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Interrelations between BID and cyclophilins in the mitochondrial apoptotic pathway.

Gillick, K.; (2006) Interrelations between BID and cyclophilins in the mitochondrial apoptotic pathway. Doctoral thesis , University of London. Green open access

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The BH3-only BCL-2 protein BID is thought, in some cases, to be responsible for the initiation of cytochrome c release from mitochondria during apoptosis. In order to study this process in an in vitro system, the rat Bid gene was cloned, recombinant BID purified, and truncated BID (tBID) produced. Additionally, reliable procedures were developed for the isolation of intact mitochondria from a rat neuroblastoma cell line and for assay of cytochrome c release from the isolated mitochondria. The involvement of the permeability transition (widely regarded as a mechanism for mitochondrial permeabilisation and/or remodelling during cytochrome c release) was investigated. However, using mitochondrially entrapped fluorophores and agents that promote the transition (Ca2+, overexpression of mitochondrial cyclophilin D), BID-induced cytochrome c release was in fact found to be independent of permeability transition. Nevertheless, CSA, a potent inhibitor of cyclophilins and the permeability transition, did inhibit cytochrome c release. To resolve the action of CSA, a kinetic model of tBID-induced cytochrome c release was formulated from first principles. This considered tBID-induced oligomerisation and auto-oligomerisation of the BAK protein into BAK pores and efflux of cytochrome c via these pores. The model was based on previously published data and the presence of BAK, but not BAX, in the isolated mitochondria. Analysis according to this model indicated that the formation of pores, rather than efflux through pores, was inhibited by CSA. Direct measurements of tBID-induced change in BAK conformation (an essential step in oligomerisation) by trypsin cleavage confirmed that CSA inhibited this step. As potential targets of CSA, the isolated mitochondria contained both cyclophilin D (mitochondrial matrix) and residual cyclophilin A (a cytosolic enzyme). However neither cyclophilin D (overexpression) nor cyclophilin A (addition of recombinant enzyme) stimulated cytochrome c release. Moreover, DB25, a CSA analogue that inhibits the catalytic activity of both cyclophilins, but does not allow interaction with downstream targets such as calcineurin, was ineffective, and instead had a stimulatory effect on cytochrome c release. It appeared therefore that the cyclophilin-CSA complex may be the inhibitory species. However inhibition was not attributable to calcineurin inhibition, as judged by 32P labelling and the effects of Ca2+. It is concluded that the cyclophilin-CSA complex may inhibit BAK conformational change by a novel mechanism. Two other novel findings arose in the course of the study, namely a 54 kD proline-rich cyclophilin D binding protein (identified by pull-downs and mass spectrometry) and phosphorylation of a (non- identified) 17 kD protein in cyclophilin D overexpressing mitochondria.

Type: Thesis (Doctoral)
Title: Interrelations between BID and cyclophilins in the mitochondrial apoptotic pathway.
Identifier: PQ ETD:592843
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: https://discovery.ucl.ac.uk/id/eprint/1445519
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