol kinase, and the peripheral ben-zodiazepine receptor, as well as BCL-2 familyproteins (30,32). However, caution should beexercised in considering the various reports ofprotein association with VDAC, given its largeexcess over other proteins in the outer mito-chondrial membrane.

VDAC exists in an open configurationthat permits the free exchange of most me-tabolites of molecular mass up to 5 kDa insize. At a voltage smaller than 30 mV, thepore has a diameter of 2.5-3 nm and is in the

anion, high conducting state, referred to asthe open state. This open state permits thepassage of ATP-4, HPO4

-2, succinate-2, andother negatively charged molecules. Above30 mV, the diameter decreases to ~1.8 nm,the conductance decreases to 2 nS and selec-tivity changes to cations. In this closed state,VDAC favors the flux of small cations suchas Ca2+, K+, and Na+, but is impermeable tothe respiratory substrates ATP and ADP (30).It is important to mention that the VDACconductance states measured in a planar li-pid membrane system vary depending on thesalt and lipid concentrations (30,32).

The idea that VDAC, ANT and Cyclo Dare the core components of the permeabilitytransition pore (33,34) has been further ex-plored recently using gene knockout andshRNA strategies (21). Silencing VDAC1expression diminished cell growth and mito-chondrial ATP synthesis. On the other hand,the basic properties of the PTP did not changein VDAC1-/- mitochondria. Thus, the par-ticipation of VDAC in PTP composition re-mains an open question (35,36).

It is now becoming clearer how the BCL-2 family proteins interfere with the channelactivity of VDAC in vitro and in vivo (3,32).Experiments with VDAC channels reconsti-tuted into the lipid matrix demonstrated thatanti-apoptotic BCL-xL promotes the mainte-nance of VDAC in a physiological openstate (3,32). One study has shown that pro-apoptotic tBID induces VDAC closure, whileBAX does not affect the conductance of thischannel (32). tBID could affect VDAC con-ductance indirectly through the lipid envi-ronment surrounding VDAC (32) and notthrough a direct physical interaction (33,34).VDAC in the closed state favors permeabil-ity to cations, like Ca2+, K+ and Na+ (30).

BCL-2 family proteins and themitochondrial apoptosis-inducedchannel

Recent reports have suggested that a new

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channel, known as MAC, is a pathway forcytochrome c release (8). The pharmacolo-gical and electrophysiological properties ofthis channel have been reproduced in yeastand human cell models of apoptosis (37,38).The channel conductance (3.3 and 4.5 nS)was first detected by applying patch-clamptechniques to mitochondria isolated fromhematopoietic FL5.12 cell lines, derived fromWEHI-3B cells, upon withdrawal of IL-3,and reproduced in outer mitochondrial mem-branes of yeast expressing human BAX (37).MAC electrophysiological activity is in-creased by BAX oligomerization in the outermembrane and is prevented by overexpres-sion of BCL-2, but not by cyclosporin A(37). More important, the addition of cyto-chrome c interferes with the largest conduc-tance state of this voltage-dependent chan-nel, estimated to be ~3.0-4.0 nm in diameter(37,39). The molecular identification of MAChas yet to be fully determined. Nonetheless,there is considerable evidence that oligo-meric BAX/BAX, BAX/BAK or/and BAK/BAK are components of MAC (8,39).

Mitochondrial membranes change theirlipid composition under various apoptoticstimuli (1,6). Recently, a lipid channel com-posed of the lipid ceramide and named cera-mide channel has also been implicated inapoptogenic factor release from mitochon-dria (9). Ceramides differ from other lipidsin that they can form intermolecular hydro-gen bonds to produce columns of ceramideresidues. Such columns form ceramide chan-nels with multiple conductance states andcapable of releasing proteins of up to 60 kDa(9). The specificity of such lipid channels isclearly very limited and, again, awaits con-vincing evidence for its real role during physi-ological apoptosis.

