The Permeability Transition Pore Complex: A Target for Apoptosis Regulation by Caspases and Bcl-2-related Proteins

1261 J. Exp. Med.  The Rocke elle Uni e si y P ess • 0022-1007/98/04/1261/11 $2.00 Volume 187, Numbe 8, Ap il 20, 1998 1261–1271 h p://www.jem.o g The Pe meabili y T ansi ion Po e Complex: A Ta ge o Apop osis Regula ion by Caspases and Bcl-2– ela ed P o eins By Isabel Ma zo, * Ca he ine B enne , * Naou al Zamzami, * San os A. Susin, * Gisela Beu ne , ‡ Die e B diczka, ‡ René Rémy, § Zhi-Hua Xie, i John C. Reed, i and Guido K oeme * F om he * Cen e Na ional de la Reche che Scien i ique, Uni é P op e de Reche che 420, F-94801 Villejui , F ance; he ‡ Facul y o Biology, Uni e si y o Kons anz, D-78434 Kons anz, Ge many; he § Cen e Na ional de la Reche che Scien i ique, Uni e si é de Pa is 11, F-91405 O say, F ance; and i The Bu nham Ins i u e, La Jolla, Cali o nia 92037 Summa y Ea ly in p og ammed cell dea h (apop osis), mi ochond ial memb ane pe meabili y inc eases. This is a leas in pa due o opening o he pe meabili y ansi ion (PT) po e, a mul ip o ein complex buil up a he con ac si e be ween he inne and he ou e mi ochond ial mem- b anes. The PT po e has been p e iously implica ed in clinically ele an massi e cell dea h in- duced by oxins, anoxia, eac i e oxygen species, and calcium o e load. He e we show ha PT po e complexes econs i u ed in liposomes exhibi a unc ional beha io compa able wi h ha o he na u al PT po e p esen in in ac mi ochond ia. The PT po e complex is egula ed by hiol- eac i e agen s, calcium, cyclophilin D ligands (cyclospo in A and a nonimmunosupp es- si e cyclospo in A de i a i e), ligands o he adenine nucleo ide ansloca o , apop osis- ela ed endop o eases (caspases), and Bcl-2–like p o eins. Al hough calcium, p ooxidan s, and se e al ecombinan caspases (caspases 1, 2, 3, 4, and 6) enhance he pe meabili y o PT po e-con ain- ing liposomes, ecombinan Bcl-2 o Bcl-X L augmen he esis ance o he econs i u ed PT po e complex o po e opening. Mu a ed Bcl-2 p o eins ha ha e los hei cy op o ec i e po- en ial also lose hei PT modula o y capaci y. In conclusion, he PT po e complex may cons i- u e a c oss oad o apop osis egula ion by caspases and membe s o he Bcl-2 amily. T wo di e en majo changes in mi ochond ial mem- b ane pe meabili y ha e been obse ed du ing he e - ec o phase o apop osis. On he one hand, he elec o- chemical g adien buil up on he mi ochond ial inne memb ane dissipa es ea ly du ing apop osis (1–4). On he o he hand, apop ogenic p o eins ha no mally a e seques- e ed in mi ochond ia a e eleased ia he ou e mi ochon- d ial memb ane. Such p o eins include cy och ome c (5–7) and apop osis inducing ac o (AIF) 1 (8, 9). The p o oon- cogene p oduc Bcl-2 p e en s he pe meabili y inc ease in bo h mi ochond ial memb anes (4, 6–10). Based on he simila i y o he e ec s o Bcl-2 and pha macological inhib- i o s o he mi ochond ial pe meabili y ansi ion (PT) po e, we ha e ad anced he hypo hesis ha opening o he PT po e migh be (co-) esponsible o he apop osis-associ- a ed changes in mi ochond ial memb ane unc ion (2, 4, 8, 11). In isola ed mi ochond ia, opening o he PT po e en- ails bo h he dis up ion o he inne mi ochond ial ans- memb ane po en ial ( Dc m ) (12, 13) and he elease o he apop ogenic p o eins AIF (8, 9) and cy och ome c (14, 15), sugges ing ha he PT po e may ha e an impo an ole in cell dea h con ol. Mo eo e , opening o he PT po e has been implica ed in clinically ele an massi e cell dea h o hepa ocy es, neu ons, and myoca diocy es induced by hepa o- oxins, exci o oxins, calcium, eac i e oxygen species, and anoxia (3, 4, 12, 13, 16–18 and e e ences ci ed he ein). I he mi ochond ion ul illed a majo ole in apop osis con ol, i should be capable o in eg a ing e y di e en p oapop o ic signal ansduc ion and damage pa hways. In his con ex , i appea s impo an ha he PT po e is a dy- namic mul ip o ein complex loca ed a he con ac si e be- ween he inne and he ou e mi ochond ial memb anes, one o he c i ical si es o me abolic coo dina ion be ween he cy osol, he mi ochond ial in e memb ane space, and 1 Abb e ia ions used in his pape : Dc m , mi ochond ial ansmemb ane po- en ial; Ac-DEVD.cmk, ace yl-Asp-Glu-Val-Asp-chlo ome hylke one; A-YVAD.cmk, ace yl-Ty -Val-Ala-Asp-chlo ome hylke one; AIF, apop- osis-inducing ac o ; ANT, adenine nucleo ide ansloca o ; A , a ac ylo- side; diamide, diazenedica boxylic acid bis 5 N , N -dime hylamide; DiOC 6 (3), 3,3 9 dihexyloxaca bocyanine iodide; PT, pe meabili y ansi ion; PTPC, PT po e complex; RT, oom empe a u e; VDAC, ol age-dependen anion channel; Z-VAD. mk, N -benzyloxyca bonyl-Val-Ala-Asp- luo- ome hylke one. I. Ma zo and C. B enne con ibu ed equally o his wo k. