Mitochondrial P450 Research Articles

Comprehensive analysis of the overexpressed cytochrome P450-based insecticide resistance mechanism in Spodoptera litura

Cytochrome P450s play critical roles in the metabolic resistance of insecticides in insects. Previous findings showed that enhanced P450 activity was an important mechanism mediating indoxacarb resistance, and multiple P450 genes were upregulated in indoxacarb resistant strains of Spodoptera litura. However, the functions of these P450 genes in insecticide resistance remain unknown. Here, the P450 inhibitor PBO effectively decreased the resistance of S. litura to indoxacarb. Ten upregulated P450 genes were characterized, all of which were overexpressed in response to indoxacarb induction. Knockdown of nine P450 genes decreased cell viability against indoxacarb, and further silencing of three genes (CYP339A1, CYP340G2, CYP321A19) in larvae enhanced the sensitivity to indoxacarb. Transgenic overexpression of these three genes increased resistance to indoxacarb in Drosophila melanogaster. Moreover, molecular modeling and docking predicted that these three P450 proteins could bind tightly to indoxacarb and N-decarbomethoxylated metabolite (DCJW). Interestingly, these three P450 genes may also mediate cross-resistance to chlorantraniliprole, λ-cyhalothrin and imidacloprid. Additionally, heterologous expression and metabolic assays confirmed that three recombinant P450s could effectively metabolize indoxacarb and DCJW. This study strongly demonstrates that multiple overexpressed mitochondrial and microsomal P450 genes were involved in insecticide resistance in S. litura.

Selectivity of osilodrostat as an inhibitor of human steroidogenic cytochromes P450

Osilodrostat (LCI699) is a potent inhibitor of the human steroidogenic cytochromes P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 is FDA-approved for the treatment of Cushing disease, which is characterized by chronic overproduction of cortisol. While phase II and III clinical studies have proven the clinical efficacy and tolerability of LCI699 for treating Cushing disease, few studies have attempted to fully assess the effects of LCI699 on adrenal steroidogenesis. To this end, we first comprehensively analyzed LCI699-mediated inhibition of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. We then studied LCI699 inhibition using HEK-293 or V79 cells stably expressing individual human steroidogenic P450 enzymes. Our studies using intact cells confirm the potent inhibition of CYP11B1 and CYP11B2 with negligible inhibition of 17-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, partial inhibition of the cholesterol side-chain cleavage enzyme (CYP11A1) was observed. To calculate the dissociation constant (Kd) of LCI699 with the adrenal mitochondrial P450 enzymes, we successfully incorporated P450s into lipid nanodiscs and carried out spectrophotometric equilibrium and competition binding assays. Our binding experiments confirm the high affinity of LCI699 to CYP11B1 and CYP11B2 (Kd ≈ 1 nM or less) and much weaker binding for CYP11A1 (Kd = 18.8 μM). Our results confirm the selectivity of LCI699 for CYP11B1 and CYP11B2 and demonstrate partial inhibition of CYP11A1 but not CYP17A1 and CYP21A2.

ALAS1 associated with goat kidding number trait was regulated by the transcription factor ASCL2 to affect granulosa cell proliferation.

ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.

LBODP008 Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Fibroblasts, IPS Cells And Model Mice

Abstract Background Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s. Microsomal P450s include 21-hydroxylase and 17α-hydroxylase/17-lyase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, is the most common cause of CAH and results in underproduction of glucocorticoids and mineralocorticoids, and overproduction of androgens. 11β-hydroxylase deficiency (11β-OHD), which is a defect of a mitochondrial P450, is the second common cause of CAH. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Intravenous adeno-associated virus (AAV)-mediated administration of human CYP21A2 into adult 21-OHD patients has been reported at the ENDO 2021 meeting. In this study, we examined the effects of induction of causative genes with an AAV vector into fibroblasts from patients and iPS cells and adrenal glands of model mice. Methods Fibroblasts from CAH patients were subjected to primary culture. The cells were infected with a serotype-2 AAV vector (AAV2) containing defected genes and cultured in steroid substrate-containing medium for 24 hours. We measured steroid metabolites in the medium by liquid chromatography tandem mass spectrometry and evaluated gene expressions by RT-PCR. In addition, iPS cells were established from fibroblasts of 11β-OHD and differentiated into adrenocortical cells with retinoic acid and cAMP. They were infected with a serotype-9 AAV vector (AAV9) containing CYP11B1 cDNA. Steroid metabolites in the culture medium were measured and gene expressions were evaluated by RT-PCR. 11β-OHD model mice were made by a gene-editing method. We injected AAV9 containing Cyp11b1 cDNA into the adrenal gland and measured serum DOC and corticosterone levels before and every 4 weeks after injection. Results AAV2 infected fibroblasts from 21-OHD and 17α-hydroxylase/17-lyase deficiency can metabolite steroid precursors but failed to convert DOC to corticosterone in 11β-OHD. Differentiation into adrenocortical cells from iPS cells of 11β-OHD was confirmed by expression of CYP21A2. AAV9 gene induction of adrenal cortical cells successfully induced 11β-hydroxylase activity. 11β-OHD mice also showed improved steroid synthesis after AAV9 adrenal induction. Conclusion These results indicate that extra-adrenal induction of CYP21A2 may ameliorate steroid metabolism in 21-OHD patients. In contrast, 11β-OHD needs AAV9 adrenal induction to treat the steroid abnormality. Our results suggest that specific gene therapeutic strategies are needed to be adapted for each type of CAH. Presentation: No date and time listed