Role of caspases in early and delayedmitochondrial dysfunction

Caspases may be involved both in theearlier events of apoptosis, such as cyto-

chrome c release, and in the delayed mito-chondrial events that include loss of mito-chondrial transmembrane potential and in-hibition of electron flow in the respiratorychain (40). Previous studies have shown thatincubation of isolated mitochondria with re-combinant human caspases promotes mem-brane permeabilization and the release ofcytochrome c and Smac/Diablo into the cy-tosol (41,42). Uncleaved forms of caspase-2efficiently insert into mitochondrial mem-branes and release cytochrome c bound toanionic phospholipid cardiolipin (42). Simi-larly, caspase-3 enters the intermembranespace and cleaves the 75-kDa subunit(NDUF1) of complex I of the electron trans-port chain of isolated mitochondria (43).Electron transport by complexes I and II isreduced by 88 and 94%, respectively. How-ever, the treatment does not affect oxygenconsumption by complex IV (43). Interest-ingly, treatment with z-VAD-fmk, a pancas-pase inhibitor, preserved electron transportchain functionality but failed to inhibit cyto-chrome c release. Additional studies on in-tact cells and isolated mitochondria havealso shown that z-VAD-fmk was able toinhibit the release of Smac/Diablo, HtrA2/Omi, AIF, and Endo G, but could not inhibitthe release of cytochrome c (28). These re-sults agree with a recent study (44) that usedsingle cell analysis to show that cytochromec release is independent of caspase-3 activ-ity, but that active caspase-3 is required forthe sustained loss of the mitochondrial trans-membrane potential.

A recent study by Lakhani and colleagues(16) has extended the major controversiesabout the role of executioner caspases inmitochondrial homeostasis. These investi-gators generated double knockout mice fortwo highly related effector caspases, cas-pase-3 and -7. Embryonic fibroblasts andthymocytes derived from mice lacking bothenzymes exhibited resistance to drugs thatinduce the intrinsic (mitochondrial) and ex-trinsic (membrane death receptor) pathways

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to apoptosis (16). In all conditions studied,the cells displayed a pronounced delay incytochrome c release and translocation ofBAX to the outer membrane. The mitochon-drial membrane potential was unaffected.Overall, the results of this study led theauthors to conclude that caspase-3 and -7 areimportant mediators for mitochondrial eventsin apoptosis.

CD95 (Fas)- and TNF-mediated apopto-sis occurs due to a cascade of morphologicaland biochemical events that include recep-tor membrane internalization, death-induc-ing signaling complex assembly and auto-proteolytic cleavage of caspase-8 insidedeath-signaling vesicles (45). Activated cas-pase-8 can either activate executioner cas-pase-3, or cleave BID, generating tBID, thatmoves to mitochondria, promoting cyto-chrome c release (45). Increasing evidencenow indicates that an increase of endocyticvacuoles is involved not only in receptorinternalization (45), but also in Golgi andmitochondrial intercommunication (46). Thisprovides further support for a model in whichcaspase-mediated cleavage of mitochondrialproteins could be involved in mitochondrialdysfunction and in the regulation of mito-chondrially mediated cell death.

The search for new caspase substratesrepresents a remarkable challenge and de-pends largely on biochemical methods suchas gel electrophoresis, chromatography andmass spectrometry. Usually, this work cantake years. Thus, bioinformatic tools for aquickly search of internal cleavage sites inprotein sequences represent an innovativeapproach. CaSPredictor is a software thatuses a novel methodology for characterizingcleavage sites in protein sequences (47).This program uses a scoring scheme thatincorporates the position-dependent aminoacid at the caspase-pentapeptide cleavagesite and the position-independent proline (P),glutamic acid (E), serine (S), and threonine(T) (PEST) amino acids, Glu or Asp (D/E),Asn (N) and Glu (Q) at the right and left

flanking of an aspartate residue within a 35-amino acid extension. Moreover, the algo-rithm uses a BLOSUM 62 substitution ma-trix to find biological similarity betweenamino acids not annotated in its database(47). The prediction accuracy of CaSPredictorwas estimated at 83%, as assessed by ROCanalysis. In a large-scale analysis, we identi-fied 1600 predicted caspase substrates witha score >0.57, with 60% sensitivity and 97%specificity (47).

Given that most transport systems areinvolved in the translocation of ions, me-tabolites and proteins across mitochondrialcompartments, our investigation attemptedto identify those proteins in the ion channelfamily, ABC transporter families and thetranslocase in the inner (Tim) and outer (Tom)mitochondrial membrane family with poten-tial cleavage sites for caspases (Table 1).Some protein candidates are structurally andfunctionally linked to apoptosis via TNFreceptor-associated protein, Fas-associatedprotein with a death domain and CARD.These observations raised unexplored possi-bilities that their cleavage may be relevant todiverse mitochondrial dysfunctions, includ-ing membrane permeabilization and poreformation, since some of them have the abil-ity to bind and transport proteins across mem-branes.

