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

Développement d'approches prédictives pour l'ingénierie des protéines par évolution dirigée et application au développement d'une thérapie anticancéreuse

Le souhait de réduire des effets secondaires associés aux anticancéreux a mené à considérer l utilisation de prodrogues activables au site d action, comme la cyclophosphamide (CPA). La CPA est activée majoritairement par le CYP2B6 humain avec une faible efficacité, obligeant à l utilisation de concentrations importantes de prodrogue. Celles-ci peuvent être réduites par transfection au niveau de la tumeur d un gène codant pour un P450 optimisé, possédant une efficacité catalytique élevée vis-à-vis de la CPA tout en étant le moins immunogène possible. Pour ce faire, en partant de la modélisation du CYP2B6 et du CYP2B11, forme canine à bas Km pour l'activation de la CPA, un gène synthétique du CYP2B11 pour l'expression dans la levure a été dessiné et divisé en 15 "modules" structuraux. Quinze chimères à points de jonction définis entre les deux CYP2Bs ont ensuite été construites par échange de ces modules, via une méthode originale de génération de banques ordonnées de chimères, SIGNAL, indépendante de l'homologie de séquence des enzymes parentaux. SIGNAL, à mi-chemin entre les processus classiques d'évolution dirigée et de mutagenèse dirigée, nous a permis, après analyse fonctionnelle des chimères, de mieux comprendre le mécanisme de métabolisation de la CPA par les deux enzymes et d'identifier des modules structuraux jouant potentiellement un rôle important dans la haute affinité du CYP2B11. En parallèle, la mise au point d'un système de sélection à haut débit des variants les plus efficaces pour l'activation de la CPA dans la levure, basé sur le principe du gène rapporteur, a été débutée, afin de pouvoir raffiner l'optimisation du P450 par un processus de mutagenèse aléatoire.One of the possibilities considered to reduce the side effects associated to chemotherapeutic treatments was to use cytotoxic prodrugs, like cyclophosphamide (CPA), activated directly into the tumors. CPA is mainly activated with a low efficiency by human CYP2B6, so large doses of prodrug are used to induce a response. The transfection of an optimized CYP gene, possessing a high catalytic efficiency towards CPA while presenting a low immunogenicity, is a route to lower active doses. For this purpose, the canine CYP2B11, which exhibits a low Km for CPA activation, was modelled and divided into 15 structural "modules". A synthetic CYP2B11 gene was designed to permit a good expression in yeast and to swap defined modules between the human and canine forms. An original sequence independent method, SIGNAL, was developed, which allowed the construction of 15 chimeras with defined junction points. This ordered library, halfway between random directed evolution and defined directed mutagenesis, permitted the generation of targeted diversity, which do not need a high-throughput approach for functional analysis. Resulting information highlighted critical segments for CPA metabolism, which are potentially involved in the high CYP2B11 affinity. The P450 functional optimization could be further enhanced by a random mutagenesis process, which requires, in contrast, a high throughput screening system. Its development in S.cerevisiae was initiated using a reporter gene method.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Ordered chimerogenesis applied to CYP2B P450 enzymes

Background: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. Methods: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifiuoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. Results: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. Conclusions: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. General significance: The result provides a new basis for determining the structural elements that control functions in complex enzymes

Inhibition of Rat Liver Estrogen 2/4-Hydroxylase Activity by Troleandomycin: Comparison with Erythromycin and Roxithromycin1

ABSTRACT Administratio

Développement de stratégies de protection d’objets métallique peints : cas des collections scientifiques et techniques

International audienc