Yeast expressed cytochrome P450 2D6 (CYP2D6) exposed on the external face of plasma membrane is functionally competent

ABSTRACT CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma membrane in addition to the regular microsomal location. Present work demonstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner face of plasma membrane. Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH-and cumene hydroperoxide-dependent dextromethorphan demethylation and NADPH-cytochrome c activity confirming previous observations in human hepatocytes. CYP2D6 found on the outside of plasma membrane (by differential immuno-inhibition and acidic shift assays on transformed spheroplasts) was catalytically competent at the cell surface for NADPH-supported activities. Anti-yeast P450-reductase antibodies inhibited neither CYP2D6 nor P450-reductase activities upon incubation with intact spheroplasts. In contrast, both activities were inhibited on isolated plasma membrane fragments. This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase. This finding was confirmed by immunostaining in confocal microscopy. Finally, gene deletion of P450-reductase caused a complete loss of plasma membrane NADPH-supported CYP2D6 activity, which suggests that the reductase participates to some degree in the transmembrane electron transfer chain. This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism

Funding Information: F.E. and M.K.: UID/BIM/0009/2020 of the Portuguese Fundação para a Ciência e a Tecnologia (FCT) and HLTH-2022-STAYHLTH-02/grant agreement 101095679 of the European Horizon´s research and innovation program. Publisher Copyright: © 2023 by the authors.A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD–CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.publishersversionpublishe

The Sterolgene v0 cDNA microarray: a systemic approach to studies of cholesterol homeostasis and drug metabolism

<p>Abstract</p> <p>Background</p> <p>Cholesterol homeostasis and xenobiotic metabolism are complex biological processes, which are difficult to study with traditional methods. Deciphering complex regulation and response of these two processes to different factors is crucial also for understanding of disease development. Systems biology tools as are microarrays can importantly contribute to this knowledge and can also discover novel interactions between the two processes.</p> <p>Results</p> <p>We have developed a low density Sterolgene v0 cDNA microarray dedicated to studies of cholesterol homeostasis and drug metabolism in the mouse. To illustrate its performance, we have analyzed mouse liver samples from studies focused on regulation of cholesterol homeostasis and drug metabolism by diet, drugs and inflammation. We observed down-regulation of cholesterol biosynthesis during fasting and high-cholesterol diet and subsequent up-regulation by inflammation. Drug metabolism was down-regulated by fasting and inflammation, but up-regulated by phenobarbital treatment and high-cholesterol diet. Additionally, the performance of the Sterolgene v0 was compared to the two commercial high density microarray platforms: the Agilent cDNA (G4104A) and the Affymetrix MOE430A GeneChip. We hybridized identical RNA samples to the commercial microarrays and showed that the performance of Sterolgene is comparable to commercial arrays in terms of detection of changes in cholesterol homeostasis and drug metabolism.</p> <p>Conclusion</p> <p>Using the Sterolgene v0 microarray we were able to detect important changes in cholesterol homeostasis and drug metabolism caused by diet, drugs and inflammation. Together with its next generations the Sterolgene microarrays represent original and dedicated tools enabling focused and cost effective studies of cholesterol homeostasis and drug metabolism. These microarrays have the potential of being further developed into screening or diagnostic tools.</p

[C7GC4]4 Association into supra molecular i-motif structures

The self-associative properties of cytidine-rich oligonucleotides into symmetrical i-motif tetramers give to these oligonucleotides the capacity of forming supramolecular structures (sms) that have potential applications in the nanotechnology domain. In order to facilitate sms formation, oligonucleotides containing two cytidine stretches of unequal length (CnXCm) separated by a non-cytidine spacer were synthesized. They were designed to associate into a tetramer including an i-motif core built by intercalation of the C·C+ pairs of the longer C stretch with the two dangling non-intercalated strands of the shorter C stretch at each end. Gel filtration chromatography shows that the non-intercalated C-rich ends give to this structure the capacity of forming extremely stable sms. Using C7GC4 as a model, we find that the sms formation rate varies as the oligonucleotide concentration and increases at high temperature. Competitively with the tetramer involved in sms elongation, CnXCm oligonucleotides form i-motif dimers that compete with sms elongation. The dimer stability is strongly reduced when the pH is moved away from the cytidine pK. This results in an equilibrium shift towards the tetramer and in the acceleration of the sms formation rate. The chromatograms of the sms formed by C7GC4 indicate a broad distribution. In a 1.5 mM solution incubated at 37°C, the equilibrium distribution is centered on a molecular weight corresponding to the assembly of nine tetramers and the upper limit corresponds to 80 tetramers. The lifetime of this structure is about 4 days at 40°C, pH 4.6