The plasma membrane Ca2+ pump(PMCA), Na+/Ca2+ exchanger (NCX) andH+/Ca2+ uniporter operate in Ca2+ extrusionand control neuronal cell death (48,49). Pro-teolytic cleavage and inactivation of theseplasma membrane proteins has been demon-strated in two neuronal cell death modelsinduced by prolonged overstimulation of theglutamate receptor subtypes (NMDA andAMPA) that lead to Ca2+ and Na+ influx andoverload in neurons (48,49). The AMPAreceptor is an example of several receptorfamilies that are regulated physiologicallyby caspases (50). PMCA is cleaved andinactivated by caspases in neurons undergo-ing excitotoxicity (48). Expression of mu-

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Table 1. Identification, structural properties and localization of cation and anion channels, ATP-binding cassette (ABC) transporters and thetranslocases in the inner (Tim) and outer (Tom) mitochondrial membrane family containing potential cleavage sites for caspases and the apoptosisinteraction domain.

Protein (GeneBank ID) Cleavage site Cellular localization Domain Apoptosis interactiondomain

Voltage-dependent calcium channel α 1G subunit 1221ESQDV Plasma membrane Calcium channel No(AAD29401)

Neuronal calcium channel α 1A subunit (AAB61613) 2421DEADG Plasma membrane Calcium channel No

Voltage-dependent L-type channel, ß-1 subunit, 498DTFDA Cytoplasm Calcium channel AnkyrinCAB1 (Q06421)

Chloride intracellular channel protein 4, mtCLIC, 161DEIDE Mitochondrial Chloride channel CARD-8(CAH70045.1)

Potassium voltage-gated channel, subfamily G, KCNJ7 187DALDS Mitochondrial K Tetra No(CAI23445.1)

Potassium channel, subfamily T, Kca4.1 (Q5JUK3) 845DNLDS Plasma membrane K Tetra No

ABC transporter (ABCA1), cholesterol and phospholipid 1292DPNDS Plasma membrane ABC transporter FADDefflux (O95477)

ABC transporter (ABCA3), chemical and metabolite 197TSPDG Plasma membrane ABC transporter ICADefflux (Q99758)

ABC transporter (ALDP/ABCD1), adrenoleukodystrophy 358SESDA Plasma membrane ABC transporter CARD-4protein (P33897)

ABC transporter (hALDR/ABCD2), adrenoleukodystrophy 565DSVDD Plasma membrane ABC transporter CARD-4protein (Q99758)

Cystic fibrosis transmembrane conductance regulator 1272VSWDS Plasma membrane ABC transporter TRAF-3(CFTR/ABCC7) (P13569)

ABC transporter (ABCC1), multidrug resistance- 1081DTVDS Plasma membrane ABC transporter TRAF-3,-5associated protein 1 (P33527)

ABC transporter (ABCB7), iron transport to mitochondria 637SSLDS Mitochondrial ABC transporter v-FLIP(O75027)

Multidrug protein resistance-3 (ABCB4), P-glycoprotein-3 683VETDG Plasma membrane ABC transporter CARD-4(P21439)

Translocase of outer mitochondrial membrane 22 kDa, 184EELDE Mitochondrial No NoTom22 (Q9NS69)

Translocase of outer mitochondrial membrane 70 kDa, 538IEIDN Mitochondrial Tetratricopeptide NoTom70 (O94826)

Translocase of inner mitochondrial membrane 50 kDa, 203DEFDN Mitochondrial Ctd phosphatase NoTim50 (AAT01208.1)

These proteins were identified in a large-scale analysis of 9800 human proteins using the CasPredictor software. Database available at http://icb.usp.br/~farmaco/jose. Domains for caspase-protein substrate interactions are indicated in the table. No means that interaction domain has notbeen found. CARD = caspase recruitment domain; FADD = Fas-associated protein with a death domain: ICAD = inhibitor of caspase-activatedDnase; TRAF = TNF receptor-associated protein: v-FLIP = viral FLICE inhibitory protein.

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tant forms of human PMCA4 that lack thecaspase cleavage site prevents the delayedrise of Ca2+ or delayed Ca2+ deregulation(48). Bano and colleagues (49) have alsoshown that both calpains and caspases cancleave NCX during excitotoxicity in neu-ronal cell lines. The broad-spectrum caspaseinhibitor z-VAD-fmk moderately reducedthe caspase-mediated cleavage of NCX3 anddelayed Ca2+ deregulation that causes ne-crotic cell death (49). Therefore, caspasesappear to be involved in the control of trans-membrane Ca2+ channels and pumps andindirectly in the increase of intracellular Ca2+

pools that may precede the opening of mito-chondrial death decision pores.

