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The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics. Numerous CYP allelic variants have been identified; the functional characterisation of these variants is required for their application in precision medicine. Results from heterologous expression systems using mammalian cells can be integrated in in vivo studies; however, other systems such as E. coli , bacteria, yeast, and baculoviruses are generally used owing to the difficulty in expressing high CYP levels in mammalian cells. Here, by optimising transfection and supplementing conditions, we developed a heterologous expression system using 293FT cells to evaluate the enzymatic activities of three CYP isoforms (CYP1A2, CYP2C9, and CYP3A4). Moreover, we established co-expression with cytochrome P450 oxidoreductase and cytochrome b 5 . This expression system would be a potential complementary or beneficial alternative approach for the pharmacokinetic evaluation of clinically used and developing drugs in vitro.

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

The high abundance and molecular versatility of iron have led to its universal presence in biological systems, yet its absorption is exceptionally challenging. Animals and yeasts use divalent metal transporters to import iron, but yeasts also employ the multicopper oxidase Fet3p for high-affinity iron uptake when iron-starved. Using long-term iron depletion in Drosophila , we identified four components involved in iron absorption: Multicopper oxidase-4 (Mco4), a Fet3p ortholog, is essential for surviving iron starvation, whereas the cytochrome b561 enzymes Fire (Ferric Iron Reductase) and Fire-like, as well as cytochrome b5 protein Firewood, are required for iron absorption under normal conditions. This study reports the presence of a high-affinity iron uptake system in an animal, a cytochrome b5 electron donor for ferric iron reduction, and intestinal ferric reductases, and provides a valuable resource for further exploration of genes involved in iron homeostasis, transport, and absorption. Although iron is essential, its absorption is inefficient; this study uncovers distinct iron uptake strategies in Drosophila , including a previously unrecognized high-affinity system activated during iron starvation.

Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the K approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.

Key messageA candidate gene cytochrome b5 for the major QTL qSRMP9 for rice seed reserve mobilization was validated during seed germination using a genome-wide association study approach.Seed reserve mobilization plays important roles in the early seedling growth in rice. However, the genetic basis underlying this process is poorly understood. In this study, the genetic architecture of variation in seed reserve mobilization during seed germination was studied using a genome-wide association study approach in rice. Three quantitative trait loci (QTL) including qSRMP6, qSRMP9, and qSRMP12 for seed reserve mobilization percentage were identified. In which, the candidate gene cytochrome b5 (OsCyb5) for the major QTL qSRMP9 was validated. Disruption of this gene in Oscyb5 mutants reduced the seed reserve mobilization and seedling growth compared with wild-type (WT) in rice. There were no significant differences of grain size, starch, protein and total soluble sugar content in the mature grains between Oscyb5 mutants and WT. However, the α-amylase activity in the germinating seeds of Oscyb5 mutants was significantly decreased compared to that of WT, and then, the starch and sugar mobilization and the glucose accumulation during seed germination were significantly decreased in Oscyb5 mutants. Two elite haplotypes of OsCyb5 associated with the higher seed reserve mobilization percentage and its elite single nucleotide polymorphism variations were mainly existed in the INDICA and AUS accessions. The natural variation of OsCyb5 contributing to seed reserve mobilization might be useful for the future rice breeding.

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.

Flavonoids are important secondary metabolites in many fruits, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. Hydroxylation of the B-ring of flavonoids is catalyzed by flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), and may also require cytochrome b₅. We report the identification of genes encoding F3'H, F3'5'H, and a putative cytochrome b₅ from grapevine (Vitis vinifera L. cv Shiraz) and their transcriptional regulation in fruit. Functionality of the genes VvF3'H and VvF3'5'H1 was demonstrated by ectopic expression in petunia (Petunia hybrida), which altered flower color and flavonoid composition as expected. VvF3'H was expressed in grapes before flowering, when 3'-hydroxylated flavonols are made, and all three genes were expressed after flowering, when proanthocyanidins (PAs) are synthesized. In berry skin, expression of all three genes was low at the onset of ripening (véraison) but increased after véraison concomitant with the accumulation of 3'- and 3',5'-hydroxylated anthocyanins. VvF3'H and VvCytoB5 were expressed in seeds but not VvF3'5'H1, consistent with the accumulation of 3'-hydroxylated PAs in this tissue. VvCytoB5 expression was correlated with expression of both VvF3'H and VvF3'5'H1 in the different grape tissues. In contrast to red grapes, where VvF3'H, VvF3'5'H1, and VvCytoB5 were highly expressed during ripening, the expression of VvF3'5'H1 and VvCytoB5 in white grapes during ripening was extremely low, suggesting a difference in transcriptional regulation. Our results show that temporal and tissue-specific expression of VvF3'H, VvF3'5'H1, and VvCytoB5 in grapes is coordinated with the accumulation of the respective hydroxylated flavonols and PAs, as well as anthocyanins. Understanding the regulation of flavonoid hydroxylases could be used to modify flavonoid composition of fruits.

