Interrelations between Bid and Cyclophilins in the Mitochondrial Apoptotic Pathway

T he B H 3-only B C L -2 protein B ID is thought, in som e cases, to be responsib le fo r the initiation o f cytochrom e c release from m itochondria during apoptosis. In o rder to study this process in an in vitro system , the ra t B id gene w as cloned, recom binant B ID purified , and truncated B ID (tB ID ) produced. A dditionally , reliable p rocedures w ere developed fo r the isolation o f in tact m itochondria from a rat neu rob lastom a cell line and fo r assay o f cy tochrom e c release from the iso lated m itochondria. T he involvem ent o f the perm eability transition (w idely regarded as a m echanism for m itochondrial perm eabilisation and/or rem odelling during cy tochrom e c release) w as investigated. H ow ever, using m itochondrially entrapped fluorophores and agents that prom ote the transition (C a2+, overexpression o f m itochondrial cyclophilin D), B ID -induced cytochrom e c release w as in fact found to be independent o f perm eability transition. N evertheless, C SA , a potent inh ib ito r o f cyclophilins and the perm eability transition , did inhibit cy tochrom e c release. T o resolve the action o f C SA , a kinetic m odel of tB ID -induced cy tochrom e c release w as form ulated from first principles. T his considered tB ID -induced oligom erisation and au to-oligom erisation o f the B A K protein into B A K pores and efflux o f cytochrom e c via these pores. T he m odel w as based on prev iously published data and the presence o f B A K , but not B A X , in the iso lated m itochondria. A nalysis according to this m odel indicated that the form ation o f pores, rather than efflux through pores, w as inhibited by C SA . D irect m easurem ents o f tB ID -induced change in B A K conform ation (an essen tia l step in o ligom erisation) by trypsin cleavage confirm ed that C SA inhibited this step. A s potential targets o f C SA , the iso lated m itochondria contained both cyclophilin D (m itochondrial m atrix ) and residual cyclophilin A (a cytosolic enzym e). H ow ever neither cyclophilin D (overexpression) nor cyclophilin A (addition o f recom binant enzym e) stim ulated cy tochrom e c release. M oreover, D B25, a C SA analogue tha t inhibits the catalytic activity o f both cyclophilins, but does not allow interaction w ith dow nstream targets such as calcineurin, w as ineffective, and instead had a stim ulatory effect on cytochrom e c release. It appeared therefore that the cyclophilin-C SA com plex m ay be the inhibitory species. H ow ever inhibition w as not a ttributable to calcineurin inhibition, as ju d g ed by 32P labelling and the effects o f C a2+. It is concluded that the cyclophilin -C SA com plex m ay inh ib it B A K conform ational change by a novel m echanism . T w o o ther novel findings arose in the course o f the study, nam ely a 54 kD pro line-rich cyclophilin D b ind ing p ro tein (identified by pull-dow ns and m ass spectrom etry) and phosphory lation o f a (non­ iden tified) 17 kD protein in cyclophilin D overexpressing m itochondria.