Adeno-Associated Virus-Mediated Gene Therapy for Patients' Fibroblasts, Induced Pluripotent Stem Cells, and a Mouse Model of Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration of Cyp21a1 ameliorates steroid metabolism in 21-hydroxylase-deficient mice. However, the effectiveness of this approach for mitochondrial P450 has not yet been evaluated. In this study, primary fibroblasts from patients with 21-hydroxylase deficiency (CYP21A2D) (n = 4), 17α-hydroxylase/17,20 lyase deficiency (CYP17A1D) (n = 1), and 11β-hydroxylase deficiency (CYP11B1D) (n = 1) were infected with adeno-associated virus type 2 (AAV2) vectors. Steroidogenic enzymatic activity was not detected in the AAV2-infected CYP11B1D fibroblasts. Induced pluripotent stem cells (iPSCs) of CYP11B1D were established and differentiated into adrenocortical cells by induction of the NR5A1 gene. Adrenocortical cells established from iPSCs of CYP11B1D (CYP11B1D-iPSCs) were infected with an AAV type 9 (AAV9) vector containing CYP11B1 and exhibited 11β-hydroxylase activity. For an in vivo evaluation, we knocked out Cyp11b1 in mice by using the CRISPR/Cas9 method. Direct injection of Cyp11b1-containing AAV9 vectors into the adrenal gland of Cyp11b1-deficient mice significantly reduced serum 11-deoxycorticosterone/corticosterone ratios at 4 weeks after injection and the effect was prolonged for up to 12 months. This study indicated that CYP11B1D could be ameliorated by gene induction in the adrenal glands, which suggests that a defective-enzyme-dependent therapeutic strategy for CAH would be required. Defects in microsomal P450, including CYP21A2D and CYP17A1D, can be treated with extra-adrenal gene induction. However, defects in mitochondrial P450, as represented by CYP11B1D, may require adrenal gene induction.

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.

Involvement of CYP2 and mitochondrial clan P450s of Helicoverpa armigera in xenobiotic metabolism

Insect CYP2 and mitochondrial clan P450s are relatively conserved genes encoding enzymes generally thought to be involved in biosynthesis or metabolism of endobiotics. However, emerging evidence argues they have potential roles in chemical defense as well, but their actual detoxification functions remain largely unknown. Here, we focused on the full complement of 8 CYP2 and 10 mitochondrial P450s in the generalist herbivore, Helicoverpa armigera. Their varied spatiotemporal expression profiles were analyzed and reflected their specific functions. For functional study of the mitochondrial clan P450s, the redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), were identified from genomes of eight insects and an efficient in vitro electron transfer system of mitochondrial P450 was established by co-expression with Adx and AdR of H. armigera. All CYP2 clan P450s and 8 mitochondrial P450s were successfully expressed in Sf9 cells and compared functionally. In vitro metabolism assays showed that two CYP2 clan P450s (CYP305B1 and CYP18A1) and CYP333B3 (mito clan) could epoxidize aldrin to dieldrin, while CYP305B1 and CYP339A1 (mito clan) have limited but significant hydroxylation capacities to esfenvalerate. CYP303A1 of the CYP2 clan exhibits high metabolic efficiency to 2-tridecanone. Screening the xenobiotic metabolism competence of CYP2 and mitochondrial clan P450s not only provides new insights on insect chemical defense but also can give indications on their physiological functions in H. armigera and other insects.

