PubMed Health⌕ Search

Biomedical subjects

S Virji

Publications and source records attributed to S Virji.

8 recordsLinked to original sources
PubMed1999

Evidence that cyclophilin-A protects cells against oxidative stress.

Cyclophilin-A is the cytosolic isoform of a family of peptidylproline cis-trans-isomerases that bind cyclosporin A. This study investigates the role of cyclophilin-A in necrotic cell death, induced by 'chemical ischaemia' and by t-butylhydroperoxide. An 18-mer antisense phosphorothioate oligodeoxynucleotide was used to target a translated region of cyclophilin-A mRNA in rat neonatal cardiomyocytes. After a 24 h exposure to the oligonucleotide, the amount of cyclophilin-A in the cells was decreased by at least 93% as judged by immunological and enzymic criteria. For the enzyme assays, peptidyl proline cis-trans-isomerase activity was measured fluorimetrically in small (10 microl) volumes of cell extract. Immunoblots were developed with a polyclonal anti-cyclophilin-A antibody after sample isoelectric focusing and SDS/PAGE. Cyclophilin-A suppression had no effect on cyanide-plus-2-deoxyglucose-induced cell death. However, cyclophilin-A-suppressed cells were markedly more sensitive to t-butylhydroperoxide. Cyclosporin A conferred some resistance to the peroxide in both types of cell, but protection was greater in cyclophilin-A-suppressed cells, where cyclosporin A increased the survival time 2-fold. It is concluded that two cyclophilin isoforms are involved, in quite different ways, in peroxide-induced cell death. Cyclophilin-A has a protective role. Another isoform, possibly mitochondrial cyclophilin-D, has a deleterious role, such that blockade by cyclosporin A leads to protection.

Animals↗
PubMed1999

The mitochondrial permeability transition pore.

This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.

Animals↗
PubMed1999

Import and processing of heart mitochondrial cyclophilin D.

Cyclophilins are a family of cyclosporin-A-binding proteins which catalyse rotation about prolyl peptide bonds. A mitochondrial isoform in mammalian cells, cyclophilin D, is a component of the permeability transition pore that is formed by the adenine nucleotide translocase and the voltage-dependent anion channel at contact sites between the inner and outer membrane. This study investigated the submitochondrial location of cyclophilin D by following the fate of radiolabelled protein following import. Precursor [(35)S]cyclophilin D was expressed in vitro from a PCR-generated cDNA. The precursor was imported by rat heart mitochondria and processed in a single step to a 21-kDa protein that was identical (SDS/PAGE) to an in vitro expressed mature protein and a cyclophilin D purified from rat heart mitochondria. No further modification of the mature protein could be demonstrated. Fractionation of mitochondria following import established that cyclophilin D locates only to the matrix. It is concluded that cyclophilin D binding to the permeability transition pore must occur at the inner face of the mitochondrial inner membrane.

Animals↗
PubMed1998

Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore.

A cyclophilin-D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore. A cDNA encoding cyclophilin-D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells. The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibition by cyclosporin A. The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix. The matrix selectively bound 32-kDa proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound. The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and the adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus-purified voltage-dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. These properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes.

Animals↗
PubMed1996

Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress.

Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.

Adenosine Diphosphate↗