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.

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.

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.

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.

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.

Risk factors associated with preterm deliveries among racial groups in a national sample of married mothers.

Seven sociodemographic and behavioral factors that may explain the increased risk of preterm deliveries among black women were examined using data from a national sample of 5823 married mothers who responded to the 1980 National Natality Survey (NNS) Questionnaire. There was a twofold increase in the rate of preterm deliveries among black women. Additionally, there was a significant decrease (by 1 week) in the mean gestational age in black mothers (p less than 0.0001) compared with white mothers. The two groups were similar with respect to smoking and age; however, there were significant differences between the two groups with respect to other risk factors. Black women had a higher rate of heavy alcohol use, significantly fewer prenatal visits, prenatal care was started later during pregnancy (p less than 0.0001) and were less educated compared with white women. The odds ratio (OR) for race adjusted for the risk factors was 1.56 (95% confidence interval (CI) equals 1.21, 2.01). All other risk factors except education had adjusted ORs greater than 1. Those risk factors that were more strongly associated with the risk of preterm births included weight gain (OR, 2.10; 95%, 1.79, 2.47), number of prenatal visits (OR, 3.37; 95% CI, 2.87, 3.95) and smoking (OR, 1.34; 95% CI, 1.13, 1.59). We conclude that race is an independent risk factor for preterm deliveries. Additionally, it is shown here that the risk of preterm deliveries is attributable to health behaviors that are amendable to change.

The relationship between alcohol consumption during pregnancy and infant birthweight. An epidemiologic study.

Heavy alcohol consumption during pregnancy has been consistently associated with inhibited intra-uterine growth; however, the effect of social drinking is not yet clear. The relationship between moderate drinking and low birth weight (less than 2,500 g) among a nationally representative sample of white married mothers who gave birth to singletons infants in the National Natality Survey (1980) is analysed here. Alcohol consumption during pregnancy was significantly associated with birth weight (p less than 0.02). Moreover, there was a gradient of risk in low birth weight associated with increasing amount of alcohol ingestion during pregnancy. There was a significant association between the mean birth weight of the singletons across different categories of alcohol intake (p less than 0.0001). The difference between the mean birth weight of the singletons among moderate drinkers compared with nondrinkers was also statistically significant (p less than 0.005). These relationships remained after simultaneously adjusting effects of the confounding variables gestational age, parity, smoking, weight gain, maternal age and education in multiple regression analyses. These findings confirm earlier reports of a relationship between alcohol use during pregnancy and decreased birth weight. Additionally, it is shown here that for moderate alcohol use during pregnancy, there is an adverse effect on the birth weight.

The relationship between occupational classification and low birth weight in a national sample of white married mothers.

The relationship between occupational classification and Low Birth Weight singletons (LBW; less than 2500 g) was studied in a sample of white married mothers employed in the National Natality Survey 1980 (N = 3300). Univariate analyses included relationship between birth weight and occupation, smoking, alcohol, parity, age, education, prenatal care, weight gain during pregnancy, gestation and sex of the singleton. Occupation was significantly associated with LBW (P less than 0.05). The LBW rate was higher among blue collar workers (162 per 1000) compared to white collar workers (132 per 1000). The mean birth weight of singletons among blue collar workers was also significantly lower compared to those among white collar workers (P less than 0.057). However, after adjusting simultaneously for the effect of the confounding variables in multiple regression analysis, occupation was no longer a significant predictor of LBW. The extent to which the validity problems may limit the interpretation of the study are discussed.