Tanveer, Ahmed; (1998) A cyclophilin-dependent mitochondrial pore. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The immunosuppressant Cyclosporin A (CsA) has been shown to protect against cell injury caused by ischaemia and reperfusion. A mitochondrial lesion in the form of a Ca2+ sensitive non-selective inner membrane pore is reputed to contribute to the progression of this form of injury and the pore is blocked by CsA. This study was aimed at resolving the CsA receptor of the pore to establish whether this protein was novel or whether it was already recognized. Mitochondrial CsA binding proteins were identified using a tritiated derivative of CsA, 3H[PA-CS], which contains a photoactive diazirine group at position 8 of the molecule. Many components were photolabelled. In order to identify the relevant protein use was made of the fact that CsA binding to mitochondrial membranes was potentiated by ADP and depressed by Ca2+. Heart mitochondria were photolabelled in the presence of ADP (2mM) or Ca2+ (>100μM). Fractionation of mitochondrial membranes extracted in detergent revealed components between 11 and 25kDa exhibiting ADP/Ca2+-sensitive labelling. ADP increased 3H[PA-CS] labelling of this fraction whilst Ca2+ reversed it. The ADP/Ca2+-sensitive labelled component was purified to a single band on SDS-PAGE migrating at approximately 22kDa Identical proteins were purified from sheep heart and rat liver mitochondria The 22kDa protein displayed peptidylprolyl cis trans isomerase (PPIase) activity suggesting it belonged to the cyclophilin (CyP) family of proteins. The protein displayed a Ki of 8nM for CsA and a kcat/Km of 5.8μM1 s-1. This agrees well with other known cyclophilins. Partial amino acid sequencing of the protein revealed sequence similarity with human cyclophilin D (CyPD). CyPD was prone to proteolytic attack during purification However removal of the outer mitochondrial membrane with low concentrations of digitonin and the subsequent treatment of the mitoplasts with 0.5M [NaCl] resulted in increased stability with an almost 10 fold increase in protein yield The localisation of CyPD was investigated using fractionated mitochondria This showed that it is localised in the matrix space Though CyPD is photolabelled in an ADP/Ca2+-sensitive manner, the [3H]CsA binding capacity of CyPD was unaffected by ADP [2mM] or Ca2+ [>100μM]. Also the PPIase activity of the protein was not influenced by high concentrations of ADP or Ca2+. This, as well as photolabelling studies, suggested that CyPD binds to another component in intact mitochondria that is sensitive to ADP and Ca2+, and that this interaction confers the same sensitivity to CyPD-CsA interaction The occasional elution of the ADP/Ca2+ sensitive component at 29kDa, rather than 22kDa, suggests that the target might be a low molecular weight protein. However a CyPD affinity column failed to retain any specifically interacting target protein from inner membrane extracts. These findings provide firm evidence that CyPD interacts with the Ca2+ sensitive pore and that it might be involved in pore regulation.

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