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1262 Pe meabili y T ansi ion Po e Complex in Apop osis he ma ix. The PT po e pa icipa es in he egula ion o ma ix Ca 2 1 , pH, Dc m , and olume and unc ions as a Ca 2 1 -, ol age-, pH-, and edox-ga ed channel wi h se - e al le els o conduc ance and li le i any ion selec i i y (12, 13, 19). Al hough he exac composi ion o he PT po e complex (PTPC) is unknown, i is hough o in ol e p o eins om he cy osol (hexokinase), he ou e mem- b ane ( ol age-dependen anion channel [VDAC]), he in- ne memb ane ( he adenine nucleo ide ansloca o [ANT]), and he ma ix (cyclophilin D) (12, 13, 20–23). As a conse- quence, he PT po e complex con ains mul iple a ge s o endogenous egula o s. In in ac cells and isola ed mi o- chond ia, PT po e opening is induced by se e al p oapop- o ic second messenge s: Ca 2 1 , p ooxidan s, ni ic oxide, ce amide, and caspase 1 (1, 2, 8, 9, 12, 13, 19, 24–27). Mo eo e , i is egula ed by he an iapop o ic oncop o eins Bcl-2 and Bcl-X L , which s abilize mi ochond ial mem- b anes (4, 8, 9, 28–31), and by he p oapop o ic Bcl-2 ana- logue Bax, which dis up s he Dc m (32). I has been unclea whe he hese e ec o s speci ically ac on PTPC, a ec o he mi ochond ial s uc u es no as- socia ed wi h PTPC (6, 7), o a he nonspeci ically pe - u b memb ane pe meabili y, as his has been sugges ed o membe s o he Bcl-2 amily (32–35). To dis inguish hese possibili ies, we pu i ied p o ein complexes con aining PTPC, econs i u ed hem in liposomes, and c ea ed a educed ex- pe imen al sys em ha sha es p ope ies o he PT po e s udied in in ac mi ochond ia o cells. Biochemical and unc ional da a indica e ha PTPC en iched om b ain homogena es con ain he p oapop o ic Bcl-2 homologue Bax (bu no Bcl-2 and Bcl-X L ), in addi ion o p o eins p e iously sugges ed o pa icipa e in he egula ion o PT (ANT, VDAC, cyclophilin D, and hexokinase). The mem- b ane pe meabili y o PTPC liposomes was enhanced by se e al induce s o PT including Ca 2 1 , p ooxidan s, and e- combinan caspases. Recombinan Bcl-2 and Bcl-X L ac as inhibi o s o PT po e opening in his a i icial sys em. Thus, PTPC cons i u es he a ge o mul iple apop osis egula- o s, emphasizing i s p obable cen al ole in cell dea h con- ol. Ma e ials and Me hods Ma e ials. Recombinan human Bcl-X L (1–209), Bcl-2 (1– 218), mu an Bcl-2 (Gly145Ala), and Bcl-2 Da 5/6 ( D 143–184), all lacking he hyd ophobic ansmemb ane domain ( D 219–239 in he case o Bcl-2; D 210–230 o Bcl-X L ) and agged NH 2 e - minally wi h His 6 , we e p oduced and pu i ied as desc ibed (34). Recombinan caspases we e p oduced as ac i e enzymes (36, 37). Caspase ac i i y is de ined as amoun o enzyme equi ed o clea e 1 m mol o he luo ogenic subs a e Ac-DEVD.amc (ace yl-Asp-Glu- Val-Asp-aminome hylcouma in; caspases 3 and 6), Ac-YVAD.amc (ace yl-Ty -Val-Ala-Asp-aminome hylcouma in; caspase 1), o Ac-WEHD.amc (ace yl-T p-Glu-His-Asp-aminome hylcouma in; caspase 4) pe hou . Caspase subs a es and inhibi o s (Ac-DEVD. cmk [ace yl-Asp-Glu-Val-Asp-chlo ome hylke one], Ac-YVAD. cmk [ace yl-Ty -Val-Ala-Asp-chlo ome hylke one]) we e pu chased om Bachem (Basel, Swi ze land). All emaining eagen s we e om Sigma Chemical Co. (S . Louis, MO), unless speci ied di e en ly. Recons i u ion o PTPCs in Liposomes. PTPCs we e pu i ied and econs i u ed in liposomes ollowing published p o ocols (22), wi h se e al modi ica ions (Fig. 1 A ). In b ie , ou Wis a (Phila- delphia, PA) a b ains (3–4-mo-old males, s o ed a 2 80 8 C) we e homogenized in bu e 1 (1 mM a -mono hioglyce ol, 10 mM glucose, pH 8.0, 40 ml; sample 1 in Fig. 1) and cen i uged wice (15 min, 12,000 g , 4 8 C) o esuspend he pelle i s in bu e 1 alone, and hen in bu e 1 plus 0.5% T i on X-100 (Boeh inge Mannheim, Indianapolis, IN) o 30 min a oom empe a u e (RT) while s i ing. Supe na an s (40 min, 50,000 g , 4 8 C) o his mix u e, he T i on-soluble p o ein ac ion (sample 2 in Fig. 1), we e mixed wi h 17 g DE52 esin p e iously equili- b a ed wi h bu e 2 (1.5 mM Na 2 HPO 4 , 1.5 mM K 2 HPO 4 , 100 mM glucose, 1 mM di hioe y hi ol, pH 8.0). These beads we e packed in o an FPLC column (XK16/20; Pha macia Bio ech, Uppsala, Sweden) and elu ed wi h bu e 2 supplemen ed wi h 50 mM KCl (bu e 3) o 400 mM KCl (bu e 4). A e equilib a- ion wi h bu e 3 (0.8 ml/min, 6 ml), elu ion was pe o med on a linea g adien om 50 o 400 mM KCl (bu e s 3 e sus 4), ol- lowed by de e mina ion o hexokinase ac i i y (sample 3 in Fig. 1). Lipid esicles we e p epa ed by mixing 300 mg phospha idyl- choline and 60 mg choles e ol in 3 ml chlo o o m, e apo a ion o he chlo o o m unde ni ogen, and esuspension in 3 ml 125 mM suc ose 1 10 mM Hepes (pH 7.4) 1 0.3% n -oc yl- b - d -py ano- side by o exing (90 min, RT). These esicles (6 ml) we e incu- ba ed wi h 6 ml o PTPC-con aining solu ion du ing 20 min a RT and dialyzed o e nigh (4 8 C) agains 125 mM suc ose 1 10 mM Hepes (pH 7.4). In se e al expe imen s, ecombinan Bcl-2, Bcl-X L , o mu a ed Bcl-2 p o eins we e added du ing he dialysis s ep a a dose co esponding o 5% o he o al PTPC p o eins, as de e mined in each expe imen . Liposomes eco e ed om dialy- sis we e ul asonica ed (120 W, Ul asonic P ocesso ; Bioblock, Illki ch, F ance) du ing 7 s in 5 mM mala e and 10 mM KCl, cha ged on a Sephadex G50 column (C16/20; Pha macia Bio- ech), and elu ed wi h 125 mM suc ose 1 10 mM Hepes (pH 7.4, 0.8 ml/min) (Fig. 1 C ). P o eins we e ex ac ed om he lipo- some p epa a ion (1 ml) by mixing wi h 2 ml 880 ml KCl 1 6 ml chlo o o m/me hanol (2:1 ol/ ol) and eco e ed om he in e - phase a e s anda d me hods (38), ollowed by esuspension in 0.1% SDS (sample 4 in Fig. 1). They we e hen p ecipi a ed wi h 80% ( ol/ ol) ace one o wo-dimensional elec opho esis. A mean o 1.86 6 0.24 m g p o ein/mg lipid (X 6 SD, n 5 5) was eco e ed om p o eoliposomes. In se e al expe imen s, pu i ied a cy och ome c (25 m g/ml, co esponding o 500 ng cy och ome c /mg