Confrontation of AlphaFold2 models with cryo-EM and crystal structures enlightens alternate geometries of the CYP102A1 multidomain protein

Abstract Large range structural dynamics plays a critical role for the function of electron transfer proteins. This information is generally not available from crystallographic structures, while cryo-electron microscopy (cryo-EM) can provide some elements but frequently with a degraded spatial resolution. Recently, AlphaFold-based structural modelling was extended to the prediction of protein complexes. In this work, bacterial CYP102A1 from Priestia megaterium was used as a test case to evaluate the capability of AlphaFold2 to predict alternative structures critical for catalysis. CYP102A1 monooxygenase, a NADPH-supported fatty acid hydroxylase, works as a soluble homodimer, each monomer harboring two flavins (FAD and FMN) and one heme cofactors. Large conformational changes are required during catalytic cycle to allow successive electron transfers from FAD to FMN and finally heme iron. We used the recently released AlphaFold2_advanced notebook (AF2A), to predict the possible alternate conformations supporting electron transfers in CYP102A1 homodimer. Challenging AF2A-derived models with previously reported experimental data revealed an unforeseen domain connectivity of the diflavin reductase part of the enzyme. Intermolecular crossed complex constitutes a novel type of structural organization never previously described. The predicted formation within the dimer of a stable complex between the heme containing domains was challenged and found consistent with uninterpreted features of reported crystallographic structures and cryo-EM imaging. The particularly efficient CYP102A1 catalytic mechanism was revisited to the light of the new evidenced connectivity in which the FMN-binding domain of each monomer oscillates on themselves to alternatively receive and transfer electrons without needing large structural change in the dimer. Such model was found explanatory for previously contradictory reported biochemical data. Possibility to mimic CYP102A1 structural organization into bicomponent eukaryotic P450 systems was evaluated by designing and modeling in silico synthetic reductase domains built from composite sequence segments from P. megaterium and human origins. More generally, this work illustrates how the ability of AF2A to predict alternate complex structures can enlighten and explain conformational changes critical for bio-assemblies

Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions

International audienceConformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently successfully extended to the prediction of protein complexes but its capability to address large conformational changes involved in catalysis remained obscure. We used bacterial CYP102A1 monooxygenase homodimer as a test case to design a competitive modelling approach (CMA) for assessing alternate conformations of multi-domain complexes. Predictions were confronted with published crystallographic and cryo-EM data, evidencing consistencies but also permitting some reinterpretation of experimental data. Structural determinants stabilising the new type of domain connectivity evidenced in this bacterial self-sufficient monooxygenase were analysed by CMA and used for in silico retro-engineering applied to its eukaryotic bi-component counterparts

GENES CODANTS POUR LA CYTOCHROME P450 REDUCTASE DU CHAMPIGNON RHIZOPUS NIGRICANS. CLONAGE PAR L'EXPRESSION DANS LA LEVURE ET CARACTERISATION