Binding of cytochrome P450 27C1, a retinoid desaturase, to its accessory protein adrenodoxin

Of the 57 human cytochrome P450 (P450) enzymes, seven are mitochondrial: 11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1. Mitochondrial P450s utilize an electron transport system with adrenodoxin (Adx) and NADPH-adrenodoxin reductase (AdR). AdR reduces Adx, which then transfers electrons to the P450. The interactions between proteins in the mitochondrial P450 system are largely driven by electrostatic interactions, though the specifics vary depending on the P450. Unlike other mitochondrial P450s, the interaction between P450 27C1, a retinoid 3,4-desaturase expressed in the skin, and Adx remains largely uncharacterized. In this work, we utilized an Alexa Fluor 488 C5 maleimide-labeled Adx to measure binding affinities between Adx and P450 27C1 or AdR. Both proteins bound Adx tightly, with Kd values < 100 nM, and binding affinities decreased with increasing ionic strength, supporting the role of electrostatic interactions in mediating these interactions. Cross-linking mass spectrometry and computational modeling were performed to identify interactions between P450 27C1 and Adx. While the residues of Adx identified in interactions were consistent with studies of other mitochondrial P450s, the binding interface of P450 27C1 was quite large and supported multiple Adx binding positions, including ones outside of the canonical Adx binding site. Additionally, Adx did not appear to be an allosteric effector of P450 27C1 substrate binding, in contrast to some other mitochondrial P450s. Overall, we conclude that P450-Adx interactions are P450-specific.

Resurrection and characterization of ancestral CYP11A1 enzymes.

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6–200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

SUN-219 Electron Transport Chain Complex 2 in Mitochondrial Pregnenolone Synthesis

The mitochondrial P450 family of enzymes (SCC), which require the electron transport chain (ETC) complexes III, IV and V, initiate steroidogenesis by cleaving the sidechain of cholesterol to synthesize steroid hormones, an essential component for mammalian survival. SCC is required for full-term gestation, and aberrant expression may cause pseudohermaphroditism, breast cancer or polycystic ovary syndrome. Complex II or succinate dehydrogenase (quinone) is shared with the TCA cycle and has no proton pumping capacity and no known role in steroid synthesis. We now show that succinate is an intermediate metabolite in the TCA cycle and plays a central role physiologically. Specifically, complex II is required for SCC activation, where the proton pump facilitates an active intermediate state conformation at the matrix, so that in the presence of succinate, ATP can add phosphate. A longer intermediate equilibrium state generates a transient stabilization to enhance the binding of phosphate anions in the presence of succinate anions, resulting in higher enthalpy and activity. An inhibition of the processing at the intermediate state stops phosphate addition and activity. We further describe that phosphate circulation brings the molten globule, an intermediate, to an active folded state. This is the first report showing that an intermediate state activated by succinate facilitates ETC complex II interaction with complexes III and IV for metabolism.

Adrenodoxin Modulates Substrate Binding in the Vitamin‐D Metabolizing Human Cytochrome P450 24A1

Vitamin‐D insufficiency is associated with rickets, osteomalacia, cardiovascular disease, and chronic kidney disease and affects nearly 50% of the population. In humans, vitamin D is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans hydroxylates either the carbon‐23 or carbon‐24 position of 1,25‐(OH)2D3 followed by a series of reactions to produce additional metabolites. The human isoform of CYP24A1 is not easily expressed and purified recombinantly, likely due to the labile nature of the enzyme. Therefore, most current structural studies are based on the rat isoform, which does not display carbon‐23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a fusion construct of human CYP24A1 with its reduction‐oxidation protein partner adrenodoxin (Adx). The fusion product contains a short linker sequence connected to a cleavable tobacco etch virus protease recognition site, thus allowing for production and then separation of human CYP24A1 from Adx. This construct provides a platform for interrogating the structural effects of an Adx‐CYP24A1 complex on substrate, analog, and inhibitor binding in CYP24A1. Notably, we’ve observed that when Adx is present, titration with 1,25(OH)2D3 primarily populates a low‐spin state. This effect is removed upon cleavage and separation of Adx. Our interpretation is that the presence of adrenodoxin stabilizes a closed form of CYP24A1 that prevents water from departing the active site as the sixth ligand. This finding points toward close structural coupling between substrate binding and redox partner recognition in mitochondrial P450 enzymes.Support or Funding InformationR00GM112862

A New Risk Variant for Multiple Sclerosis at 11q23.3 Locus Is Associated with Expansion of CXCR5+ Circulating Regulatory T Cells.

Genome-wide association studies and meta-analysis have contributed to the identification of more than 200 loci associated with multiple sclerosis (MS). However, a proportion of MS heritability remains unknown. We aimed to uncover new genetic variants associated with MS and determine their functional effects. For this, we resequenced the exons and regulatory sequences of 14 MS risk genes in a cohort of MS patients and healthy individuals (n = 1070) and attempted to validate a selection of signals through genotyping in an independent cohort (n = 5138). We identified three new MS-associated variants at C-X-C motif chemokine receptor 5 (CXCR5), Ts translation elongation factor, mitochondrial (TSFM) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Rs10892307 resulted in a new signal at the CXCR5 region that explains one of the associations with MS within the locus. This polymorphism and three others in high linkage disequilibrium mapped within regulatory regions. Of them, rs11602393 showed allele-dependent enhancer activity in the forward orientation as determined by luciferase reporter assays. Immunophenotyping using peripheral blood mononuclear cells from MS patients associated the minor allele of rs10892307 with increased percentage of regulatory T cells expressing CXCR5. This work reports a new signal for the CXCR5 MS risk locus and points to rs11602393 as the causal variant. The expansion of CXCR5+ circulating regulatory T cells induced by this variant could cause its MS association.

The Cytochrome P450 24A1 Interaction with Adrenodoxin Modulates Substrate Binding and Relies on Species‐variable Recognition Sites

Redox partner and accessory protein interactions are known to modulate function in mammalian cytochrome P450 enzymes by exerting a level of allosteric control, most notably demonstrated by the interaction of cytochrome b5 with P450 enzymes in the endoplasmic reticulum. However, in the inner mitochondrial membrane, in which P450 electron transfer occurs solely via interaction with the ferredoxin protein adrenodoxin (Adx), the events that modulate P450 function are not clear. In this study, we report the use of combined spectroscopic techniques to demonstrate that Adx serves to modulate substrate binding in the mitochondrial vitamin D metabolizing enzyme CYP24A1. The presence of equal molar amounts of Adx potentiates substrate binding for 25(OH)D3 and 1,24,25(OH)3D3, but not for the primary substrate 1,25(OH)2D3. We follow up on the CYP24A1‐Adx interaction by utilizing solution nuclear magnetic resonance (NMR) spectroscopy to monitor the spectra of uniformly labeled 15N Adx while titrating with unlabeled CYP24A1 bound to the P450 inhibitor clotrimazole. The pattern of NMR line broadening, combined with single and double mutants of Adx, suggest the presence of multiple recognition sites between the proteins. In order to evaluate redox partner complexes for CYP24A1 isoforms displaying distinct regioselectivity for vitamin D, all of the binding data were acquired in parallel for a carbon 23 hydroxylase, a carbon 24 hydroxylase, and a combined carbon 23/24 hydroxylase. Interestingly, disruption of the CYP24A1‐Adx complex in response to Adx mutagenesis was variable between CYP24A1 isoforms, suggesting a correlation between redox partner binding and regioselectivity and consistent with the ability of Adx to modulate substrate binding. Therefore, this work provides structural evidence in support of Adx modulation of function in a mitochondrial P450 enzyme.Support or Funding InformationThis work was supported by R00GM112862 and the State University of New YorkThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Catalytic modulation of human cytochromes P450 17A1 and P450 11B2 by phospholipid