The superfamily of ATP-binding cas-sette (ABC) transporter proteins consists ofefflux pumps that have important roles intransporting a diverse group of toxicantsincluding lipophilic cationic, anionic, andneutrally charged drugs, peroxidation prod-ucts and antigenic peptides (51,52). ABCtransporters are localized in the plasma mem-brane, endoplasmic reticulum and in or-ganelles, including peroxisomes and mito-chondria. Five mammalian mitochondrialABC transporters have been described basedon the presence of mitochondrial targetingpresequences (51,52). Members of this fam-ily are involved in the export of iron-sulfur-containing proteins such as apoproteins fromthe mitochondrial matrix to the cytosol (51).Deletions of mitochondrial ABC transport-ers disturb iron homeostasis and are lethal(53). At present, one study (54) has shownthat the proteolytic cleavage of ABCA1, atransmembrane transporter involved in cy-tosolic cholesterol efflux by calpain, in-creases its activity. An uncleavable form ofABCA1 displayed a 4-fold increase in theefflux of cholesterol, which prevented mac-rophage cell death due to cholesterol over-load (54). We noted that calpain and cas-pases have cleavage sites within the samePEST sequence (Table 1). This raises thechance that both proteases could regulate the

pumping activity of ABC transporters.Mitochondrial chloride intracellular chan-

nel protein 4 (mtCLIC) belongs to a CLICfamily of soluble globular proteins that canform ion channels in organelles and plasmamembranes similar to bacterial toxins, an-nexins and BCL-xL (55). It has been shownthat mtCLIC protein 4 is up-regulated dur-ing the cell death response to cytotoxic agentsand DNA damage. Overexpression of themtCLIC gene is sufficient to induce kerati-nocyte cell death, which is associated withthe expression of p53, loss of mitochondrialmembrane potential and cytochrome c re-lease (54). More importantly, cell death isinhibited by z-VAD-fmk, a pancaspase in-hibitor (55).

The translocase of the inner (Tim com-plex) and outer (Tom complex) mitochon-drial membranes is composed of large andsmall proteins that display an intrinsic ca-pacity to bind and transfer polypeptides intomitochondrial compartments (56). They con-tain water-filled pores that mediate translo-cation of proteins tagged with presequencesthat act as bipartite sorting signals. These N-terminal sorting signals form amphipathichelices with a hydrophobic and positivelycharged side. The sequences are first recog-nized by receptors and targeted to the pro-tein-conducting channel (Tom proteins). Thistranslocation mechanism depends on bothATP and the mitochondrial membrane po-tential (56).

The role the Tim23 complex for translo-cation of AIF, Endo G, Smac/Diablo, andHtrA2/Omi into the intermembrane mito-chondrial space has been demonstrated (56).Furthermore, a study with yeast mitochon-dria has shown that perturbations of Tim23conductance activity with synthetic peptidesand specific antibodies cause matrix swell-ing and ultimately cytochrome c release (57).Tim50 is a subunit of the Tim23 complex(56). It has been demonstrated that clones ofhuman cells and zebrafish embryos lackingthe Tim50 gene undergo mitochondrial dys-

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function and rapid apoptosis (58). Tom20and Tom22 have a negatively charged N-terminal region exposed to the cytosol thatcontains a putative cleavage site for cas-pases. This region coordinates the recogni-tion and translocation of many nuclear pro-teins into mitochondria (58). Interestingly,deletion of the Tom22 gene is lethal (56). Acomplex of approximately 400 kDa contain-ing Tom20, Tom40 and Tom70 operates inthe translocation of apocytochrome c insidethe intermembrane space, where it incorpo-rates the heme co-factor and is released asholo-cytochrome c (56). The possible con-sequences of Tom22 cleavage by caspasesare numerous. For example, it could inter-rupt cytochrome c import causing electrontransport interruption. In conclusion, Tim/Tom complexes appear to be interesting path-ways for apoptogenic factor release. Futurestudies are needed to challenge this hypo-thesis and to test how caspases can affectprotein translocation across mitochondrialmembranes.

Outer membrane rupture andmitochondrial remodeling

The occurrence of swelling is common inthe mitochondria of the cells committed todie, as a result of inner membrane perme-ability transition (20,21). Since the surfaceof the inner membrane is greater than thesurface of the outer membrane, the swellingforces the expansion of the inner membraneto the cytoplasm, causing the outer mem-brane to rupture (10). Korsmeyer’s grouphas also described the morphological fea-tures of degenerating mitochondria treatedwith tBID and crista ultrastructural remodel-ing using high-voltage electron microscopyand tomography (59). The authors proposedthat tBID is necessary for mitochondrial re-modeling and further mobilization of cyto-chrome c (~85%) that is retained in mito-chondrial crista stores. Based on their cristaremodeling, the mitochondria were classi-

fied as class I (normal) or class II, III, and IV(59). Class II mitochondria with highly in-terconnected and condensed cristae occurafter 2-5 min in cells treated with tBID, TNFand Fas and several intrinsic death stimuliincluding thapsigargin, tunicamycin and bre-feldin A (59). Class III mitochondria areswollen and their outer membrane are rup-tured. This is comparable to the type II mito-chondrial profile described by Sesso andcolleagues (10).

Finally, fusion and fission (division) aretwo morphologically and physiologicallyopposite processes within mitochondrialdynamics. It has been demonstrated that per-turbations in these processes result in mito-chondrial outer membrane permeabilizationand release of apoptogenic factors and apop-tosis (60). Cytosolic GTPases of the dynaminfamily, optic atrophy 1 and mitofusins 1 and2, are required for mitochondrial fusion, whiledynamin-related protein 1 and Fis1 are re-quired for mitochondrial fission (60).

The present overview suggests that nu-merous biomolecules that are produced bybiochemical and bioenergetic reactions in-side and outside the mitochondria, and regu-lators and effectors of apoptosis play a rolein the coordination of events involved inmitochondrial membrane permeabilizationand the release of cytochrome c, Smac/Diablo, HtrA2/Omi and AIF. An integratedmodel is depicted in Figure 1. Permeabiliza-tion of the inner or outer membrane must beaccompanied by exchange of cations, likeCa2+, K+, and Na+, and metabolites that havean important impact on mitochondrial vol-ume, bioenergetic reactions and the mito-chondrial membrane potential. The openingand release of apoptogenic proteins occurvia one of the death decision pores, namelythe PTP formed in the inner mitochondrialmembrane or a proteolipid pore formed bythe MOMP. The molecular components ofPTP and MOMP pore are variable or remainto be determined. However, cytosolic andmitochondrial proteins, including VDAC,

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ANT and pro-apoptotic proteins tBID, BAXand BAK, may participate in their assemblyand operation. The MAC and a ceramidelipid pore may also be involved in the re-lease of the apoptogenic factors. Nonethe-less, the simple possibility that apoptogenicfactor release occurs through a nonspecificmitochondrial outer membrane rupture alsoremains valid. Our understanding of the criti-cal role of caspases in mitochondrial dys-function is based on the experiments usingrecombinant caspases, their peptide inhibi-tors and mice deficient in both caspase-3 andcaspase-7. Few components of the mito-chondrial machinery have been described as

caspase substrates. One study has shownthat caspase-3 can enter the mitochondriaand cleave NDUF1, a component of com-plex I of the respiratory chain. It remainsunclear how a cytosolic protease can crossboth mitochondrial membranes to cleave amatrix-exposed subunit embedded withincomplex I. However, biochemical analysishas demonstrated that pro-caspase and ac-tive caspase (-3, -7, -8, and -9) can partiallyco-localize in the outer membrane or arereleased into mitochondria via “death sig-naling vesicles” stimulated by TNF andCD95/Fas (45,46). In this scenario, it may bepossible that caspases, especially the apical

Figure 1. Schematic representation of mitochondrial death decision pores with the biochemical ability to promotethe inner and outer mitochondrial membrane permeabilization and the release of apoptogenic factors: cytochromec, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. The four-channel/pore-forming com-plexes described are: I) a large conductance pore-forming complex, named permeability transition pore (PTP) thatis formed in the inner mitochondrial membrane in response to radical oxygen species overproduction duringbioenergetic reactions and Ca2+ overload; II) a proteolipid pore formed in response to outer mitochondrialmembrane permeabilization caused by oligomerized forms of BAX and BAK after their activation by tBID andnamed mitochondrial apoptosis-inducing channel (MAC); III) a lipid channel formed by the lipid ceramide, and IV)voltage-dependent anion channel (VDAC). Outer membrane rupture could also act as an alternative route forapoptogenic factor release. Various components of the channel- and pore-forming subunits may be cleaved bycaspases (see Table 1). Their cleavage might cause changes in their structural organization and a switch to anonspecific channel or pore with different conductance or selectivity, or turn off the channel or transport system.The validity of this mechanistic model needs to be examined experimentally.

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Acknowledgments

The authors are grateful to Alicia Kowal-towski (Departamento de Bioquímica,IQ-USP), Gustavo Pessini Amarante-Mendes(Departamento de Imunologia, ICB-USP),Mauro Degli Esposti (Faculty of Life Sci-ences, University of Manchester, UK), andTatiana Rostovtseva (Laboratory of Physi-cal and Structural Biology, NICHD-NIH)for expert reviews of the manuscript andhelpful comments.

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