lipid) was added be o e he sonica ion s ep, ollowed by wo washes on Sephadex G50 columns o emo e excess cy och ome c om he supe na an . De e mina ion o Calcein E lux om PTPC Liposomes. Liposomes we e gene a ed as desc ibed abo e wi h he sole di e ence ha sonica ion was pe o med in 8 mM calcein (Molecula P obes Inc., Eugene, OR) 1 10 mM cobal chlo ide. 200 m l liposome suspension was incuba ed o 90 min wi h di e en concen a ion o a ac yloside (A ) and/o 50 m M bongk ekic acid (gi om Hans J. Duine, Del Uni e si y, Del , The Ne he lands). The supe na an s o liposomes (4.5 3 10 6 g , 45 min, 4 8 C) we e eco - e ed, supplemen ed wi h EDTA ( inal concen a ion o 1 mM), and subjec ed o luo ome ic analysis (exci a ion a 488 nm, emission a 520 nm) in a luo escence spec ome e (F4500; Hi a- chi, Tokyo, Japan). Wes e n Blo s and Two-dimensional Elec opho esis. To al b ain ho- mogena es, T i on-soluble p o eins, PTPC p epa a ions om an- ion exchange columns, and p o eins ex ac ed om PTPC- econs i- u ed liposomes we e sepa a ed by SDS-PAGE (10–15%, 30 m g Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1263 Ma zo e al. p o ein/lane), ollowed by Wes e n blo using monoclonal an i- bodies ecognizing cy och ome c (Pha Mingen, San Diego, CA; e e ence 5), hsp60 (clone LK1; Sigma Chemical Co.), VDAC (gi om F. Thinnes, Molecula Pa hology Ins i u e o Expe i- men al Medicine, Gö ingen, Ge many), o polyclonal abbi an- ise a agains he ANT (gi om T. Wallimann, Zü ich, Swi ze - land; e e ence 22), and he NH 2 e minus o cyclophilin D (gi om Paolo Be na di, Uni e si y o Pado a, Pado a, I aly; e e - ence 20), F 1 ATPase (p o ided by P.V. Vignais, Cen e Na ional de la Reche che Scien i ique, G enoble, F ance; e e ence 1), Bcl-2 (speci ic o esidues 20–34; Calbiochem Co p., La Jolla, CA), Bcl-X L (Ab-1; Calbiochem Co p.), Bad, Bag-1, o Bax (San a C uz Bio echnology, San a C uz, CA). In one se ies o ex- pe imen s, supe na an s (3.5 3 10 5 g , 60 min, 4 8 C) o liposomes we e supplemen ed wi h bo ine se um albumin (6 m g/ml), p e- cipi a ed wi h 80% ace one (80%, o e nigh , 4 8 C, cen i uga ion: 1.4 3 10 3 60 min a 4 8 C), and examined o he elease o cy o- ch ome c. Two-dimensional elec opho esis was pe o med a e s anda d p o ocols using a Pha macia (Pisca away, NJ) ampholy e (1%, pH 3–10; e e ence 39). Cy o luo ome ic Analysis o PTPC Liposomes. 10- ml aliquo s (z107) o liposomes we e incuba ed du ing 15 min a RT in 125 mM suc ose 1 10 mM Hepes (pH 7.4) supplemen ed wi h he indi- ca ed dose o PT induce s (A , CaCl2, diazenedica boxylic acid bis 5N,N-dime hylamide [diamide], e -bu ylhyd ope oxide) and/o PT inhibi o s (bongk ekic acid; cyclospo in A, N-me hyl-Val-4- cyclospo in A [p o ided by Sandoz, Basel, Swi ze land]; and monochlo obiman [Molecula P obes]). Al e na i ely, liposomes we e incuba ed du ing 15 min a 378C in he p esence o he in- dica ed dose o ac i e o inhibi o -inac i a ed caspases. Dilu ed (1 ml) liposomes we e incuba ed wi h 3,39dihexyloca bocyanine io- dide [DiOC6(3), 80 nM, 20–30 min a RT; Molecula P obes], ollowed by analysis o DiOC6(3) e en ion in a FACS-Van age cy o luo ome e (Bec on Dickinson, San José, CA). The o wa d sca e h eshold was se a 30 (Amp 16) and he low- a e a 1,500 e en s/s. The pho omul iplye o he side sca e and FL1 we e se a 700 mV and 700–800 mV, espec i ely. The luo es- cence was exci ed wi h an A gon lase (exci a ion wa eleng h 488 nm) and analyzed in FL-1 (wa e leng h 530 6 30 nm). The o wa d and side sca e s we e ga ed on he quan i a i ely mos abundan popula ion o liposomes while excluding backg ound noise. Calib a ion wi h ca boxyla e mic osphe es (Fluo esb i e BB; Polyscience, Wa ing on, PA) o de ined diame e s was used o de e mine he diame e o liposomes ha we e ga ed on (ga e: 150 o 300 nm; mean size o liposomes: 230 6 60 nm; X 6 SD o 5 3 104 e en s). Elec on mic oscopy con i med he p esence o mos ly unilamella p o eoliposomes o he expec ed size in he PTPC liposome p epa a ion. T iplica es o 5 3 104 liposomes we e analyzed o each da a poin . Resul s we e exp essed as pe - cen o educ ion o DiOC6(3) luo escence (log scale, geome ic mean), conside ing he educ ion ob ained wi h 0.25% SDS (15 min, RT) in PTPC liposomes as 100% alue. E alua ion o Caspase E ec s on Isola ed Mi ochond ia. Pu i ied mouse li e mi ochond ia we e incuba ed in 10 mM T is-MOPS Figu e 1. En ichmen o he PTPC. (A) S eps o he pu i ica ion p ocess. Fo de ails consul Ma e i- als and Me hods. (B) Typical p o ile o an anion ex- change ch oma og aphy pe o med on T i on-solu- ble p o eins (A, 2). Hexokinase ac i i y (solid line) elu es om he DE52 esin a a KCl concen a ion (linea g adien , do ed line) o 190 6 10 mM. The mos ac i e ac ions (ba ) a e eco e ed (A, 3) and econs i u ed in liposomes as ou lined in A. Cy o- ch ome c elu es om he g adien wi h he majo p o ein peak, a 70 6 10 mM (a ow). (C) Typical p o ile o a molecula weigh ch oma og aphy pe - o med on liposomes econs i u ed wi h he ac- ions eco e ed in B (A, 3). No e ha hexokinase ac i i y accumula es in a ew ac ions o he lipo- some–p o ein mix u e. The ac ion con aining maximum hexokinase ac i i y cons i u es he PTPC liposomes (A, 4). (D) Immunochemical de ec ion o p o eins con ained in PTPC. P o eins om suc- cessi e s eps o he pu i ica ion p ocedu e (A) we e analyzed by Wes e n blo o he p esence o he in- dica ed p o eins. P o eins ex ac ed om PTPC li- posomes cons i u e he inal s ep (A, 4) o he pu i- ica ion p ocedu e. Resul s a e ep esen a i e o 22 (B and C), and wo o h ee (D) independen de e - mina ions. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1264 Pe meabili y T ansi ion Po e Complex in Apop osis 1 100 mM NH4Cl 1 10 mM EGTA (pH 7.2) du ing 30 min a RT in he p esence o di e en caspases. The supe na an (1.5 3 105 g) o hese mi ochond ia was s o ed a 2808C un il es ing o apop ogenic ac i i y on isola ed HeLa nuclei (90 min, 378C, RT). DNA agmen a ion was quan i ied by p opidium iodine s aining (10 mg/ml, >5 min a RT) and cy o luo ome ic analysis in an EPICS P oli e II (Coul e , Hialeah, FL), as desc ibed (26). Resul s we e exp essed as he pe cen age o subdiploid nuclei, a - e sub ac ion o alues ob ained wi h bu e only (,20%). Fo con ol pu poses, di e en caspase inhibi o s (Ac-DEVD.cmk, Ac-YVAD.cmk, o Z-VAD. mk (N-benzyloxyca bonyl-Val-Ala- Asp- luo ome hylke one); 100 mM inal concen a ion) we e added o he mi ochond ial supe na an 15 min be o e de e mina ion o apop ogenic ac i i y. Aliquo s o caspase- ea ed mi ochond ia we e esuspended in 400 mM manni ol, 50 mM T is (HCl, pH 7.2), 5 mg/ml BSA, 10 mM KH2PO4, and 5 mM succina e, and hen labeled wi h DiOC6(3) (100 nM, 15 min a RT) and sub- jec ed o cy o luo ome ic analysis using ca bonylcyanide m-chlo- ophenylhyd azone (CCCP; 50 mM) o A (5 mM) as posi i e con ols o maximum Dcm dis up ion. Resul s Recons i u ion o he PTPC in Liposomes. Hexokinase 1 is a cy osolic p o ein, pa o which associa es wi h he mi o- chond ial ou e memb ane whe e i binds o po in wi hin he con ac si e (22, 40–42). Taking ad an age o his ac , we aced he hexokinase ac i i y copu i ying wi h a p o ein complex ha is wa e insoluble in b ain homogena es, pa - i ions in o he i on-soluble ac ion, elu es om an anion exchange FPLC column a a ela i ely high salini y, and in- co po a es in o phospha idylcholine/choles e ol liposomes (Fig. 1 A–C). When compa ing he abundance o p o eins ex ac ed om liposomes inco po a ing hexokinase ac i i y wi h ha o he p eceding pu i ica ion s eps, i appea s ha some p o eins a e selec i ely en iched (cyclophilin D, he z60-kD iso o m o he ANT, Bax, Bag-1), whe eas some a e educed (VDAC, F1 ATPase) o elimina ed below he limi o de ec ion (Bcl-XL, Bcl-2, Bad, cy och ome c, hsp60; Fig. 1 D). As shown by wo-dimensional gel elec- opho esis, PTPC liposomes con ain a limi ed se o p o- eins whose comple e iden i ica ion is s ill in p og ess (Fig. 2). PTPC-con aining liposomes can be ea ed wi h induc- e s o PT po e opening, which cause he elease o encap- sula ed molecules such as mala e (106 dal ons) and ATP (509 dal ons) (22, 43). Simila ly, he luo och ome calcein (622 dal ons), a hyd ophilic polyanionic luo och ome p e- iously used o measu e PT po e opening in in ac cells (44), can be encapsula ed in o PTPC liposomes and hen eleased by incuba ion wi h he ANT ligand A , a po en induce o PT po e opening (Fig. 3 A). This e ec is p e- en ed by ano he ANT ligand, bongk ekic acid, which in- hibi s PT po e opening (Fig. 3 A). We ha e de eloped an- o he app oach o quan i y PT po e opening induced in PTPC liposomes. Liposomes we e equilib a ed wi h he amphophilic ca ionic luo och ome DiOC6(3) (573 dal- ons). The e en ion o DiOC6(3) luo escence was hen moni o ed in a cy o luo ome e ( ha is, in a low in which liposomes a e dilu ed and he ex e nal DiOC6(3) concen- a ion app oaches 0), whe eas ga ing on a popula ion o li- posomes wi h de ined o wa d and side sca e cha ac e is- ics (es ima ed diame e : 0.15–0.3 mm). When using his app oach, we ound an A -induced shi in DiOC6(3) e- en ion, sugges ing ha mos i no all p o eoliposomes ound in his popula ion con ain a PT po e. PT po e open- ing does no p o oke a change in he a e age size ( o wa d sca e ) o liposomes, no does i a ec hei ul as uc u e. A good co ela ion was ound be ween he elease o small molecules (ATP, mala e, calcein) measu ed in bulk expe i- men s and he DiOC6(3) elease measu ed by cy o luo om- e y (Fig. 3; e e ences 22, 43; and unpublished da a). This sugges s ha all hese molecules can be eleased h ough he PT po e, in acco d wi h i s epo ed molecula cu o o 1,500 dal ons (12). The baseline DiOC6(3) inco po a ion was he same in liposomes con aining unc ional PTPC (mean luo escence channel 593 6 53, X 6 SEM, n 5 3) as ha obse ed in con ol liposomes (mean channel 601 6 43) (Fig. 3 B), indica ing ha he PT po e is cons i u i ely closed. PTPC-con aining liposomes elease DiOC6(3) in esponse o se e al agen s ha induce PT in in ac mi o- chond ia (A , e -bu ylhyd ope oxide, Ca21, diamide), wi h inhibi o y e ec s o bongk ekic acid, monochlo obiman, cy- clospo in A, and N-me hyl-Val-4-cyclospo in A, a nonim- munosupp essi e cyclophilin D ligand no ac ing on cal- cineu in (Fig. 3 C). These indings emphasize he unc ional simila i y be ween he na u al (mi ochond ial) PTPC (8, 12) and he econs i u ed (liposomal) PTPC (Fig. 3). Mo e- o e , hey con i m ha he ANT ( a ge o bongk ekic acid and a ac yloside), cyclophilin D ( a ge o cyclospo in A and N-me hyl-Val-4-cyclospo in), and edox-sensi i e SH Figu e 2. Two-dimensional gel elec opho esis o p o eins ex ac ed om PTPC liposomes (Fig. 1 A, 4). Sil e -s ained p o eins whose abun- dance is consis en ly ( h ee expe imen s) educed upon diges ion wi h caspase 1 (1.5 U/ml) a e ma ked in ec angles. Resul s a e ep esen a i e o h ee independen expe imen s. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1265 Ma zo e al. g oups ( a ge o diamide and monochlo obiman), as well as Ca21-sensi i e si es, pa icipa e in he egula ion o he PTPC (12, 13, 20–23). E ec o Recombinan Bcl-2 and Bcl-XL on PTPC. Unde he condi ions o ac iona ion desc ibed in Fig. 1, a p oap- op o ic membe o he Bcl-2 amily (Bax) selec i ely coen- iches wi h componen s o PTPC, whe eas se e al an iapop- o ic membe s o he Bcl-2 amily do no (Bcl-XL, Bcl-2; Fig. 1 D). We he e o e in es iga ed he e ec o an iapop- o ic membe s o he Bcl-2 amily on PTPC. Recombinan Bcl-2 and Bcl-XL p o eins, as well as mu an Bcl-2 p o- eins, we e inco po a ed in o liposomes oge he wi h PTPC ia dialysis, a p ocedu e ha allows o he o ien ed, pH-independen inco po a ion o p o eins in o lipid mem- b anes (45, 46). I espec i e o he p esence o Bcl-2–like p o eins, all liposome p epa a ions consis en ly (n 5 12) main ained a simila baseline DiOC6(3) luo escence (mean luo escence channel 620 6 18 and 616 6 19 in he p es- ence o absence o Bcl-2, espec i ely, X 6 SEM, 12 inde- penden expe imen s), wi h compa able SDS- eleasable DiOC6(3) elease (Fig. 4 A) o as long as 8 h (no shown), sugges ing ha Bcl-2 does no augmen he memb ane pe - meabili y in his expe imen al sys em. Mo eo e , Bcl-2 does no pe u b he ul as uc u e o PTPC liposomes o hei p o ein composi ion (no shown). The p esence o Bcl-XL o Bcl-2 p o ec ed agains he DiOC6(3) elease in- duced by a ac yloside, e -bu ylhyd ope oxide, as well as low doses o Ca21, bu no by diamide (Fig. 4 C). Simila esul s we e ob ained, when ins ead o DiOC6(3) e en ion, calcein e lux was s udied (no shown). These e ec s co e- la e wi h he unc ional po ency o Bcl-2, which p o ec s cells agains mos PT induce s (8, 9, 47), bu no agains di- amide (8, 9). A Bcl-2 dele ion mu an lacking a pu a i e channel- o ming domain co esponding o he a5 and a6 helices, Bcl-2Da5/6, which has los i s an iapop o ic unc- ion (34), ailed o p e en he DiOC6(3) elease. In addi- ion, a Bcl-2 poin mu an in he BH1 egion, Bcl-2 (Gly145Ala), which does no in e ac wi h Bax, ailed o p o ec agains apop osis (48) and had no inhibi o y e ec on PTPC liposomes (Fig. 4 C). Al oge he , hese indings sugges ha Bcl-2 can egula e memb ane pe meabili y by ac ing on o in conce wi h PTPC. E ec o Recombinan Caspases on PTPC. Since caspases a e in ol ed a all s ages o apop osis (5, 26, 49–51), we es ed whe he caspases migh ac on PTPC. PTPC econs i u ed in liposomes we e exposed o ecombinan caspases, ol- Figu e 3. Func ion o econ- s i u ed PTPC po es. (A) Re- lease o calcein om PTPC-con- aining liposomes incuba ed wi h wo an agonis ic ANT ligands. Calcein-loaded PTPC lipo- somes we e incuba ed wi h he indica ed dose o A and/o bongk ekic acid (50 mM), ol- lowed by luo ome ic de e mi- na ion o he calcein elease in o he supe na an . (B) Cy o luo o- me ic p o ile o liposomes la- beled wi h he po en ial-sensi i e luo och ome DiOC6(3). Lipo- somes we e econs i u ed ei he in he p esence o he hexoki- nase-con aining ac ion (PTPC liposomes) o in i s absence (con- ol liposomes), ea ed wi h SDS (0.25%), A (25 mM), and/o bongk ekic acid (BA; 50 mM), ollowed by DiOC6(3) s aining and cy o luo ome ic analysis. (C) PTPC liposomes ea ed wi h PT induce s (A [25 mM], CaCl2[25 mM], diamide [500 mM] o e -bu ylhyd ope oxide [ BHP, 500 mM]) and/o PT in- hibi o s (bongk ekic acid [BA; 50 mM], cyclospo in A [CsA; 10 mM], N-me hyl-4-Val-CsA [mod. CsA; 10 mM], o monochlo obi- man [MCB; 50 mM]). Resul s a e exp essed as pe cen age (X 6 SD o iplica es) o he DiOC6(3) elease induced by 0.25% SDS. Resul s a e ep esen a i e o a leas h ee independen de e mi- na ions. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1266 Pe meabili y T ansi ion Po e Complex in Apop osis lowed by de e mina ion o he DiOC6(3) e en ion. Se e al caspases induced DiOC6(3) elease in a dose-dependen ash- ion (Fig. 5, A and B). This e ec was only ob ained in PTPC- con aining liposomes, bu no in con ol liposomes (no shown). Te apep ide inhibi o s o caspases (Ac-YVAD.cmk o caspases 1 and 4 and Ac-DEVD.cmk o caspases 2, 3, and 6) abolish caspase-induced DiOC6(3) elease, sugges - ing ha his e ec in ol es p o eolysis a he han nonen- zyma ic p o ein in e ac ions (Fig. 5, A and B). Acco dingly, wo-dimensional gel elec opho esis o p o eins ex ac ed om PTPC liposomes sugges se e al uniden i ied p o eins o be caspase 1 subs a es (Fig. 2). The same caspases ha elease DiOC6(3) om PTPC liposomes also dis up he Dcm in isola ed li e mi ochond ia (Fig. 5 C) and elease AIF, which causes isola ed nuclei o unde go DNA ag- men a ion (Fig. 5 D). Bcl-2 and Bcl-XL inco po a ed in o liposomes educe he caspase-induced DiOC6(3) elease, whe eas inac i e Bcl-2 mu an s (Bcl-2Da5/6 and Bcl- 2(Gly145Ala) ail o s abilize PTPC (Fig. 6, A and B). This Bcl-2 e ec can be a leas pa ially o e come by high caspase concen a ions. Thus, in addi ion o s abilizing PTPC liposomes exposed o A , e -bu ylhyd ope oxide and cal- cium (Fig. 4), Bcl-2, and Bcl-XL pa ially supp ess caspase- induced DiOC6(3) elease (Fig. 6). Failu e o PTPC o Release Cy och ome c. Since induc ion o PTPC in in ac mi ochond ia causes cy och ome c e- lease (14, 15; and unpublished da a), and since se e al g oups ha e sugges ed ha Bcl-2 p ima ily egula es he e- lease o cy och ome c ia he ou e mi ochond ial mem- b ane a he han PT (6, 7, 31, 52), we in es iga ed he pu- a i e ela ionship be ween PT po e opening and cy och ome c. Inco po a ion o pu i ied cy och ome c in o PTPC lipo- somes (which cons i u i ely a e de oid o cy och ome c, Fig. 1 D) does no al e hei unc ional beha io . Thus, PTPC liposomes con aining cy och ome c exhibi a no mal baseline le el o DiOC6(3) e en ion and elease DiOC6(3) in esponse o A and caspases in a Bcl-2–inhibi able ash- ion (Fig. 7 A). Al hough such liposomes con ain signi ican amoun s o SDS- eleasable cy och ome c, hey ully e ain cy och ome c when incuba ed wi h doses o A o caspase ha cause DiOC6(3) elease (Fig. 7 B). This indica es ha PTPC a e no di ec ly esponsible o he elease o cy o- ch ome c. Discussion Func ional Equi alence o Na u al and Recons i u ed PTPC: A Ta ge o Mul iple E ec o s Including Caspases. PTPCs a e o med a he mi ochond ial inne /ou e memb ane con- ac si e whe e hey unc ion as a Ca21-, ol age-, pH-, and edox-ga ed channel wi h se e al le els o conduc ance (12, 13, 19). In his wo k, we epo he unc ional analysis o PTPC en iched om b ain homogena es and econs i- u ed in liposomes. Al hough he exac molecula composi ion o PTPC emains o be de ined, he unc ional explo a ion o PTPC (Fig. 3) sugges s ha i does con ain unc ionally in e connec ed si es o in e ac ion wi h bongk ekic acid and A ( wo ligands o he ANT and pe haps o he mem- Figu e 4. E ec s o Bcl-2 on PTPC. Hexokinase-en iched ac ions (Fig. 1 A, 3) we e in- co po a ed in o liposomes by di- alysis in he p esence o absence o ecombinan Bcl-2, Bcl-2 (Gly145Ala), Bcl-2Da5/6, o Bcl-XL, ollowed by unc ional analysis. (A) Rep esen a i e luo- escence p o iles o con ol PTPC and Bcl-2 PTPC lipo- somes ea ed wi h bu e only (con ol), SDS, o A , ollowed by incuba ion wi h DiOC6(3). No e he absence o A e ec s in Bcl-2 PTPC liposomes. (B) Inco po a ion o na i e and mu- an Bcl-2 p o eins in o lipo- somes. P o eins we e ex ac ed om PTPC liposomes p epa ed in he p esence o absence o he indica ed Bcl-2 mu an , ollowed by immunochemical quan i a- ion o Bcl-2 wi h a monoclonal an ibody ha ecognizes an epi ope ( esidues 20-34) no a - ec ed by he mu a ions. (C) Func ional impac o Bcl-2 and Bcl-XL. The di e en PTPC lipo- some p epa a ions we e ea ed wi h A (25 mM), CaCl2 (25 mM), diamide (500 mM), o e -bu ylhyd ope oxide ( BHP, 500 mM) o de e mine he DiOC6(3) elease. Resul s a e ep- esen a i e o h ee o i e independen expe imen s. 100% DiOC6(3) elease was de ined as he SDS-induced educ ion o DiOC6(3) luo escence ob- se ed in PTPC liposomes gene a ed in he absence o Bcl-2 o Bcl-XL. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1267 Ma zo e al. be s o he mi ochond ial ca ie amily), cy och ome c and N-me hyl-4-Val cy och ome c ( wo ligands o he cyclo- philin D), diamide, and monochlo obimane (which ac on hiol esidues), as well as Ca21. Acco dingly, we de ec ed he ANT, cyclophilin D, and addi ional molecules p e i- ously sugges ed o associa e wi h he ANT, namely po in and hexokinase, in he PTPC (Fig. 1). In addi ion o hese molecules, PTPCs pu i y wi h Bax, Bag-1, F1-ATPase (Fig. 1 D), and se e al noniden i ied p o eins (Fig. 2) whose im- pac on PTPC emains unclea . Howe e , he unc ional da a indica e ha PTPC liposomes egula e memb ane pe - meabili y in a ashion ha esembles he PT po e ound in Figu e 5. E ec o caspases on PTPC liposomes and isola ed mi ochond ia. (A) Rep esen a- i e DiOC6(3) luo escence his- og ams ob ained a e ea men o liposomes wi h a ious caspases (1.2 U/ml o caspase 1, 10 U/ml o caspase 6) in he p esence o absence o he indi- ca ed caspase inhibi o (100 mM). (B) Dose dependency o e ec s ob ained wi h di e en ecombinan caspases on PTPC liposomes. (C) E ec o caspases on he Dcm. Mi ochond ia we e ea ed du ing 30 min wi h 5 U caspase/200 ml, ollowed by de- e mina ion o he Dcm using DiOC6(3). The p o onopho e m-chlo ophenylhyd azone (50 mM) de ined 100% Dcm dis up- ion. (D) Release o AIF in o he mi ochond ial supe na an . In ac mi ochond ia we e ea ed wi h he indica ed caspase (5 U/200 ml), ollowed by cen i uga ion and emo al o he supe na an ha was es ed o apop ogenic ac i i y on isola ed HeLa nuclei. The incuba ion was pe o med in he p esence o e apep ide inhibi o s (which inhibi caspases bu no AIF) o in he p esence o Z-VAD. mk (which inhibi s AIF) o exclude ha nuclea DNA deg ada ion is a di- ec caspase e ec . Simila esul s we e ob ained wi h mouse and a (no shown) hepa ocy e mi ochond ia. Figu e 6. E ec o Bcl-2 on he caspase induced DiOC6(3) elease obse ed in PTPC lipo- somes. (A) Rep esen a i e DiOC6(3) s aining p o iles. Lipo- somes we e gene a ed in he p esence o ecombinan Bcl-2, Bcl-XL, and he indica ed Bcl-2 mu an s, ea ed wi h 1 U caspase 1, and labeled wi h DiOC6(3) o de e mine he DiOC6(3) elease. Resul s a e ep esen a i e o a leas h ee independen de e mi- na ions. (B) Dose esponse cu es o caspase e ec s on liposomes con aining Bcl-XL, Bcl-2, o Bcl-2 mu an s. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1268 Pe meabili y T ansi ion Po e Complex in Apop osis mi ochond ia. Thus, using a numbe o di e en induce s and inhibi o s o PT, we ound an app oxima e unc ional equi alence be ween he na u al (mi ochond ial) PTPC and he econs i u ed (liposomal) PTPC (Fig. 3) in he eg- ula ion o memb ane pe meabili y. Bo h in mi ochond ia and in PTPC liposomes, a simila panel o agen s ac s o pe meabilize memb anes (Ca21, A , p ooxidan s, and dia- mide) o o s abilize memb ane unc ion (cyclospo in A, monochlo obiman, and bongk ekic acid; e e ences 8, 12, Fig. 3). Thus, he p o ocol o PTPC en ichmen and in- co po a ion in o liposomes yields a educed expe imen al sys em in which hei unc ion can be analyzed wi hou in- e e ence by o he mi ochond ial s uc u es. The equi alence be ween he na u al and he econs i- u ed PTPC also ex ends o he ac ha caspases dis up he memb ane pe meabili y in bo h PTPC liposomes (Fig. 5, A and B) and in ac mi ochond ia (Fig. 5, C and D). This sugges s, in line wi h p e ious obse a ions (5, 26, 30, 49–51), ha caspases ac as acul a i e induce s o PT (e.g., caspase-1 ac i a ed a e Fas/APO-1 c oss-linking and pe - haps caspase 3 in neu onal de elopmen ) in speci ic signal ansduc ion pa hways. The molecula a ge (s) o caspases wi hin he PTPC emain(s) o be de ined. O no e, caspases a e no only in ol ed in he ups eam p emi ochond ial phase, bu also in he downs eam pos mi ochond ial s age o apop osis, when hey a e ac i a ed as a esul o mi o- chond ial cy och ome c and AIF elease (5–7, 26). Thus, mi ochond ia and caspases may engage in a posi i e ampli- ica ion loop in which caspases cause mi ochond ial mem- b ane dis up ion, which in u n a o s he elease o caspase-ac i a ing ac o s. Bcl-2– ela ed P o eins Ac on PTPC. The da a epo ed in his pape indica e ha Bcl-2 and Bcl-XL egula e PT by di ec ly ac ing on PTPC. I has been sugges ed ha Bcl-2 and Bcl-XL would p ima ily ac on he mi ochond ial e- lease o cy och ome c (6, 7, 52), which would be an e en ups eam o (6, 31) o independen om (7) PT po e open- ing. Howe e , he PTPC econs i u ed in o liposomes do no con ain cy och ome c (Fig. 1, B and D), ye a e egu- la ed by Bcl-2 and Bcl-XL, implying ha Bcl-2/Bcl-XL a - ec ce ain mi ochond ial unc ions in a cy och ome c–inde- penden ashion. As shown in his wo k, he PTPC is no he s uc u e esponsible o cy och ome c elease (Fig. 7), in line wi h p e ious es ima ions sugges ing ha he PTPC has a molecula cu o o z1,500 dal ons (12, 13). Two specula i e possibili ies emain plausible. Fi s , he p ima y egula o y a ge o Bcl2/BclXL in he mi ochond ion could be he PTPC ha , once opened, causes cy och ome c elease in an indi ec ashion, ei he by ac i a ing a ye un- known cy och ome c–speci ic anspo e o by mechanically dis up ing he in eg i y o he ou e mi ochond ial mem- b ane, e.g., due o local dis ension o he mi ochond ial ma ix (12–14, 53). Second, Bcl2/BclXL migh a ec PTPC and cy och ome c independen ly om each o he in a pleio opic ashion. In a o o his la e hypo hesis, BclXL has been epo ed o bind o cy och ome c (52), and Bcl-2 migh in e ac wi h cy och ome c ia he mammalian CED4 homologue (54). Recombinan Bcl-2 and Bcl-XL inco po a ed in o PTPC liposomes inhibi he induc ion o PT by a a ie y o induce s: he ANT ligand a ac yloside, he p ooxidan e - bu ylhyd ope oxide, Ca21 (Fig. 4), and low doses o caspases (Fig. 6). In con as , Bcl-2 and Bcl-XL ail o p o ec PTPC liposomes agains diamide (Fig. 4 C), in line wi h he ac ha Bcl-2 is an ine icien inhibi o o diamide-induced Dcm dis up ion, bo h in cells and in isola ed mi ochond ia (8, 9). Mo eo e , Bcl-2 ails o p e en he e ec s o high doses o caspases (Fig. 6), in acco d wi h ou p e ious ob- se a ion ha Bcl-2 p esen in mi ochond ia om human CEM-C7 T lymphoma cells ails o coun e ac caspase 1–induced PT and apop osis (26). The inding ha hese Bcl-2 and Bcl-XL e ec s can be o e come by high, bu no by low, doses o caspses may esol e a con o e sy oppos- ing models in which Bcl-2 homologues comple ely ail o p e en Fas/APO-1 (caspase 1–dependen ) apop osis (26, 55–58) o , on he con a y, e icien ly coun e ac caspase 1–media ed (59) o Fas/APO-1– igge ed apop osis (30, 60). Mo eo e , he ac ha Bcl-2 mi iga es he PT in- duced by caspases ha a e b oadly in ol ed in apop osis (e.g., caspases 3 and 6) sugges s ha i can in e up a sel - ampli ying loop in which caspase e ec s on mi ochond ia a o he elease o caspase ac i a o s. We ha e in es iga ed whe he Bcl-2 ac s as an inhibi o o caspase-media ed di- ges ion o PTPC p o eins. Ou p elimina y indings indi- ca e ha Bcl-2 does no p e en he diges ion o caspase 1 subs a es (no shown), sugges ing ha i inhibi s he unc- ional consequence o caspase 1–media ed p o eolysis a he han p o eolysis i sel . I has been shown ecen ly ha caspase 3 clea es Bcl-2, he eby con e ing i om a dea h inhibi o o a dea h p omo e (61). Howe e , caspase 1 does no diges Bcl-2, a leas in he condi ions epo ed in Fig. 6 A, sugges ing he unc ional ele ance o addi ional caspase a ge s wi hin he PTPC. C ys allog aphic da a (62) and s udies o a i icial mem- b anes con aining Bcl-XL o Bcl-2 (33, 34) sugges ha Bcl-2–like p o eins cons i u e ion channels. Howe e , Bcl- XL and Bcl-2 inco po a ed in o memb anes con aining PTPC, a he han inc easing memb ane pe meabili y, s a- bilize PTPC liposomes and p e en PT po e opening. This appa en disc epancy may be explained by he composi ion Figu e 7. Cy