Unlike most of the drug-metabolizing cytochrome P450s, microsomal P450 17A1 and mitochondrial P450 11B2 catalyze sequential multi-step reactions in steroid biosynthesis. The membrane phospholipid composition might be one parameter that modulates the efficiency and processivity of specific pathways. Here we systematically examined the effects of physiologically relevant phospholipids on the catalysis of purified P450 17A1, P450 11B2, and P450 11B1 in reconstituted assay systems. Both dioleoylphosphatidylcholine (DOPC, 18:1) and dilauroylphosphatidylcholine (DLPC, 12:0) were found to be very efficient in reconstituting 17-hydroxylase and 1720-lyase reactions of P450 17A1. Phosphatidylethanolamine (PE) specifically enhanced 1720-lyase activity up to 2.4-fold in the presence of phosphatidylcholine. On the other hand, P450 11B2-catalyzed production of aldosterone from 11-deoxycorticosterone was very low and from 18-hydroxycorticosterone nil, implying low processivity. DOPC or cardiolipin, which is exclusively located in the inner mitochondrial membrane, maximized aldosterone yield. In sharp contrast, reconstitution of homologous P450 11B1 with DOPC significantly decreased corticosterone formation without affecting the synthesis of 18-hydroxycorticosterone. The intrinsic fluorescence of P450 17A1 and 11B2 increased in the presence of DOPC, DLPC and PE. Acrylamide quenching studies showed that PE decreased solvent accessibility for tryptophan in P450 17A1, as did 20:4 PC or 18:2 PC for P450 11B2. A moderately positive correlation between the proportion of high-spin substrate-bound species and catalytic activity was only observed in the presence of phosphatidylcholines with low-temperature phase transition. These results demonstrate the potential for phospholipids to regulate the activity of steroidogenic P450 activities and thereby steroid hormone biosynthetic pathways.

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH)2D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH)2D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.

Evidence for activation of nitenpyram by a mitochondrial cytochrome P450 in Drosophila melanogaster.

Nitenpyram is a member of the economically important neonicotinoid class of insecticides. The in vivo metabolism of nitenpyram is not well characterised, but cytochrome P450 activity is the major mechanism of resistance to neonicotinoids identified in insect pests, and P450s metabolise other neonicotinoids including imidacloprid. Here, we used the GAL4-UAS targeted expression system to direct RNA interference (RNAi) against the cytochrome P450 redox partners to interrupt P450 functions in specific tissues in Drosophila melanogaster. RNAi of the mitochondrial redox partner defective in the avoidance of repellents (dare) in the digestive tissues reduced nitenpyram mortality, suggesting an activation step in the metabolism of nitenpyram carried out by a mitochondrial P450. RNAi of the mitochondrial cytochrome P450 Cyp12a5, which is expressed in the digestive tissues, resulted in the same phenotype, and transgenic overexpression of Cyp12a5 increased nitenpyram sensitivity. These results suggest that in vivo metabolism of nitenpyram by the mitochondrial P450 CYP12A5 results in the formation of a product with higher toxicity than the parent compound. © 2018 Society of Chemical Industry.

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development

BackgroundThe initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.ResultsFor a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3–5 times higher than in the control plants of the wild type.ConclusionsFor the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.

Evolutionary origin of mitochondrial cytochrome P450.

Different molecular species of cytochrome P450 (P450) are distributed between endoplasmic reticulum (microsomes) and mitochondria in animal cells. Plants and fungi have many microsomal P450s, but no mitochondrial P450 has so far been reported. To elucidate the evolutionary origin of mitochondrial P450s in animal cells, available evidence is examined, and the virtual absence of mitochondrial P450 in plants and fungi is confirmed. It is also suggested that a microsomal P450 is the ancestor of animal mitochondrial P450s. It is likely that the endoplasmic reticulum-targeting sequence at the amino-terminus of a microsomal P450 was converted to a mitochondria-targeting sequence possibly by point mutations of a few amino acid residues or by an exon-shuffling/moving event shortly after animal lineage diverged from plants and fungi in the course of evolution of eukaryotes. It is suggested that the microsome-type P450 first imported into mitochondria utilized the existing ferredoxin in the matrix to receive electrons from NADPH, retained its oxygenase activity in the mitochondria, and gradually diversified to several P450s with different substrate specificities in the course of the evolution of animals.

P.1.h.028 - Investigation of the effect of post-learning sleep deprivation on hippocampal synaptic plasticity-related genes and micro-RNA in mice

This article discusses the insect CYP genes and P450 enzymes. Cytochrome P450, or CYP genes, constitutes one of the largest family of genes, with representatives in virtually all living organisms, from bacteria to protists, plants, fungi, and animals. The multiple functions of insect P450 enzymes, their complex biochemistry, and their toxicological and physiological importance are presented. Physiological functions are not restricted to one branch of the P450 evolutionary tree, and the ramifications that seem typical of “environmental response genes” are found in both microsomal and mitochondrial P450 encoding genes. The CYP3 and CYP4 clans seem the most diverse, and the smaller CYP2 and mitochondrial CYP clans seem to have proportionally more genes found as orthologs across species. There are many physiological functions for P450s that just are not known or suspected. CYP4C7 was shown to be selectively expressed in the corpora allata and to metabolize JH and its precursors to new metabolites.