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A mitochondrial cylophilin

Virji, Sukaina; (1999) A mitochondrial cylophilin. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Cyclophilins (CyPs) are a family of proteins which catalyse the cis/trans isomerisation of prolyl-peptide bonds in proteins. The activity is blocked by the immunosuppressant drug Cyclosporin A. Cellular CyPs have been implicated in the refolding and trafficking of proteins and in Ca2+ signalling pathways but their precise roles have yet to be elucidated. Mitochondrial CyP is of potential clinical significance as it may control the activity of a mitochondrial inner membrane pore relevant to certain forms of cell injury brought about by Ca2+ and oxidant stress. In the present study, purification of PPIase from the matrix and intermembrane space fractions of rat liver mitochondria yielded a 21kDa CyP (CyP21) and an 18kDa CyP respectively on SDS-PAGE. Their locations were confirmed by mitochondrial marker enzyme analyses. The peptide sequence of CyP21 was determined and used to design degenerate primers; the aim was to generate a probe by PCR to screen a cDNA library for the full length cDNA of CyP21. However, degenerate primers were unsuccessful. Accordingly, the published human mitochondrial CyP base sequence was used to design non-degenerate primers. A fragment of DNA was obtained, inserted into the plasmid vector pCR-Script, and cloned in E.coli. Dideoxy sequencing confirmed it was from the correct protein and so it was radiolabelled and used to screen a rat liver cDNA library in bacteriophage . A positive phage clone was obtained, inserted into the plasmid vector pBluescript, cloned into E.coli and sequenced to confirm its identity. The cloned cDNA was then used to construct a CyP-GST fusion protein using a pGEX plasmid vector. The fusion protein was expressed and used to prepare a CyP affinity matrix. The binding of mitochondrial constituents under various conditions relevant to activation of the mitochondrial pore was investigated. A mitochondrial inner membrane protein of approximately 32kDa molecular weight was found to bind to the CyPD part of the fusion protein in a manner insensitive to traditional pore effectors. The possible nature of this binding protein is discussed.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A mitochondrial cylophilin
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Mitochondrial cyclophin
URI: https://discovery.ucl.ac.uk/id/eprint/10102362
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