och ome c e- en ion in PTPC liposomes. Li- posomes we e gene a ed in he absence o p esence o ecombi- nan Bcl-2, ollowed by gene a- ion o a KCl-dependen ion g adien and inco po a ion o cy- och ome c du ing he sonica ion s ep. (A) E ec o SDS (0.25%), A (50 mM), o caspases 1 o 3 (1 U), as de e mined by low cy- ome y a e labeling wi h DiOC6(3). (B) Supe na an s o he liposomes ea ed as in A we e sub- jec ed o p o ein p ecipi a ion, ollowed by Wes e n blo analysis o he elease o cy och ome c. No e ha he blo has been o e exposed. The amoun o cy och ome c eleased upon SDS ea men was es ima ed o be 1 mg, and he de ec ion limi o he immunoblo is z10 ng/lane. Downloaded om h p:// up ess.o g/jem/a icle-pd /187/8/1261/1115455/97-1889.pd by Uni e sidad De Za agoza use on 27 Oc obe 2020 1269 Ma zo e al. o he a i icial memb anes, which only allow Bcl-2 o o m channels when hey con ain, in addi ion o neu al lipids (as in his pape ), an unusually high pe cen age (30– 40%) o acidic lipids (33, 34). A p esen , we canno dis- c imina e be ween he possibili ies ha he PT-inhibi o y e ec o Bcl-2 is due o in e ac ions wi h and con o ma- ional e ec s on PTPC cons i uen s, o a he due o he speci ic neu aliza ion o Bax (35, 48), a p oapop o ic mol- ecule ha is p esen in PTPC (Fig. 1 D) and a o s PT (32). I espec i e o hese possibili ies, he Bcl-2 e ec on PTPC co ela es wi h i s an iapop o ic po en ial in he sense ha mu a ions o dele ions abolishing he dea h an agonis ic po en ial o Bcl-2 also ab oga e i s PT-inhibi o y unc ion. In addi ion o i s PT-inhibi o y e ec , which may ac- coun o a leas pa o i s cy op o ec i e ac ion, Bcl-2 has u he pleio opic e ec s (4, 10). Al hough some o hese e ec s, including hose conce ning he capaci y o Bcl-2 o a ec edox egula ion o in acellula Ca21 pa i ion, may be seconda y o PT modula ion; o he s a e mo e di icul o accommoda e in a model in which he majo ac ion o Bcl-2 would be PT egula ion. This applies, in pa icula , o he pa icipa ion o Bcl-2 pa icipa ion in a mul ip o ein ensemble o “apop osome” in ol ing he mammalian CED-4 homologue(s), cy och ome c, and la ge p odomain caspases. As a possibili y, Bcl-2 could exe a dual unc ion in which i simul aneously o sequen ially ac s on PTPC and inac i a es he apop osome (10). The Cen al Execu ione o Apop osis: In ol emen o PT- PC? Changes in mi ochond ial memb ane unc ion ha e been p oposed o o m pa o he “cen al execu ione ” (63), colloquially also e e ed o as “g ea in eg a o ” o “apos a ” (2, 3, 6–8, 11). Ac i a ion o he cen al execu- ione du ing he e ec o s age would con ol he commi - men o unde go cell dea h and uni y he many p i a e in- duc ion pa hways o apop osis in o one common pa hway. The indings epo ed he ein indica e ha PTPC can con- s i u e a c oss oad a which physiological modula o s o PT (Ca21, Mg21, pH, ADP, ATP, NAD(P)H, glu a hione, ce - amide, lipid oxida ion p oduc s, e c.; e e ences 12, 13, 19; Fig. 3), caspases (Fig. 5), and Bcl-2 homologues (Fig. 4, 6) oge he in luence he a e o he cell. Thus, PTPC may si- mul aneously collec in o ma ion on he me abolic s age o he cell, signal ansduc ion pa hways, as well as on he composi ion o he Bcl-2 complex. Opening o he PT po e, which occu s almos uni e sally du ing apop osis, has le hal epe cussions including he mi ochond ial gene a ion o eac i e oxygen species, dis up ion o oxida i e phos- pho yla ion, and he mi ochond ial elease o apop ogenic p o eins necessa y o he ac i a ion o downs eam caspases and endonuclease ac i a ion (1–10, 14, 15). In conclusion, PTPC may be iden ical wi h o o m pa o he c i ical s uc u e ha in eg a es di e en apop osis induc ion pa hways, decides he a e o he cell, and coo - dina es he common dea h p og am. I his in e p e a ion is co ec , he u u e elucida ion o he exac composi ion and ine uning o PTPC should u nish in aluable clues o he unde s anding o he apop o ic p ocess. We hank D s. A. S ini asen and K. Tomaselli (Idun Pha maceu icals, La Jolla, CA) o ecombinan caspases 1, 3, and 6; N. Tho nbe y (Me ck, Rahway, NJ) o caspases 1, 2, and 4; G. Sal esen (The Bu nham Ins i- u e, La Jolla, CA) o caspases 3 and 6; D s. S. Ma sujama, C. Aimé-Sempé, and S. Takajama (The Bu nham Ins i u e) o Bcl-2 plasmid cons uc ions. Elec on mic oscopic analyses o PTPC liposomes we e pe o med by Ma ie-Ch is ine P é os (Pas eu Ins i u e, Pa is, F ance). This wo k has been suppo ed by g an s om he Agence Na ionale pou la Reche che con e le SIDA, As- socia ion pou la Reche che con e le Cance , Cen e Na ional de la Reche che Scien i ique, Fonda ion pou la Reche che Médicale, Ins i u Na ional de la San é e de la Reche che Médicale, Ligue Na ionale con e le Cance ( o G. K oeme ), Uni e si y o Cali o nia B eas Cance Resea ch P og am (g an No. IRB- 009B) and CaP-CURE Inc. ( o J.C. Reed). I. Ma zo and S.A. Susin ecei e ellowships om he Spanish Minis y o Science and om he Eu opean Commission, espec i ely. Add ess co espondence o Guido K oeme , 19 ue Guy Môque , B.P. 8, F-94801 Villejui , F ance. Phone: 33-1-49-58-35-13; Fax: 33-1-49-58-35-09; E-mail: [email p o ec ed] Recei ed o publica ion 22 Oc obe 1997 and in e ised o m 13 Janua y 1998. Re e ences 1. Zamzami, N., P. Ma che i, M. Cas edo, C. Zanin, J.-L. Va- yssiè e, P.X. Pe i , and G. K oeme . 1995. 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