CE TRAVAIL PRESENTE L'ETUDE DU ROLE DES PROTEINES REDOX ASSOCIEES AU CYTOCHROME P450, LA NADPH-CYTOCHROME P450 REDUCTASE ET LE CYTOCHROME B5, AINSI QUE LEUR IMPLICATION DANS LA REPONSE INDUITE PAR L'EXPOSITION DU CHAMPIGNON FILAMENTEUX RHIZOPUS NIGRICANS A LA PROGESTERONE. LES ARNMS SPECIFIQUEMENT PRODUITS EN REPONSE A LA PROGESTERONE ONT ETE AMPLIFIES PAR RT-PCR EN UTILISANT DES AMORCES DE SEQUENCE DEGENEREE. LES PRIMERS ONT ETE DEFINIS EN COMPARANT LES REGIONS TRES CONSERVEES DE LIAISON AU FMN ET FAD CHEZ LES REDUCTASES CONNUES. LES ADNC CORRESPONDANT AUX REGIONS CENTRALES DE DEUX NOUVELLES REDUCTASES NOMMEES CPR1-CS ET CPR2-CS ONT ETE CLONES. D'AUTRE PART, PAR CLONAGE FONCTIONNEL DANS UNE SOUCHE DE LEVURE S. CEREVISIAE DONT LE GENE CPR1 EST SOUS LE PROMOTEUR CONDITIONNEL ET LE GENE CYB5 EST INACTIVE, NOUS AVONS IDENTIFIE DEUX ADNC, DONT L'EXPRESSION PERMET D'ABOLIR L'HYPERSENSIBILITE AU KETOCONAZOLE D'UNE TELLE LEVURE : UN ADNC LONG CODANT POUR UNE REDUCTASE (CPR1-FL) QUI INCLUT LA SEQUENCE CPR1-CS ET UN ADNC CODANT POUR UN CYTOCHROME B5 (CYB5-1). L'ETUDE DE L'EXPRESSION PAR NOTHERN BLOT MONTRE QUE DEUX TRANSCRITS DE 2,1 KBP (CPR1-FL ET CPR2-CS), CORRESPONDENT AUX REDUCTASES QUE NOUS AVONS CLONEES. SEULE CPR1-FL EST FORTEMENT INDUIT (8-FOIS) LORS DE LA REPONSE A LA PROGESTERONE. PAR CONTRE L'EXPRESSION DE CPR2-CS ET CYB5-1 N'EST PAS MODIFIEE. LA LOCALISATION CELLULAIRE ET DE LA FONCTION DU PRODUIT DE CPR1-FL ONT ETE ETUDIEES DANS UNE SOUCHE DE LEVURE DONT LE GENE DE LA CPR1 ENDOGENE A ETE PREALABLEMENT INTERROMPU. CETTE ANALYSE CONDUITE EN UTILISANT L'ADNC COMPLET CPR1-FL AINSI QU'UNE VERSION TRONQUEE COMMENCANT AU PREMIER ATG TROUVE EN 5 - (CPR-S) REVELE QUE CES DEUX ADNC CODENT POUR DES PRODUITS FONCTIONNELLEMENT EQUIVALENTS LOCALISES DANS LE CYTOSOL ET LES MICROSOMES. L'ADDITION D'UN CODON ATG IMMEDIATEMENT EN AMONT DU PREMIER ATG DE L'ADNC DE CPR1-FL (CPR1-L) ACCROIT LA FRACTION DE L'ACTIVITE PRESENTE DANS LES MICROSOMES : LA RESISTANCE AU KETOCONAZOLE EST ACCRUE, ALORS QUE L'ACTIVITE NADPH-CYTOCHROME C REDUCTASE GLOBALE SE TROUVE DIMINUEE. L'ADNC DU CYB5-1 A ETE REFORMATE ET SON PRODUIT SUREXPRIME ET SURPRODUIT DANS E.COLI. PUIS SOLUBILISE PAR DES DETERGENTS ET PURIFIE. IL MONTRE UN SPECTRE D'ABSORPTION ET DES PROPRIETES REDOX TYPIQUES DU CYTOCHROME B5. DE PLUS IL MODULE FORTEMENT LES ACTIVITES TESTOSTERONE HYDROXYLASE AND ETHOXYCOUMARIN DEETHYLASE DES P450 3A4 ET 2B6 HUMAINS DE MANIERE DEPENDANTE DE LA NATURE DE LA REDUCTASE. L'ENSEMBLE DES DONNES MONTRE QUE TROIS COMPOSANTS DU SYSTEME MONOOXYGENASE DE RHIZOPUS NIGRICANS ONT ETE CLONES ET CARACTERISES. LEUR IMPLICATION DIFFERENTE EN REPONSE A LA PROGESTERONE A ETE DEMONTRE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Producing Chimeric Genes by CLERY In Vitro and In Vivo Recombination

International audienc