Cytochrome P450 comprises a group of monooxygenases that hydroxylate xenobiotics and natural compounds with diverse electron transfer systems. Here we identify a natural fusion protein of cytochrome (Cyt) b 5 and Cyt b 5 reductase (CBBR) that transfers electrons from NADH to the cytochrome P450 CYP540A2. This cytochrome P450 system hydroxylates medium-chain fatty acids (MCFAs) to generate ( R )-β-hydroxy-MCFAs with 7–12 carbon atoms. Kinetic studies of CYP540A2 mutants indicate that side chains of Ser431 and Gln542 residues bind the carboxyl moiety of MCFAs for hydroxylation at their β-carbons. Pre-steady state kinetics also indicate that a predicted linker region between the FAD- and Cyt b 5 -domains of CBBR modulates electron transfer from NADH to CYP540A2. The present study also identifies a dehydrogenase that oxidizes ( R )-β-hydroxy-MCFAs to β-oxo-fatty acids that are substrates in the general β-oxidation mechanism of fatty acid degradation. The genes encoding CBBR, CYP540A2, and ( R )-β-hydroxy-MCFA dehydrogenase are clustered in the genome of the fungus Aspergillus nidulans and related fungi. The A. nidulans genes are induced by MCFAs, and disrupting CBBR and CYP540A2 genes accumulated more intracellular decanoic acid. Our findings reveal an adaptive monooxygenase-dependent β-oxidation mechanism that alternates with conventional β-oxidation, thus allowing fungi to metabolize MCFAs. A fungal fusion protein of cytochrome b5 and its reductase transfers electrons to cytochrome P450 CYP540A2, enabling the hydroxylation of medium-chain fatty acids. This pathway provides an alternative route for β-oxidation in their metabolism.

Liposomes are employed as drug delivery vehicles offering a beneficial pharmacokinetic/distribution mechanism for in vivo therapeutics. Therapeutic liposomes can be designed to target specific cell types through the display of epitope-specific targeting peptides on their surface. The majority of peptides are currently attached by chemical modification of lipid constituents. Here we investigate an alternative and novel method of decorating liposomes with targeting ligand, using remotely and spontaneously inserting chimeric tail-anchored membrane (TA) proteins to drug loaded liposomes. An artificial TA protein chimera containing the transmembrane domain from the spontaneously inserting TA protein cytochrome b5 (Cytb5) provided a robust membrane tether for the incorporation of three different targeting moieties into preformed liposomes. The moieties investigated were the transactivator of transcription (TAT) peptide, the EGF-receptor binding sequence GE11 and the placental and tumour homing ligand CCGKRK. In all cases, TA protein insertion neither significantly altered the size of the liposomes nor reduced drug loading. The efficacy of this novel targeted delivery system was investigated using two human cell lines, HeLa M and BeWo. Short term incubation with one ligand-modified TA chimera, incorporating the TAT peptide, significantly enhanced liposomal delivery of the encapsulated carboxyfluorescein reporter. The Cytb5 TA was successfully employed as a membrane anchor for the incorporation of the desired peptide ligands into a liposomal drug delivery system, with minimal loss of cargo during insertion. This approach therefore provides a viable alternative to chemical conjugation and its potential to accommodate a wider range of targeting ligands may provide an opportunity for enhancing drug delivery.

Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities.