Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Changes in mitochondrial enzymatic activities of monocytes during prolonged hypobaric hypoxia and influence of antioxidants: A randomized controlled study

OBJECTIVES Exposure to high altitudes is associated with oxidative cellular damage due to the increased level of reactive oxygen and nitrogen species and altered activity of antioxidant systems. Subjects were submitted to prolonged hypoxia, to evaluate changes in mitochondrial enzyme activities of monocytes and their attenuation by supplementation with antioxidants. METHODS Twelve subjects were randomly assigned to receive antioxidant supplements or placebo prior to and during an expedition to Pik Lenin (7145 m). Monocytes were isolated from blood samples to determine the activity of mitochondrial enzymes cytochrome c oxidase and citrate synthase at 490 m (baseline) and at the altitudes of 3550 m, 4590 m, and 5530 m. RESULTS An increase in citrate synthase activity at all altitudes levels was observed. Hypoxia induced an increase in the activity of cytochrome c oxidase only at 4590 m. Neither citrate synthase activity nor cytochrome c oxidase activity differed between the subjects receiving antioxidant supplements and those receiving placebo. CONCLUSIONS Hypoxia leads to an increase in citrate synthase activity of monocyte mitochondria as a marker of mitochondrial mass, which is not modified by antioxidant supplementation. The increase in mitochondrial mass may represent a compensatory mechanism to preserve oxidative phosphorylation of monocytes at high altitudes.

Mimicking respiratory phosphorylation using purified enzymes

The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

p73 regulates basal and starvation-induced liver metabolism in vivo

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly

We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. What is Known: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.