Association of Cytochromes P450 1A2 and 2B4: are the Interactions between Different p450 Species Involved in the Control of the Monooxygenase Activity and Coupling?

Davydov, Dmitri R.; Петушкова, Н. А.; Bobrovnikova, E. V.; Knyushko, Tatyana V.; Dansette, Patrick M.

doi:10.1007/978-1-4615-0667-6_53

Cited by 27 publications

(42 citation statements)

References 14 publications

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“…Analysis of these effects led Backes and co-workers (13,30,35) to the conclusion that the effects of CYP2B4 and CYP2E1 on CYP1A2 are exerted through the formation of mixed oligomers, where the interactions of CPR with CYP1A2 are promoted, whereas the CPR binding to the other P450 is inhibited. This conclusion is supported by our FRET-based studies of the interactions of CPR with mixtures of CYP1A2 and CYP2B4 (6).…”

supporting

confidence: 74%

“…The most extensively studied P450-P450 pair is that of rabbit CYP1A2 and CYP2B4 enzymes (3, 6, 13, 30 -34). The co-presence of CYP2B4 in mixed systems with CYP1A2 boosts the activity of the CYP1A2 (6,30) while inhibiting CYP2B4 (3). Similar relationships were also demonstrated between CYP1A2 and the rabbit CYP2E1 (34).…”

supporting

confidence: 54%

“…Competition of different P450 species for a limited amount of CPR, which has been observed by numerous investigators (1)(2)(3)(4)(5)(6), constitutes an important element in the inter-P450 cross-talk. However, the integrative connections in microsomal monooxygenase go far beyond that and involve functional effects of physical interactions of multiple P450 molecules that result in oligomerization (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 93%

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Interactions among Cytochromes P450 in Microsomal Membranes

Davydov

Davydova

Sineva

et al. 2015

Journal of Biological Chemistry

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Background: There are multiple cytochrome P450 species co-localized in the endoplasmic reticulum. Results: Membrane-bound cytochromes P450 3A4, 3A5, and 2E1 associate into heteromeric complexes, where the properties of the individual enzymes are considerably modified. Conclusion:The properties of the P450 ensemble cannot be predicted by summation of the properties of the individual enzymes. Significance: We disclose a mechanism of regulatory cross-talk between multiple P450 species through hetero-oligomerization.

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supporting

confidence: 74%

supporting

confidence: 54%

mentioning

confidence: 93%

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Interactions among Cytochromes P450 in Microsomal Membranes

Davydov

Davydova

Sineva

et al. 2015

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

“…Our initial titrations and Job plot were limited subsets of data for the system, and thus limited the ability to assess the role of protein-protein interactions in regulating P450 activity. Similarly, reliance on the titration format and small data sets may explain why groups were not able to actually fit data from mixed P450 systems to possible complexation mechanisms (19,32,39,40).As discussed by Beechem (41), global analysis of multi-dimensional data, for example, where multiple component concentrations are simultaneously varied and the entire superset is analyzed as a whole, is always more informative about the underlying biochemical mechanism, in comparison with trying to analyze individual subsets of data separately. For this study, we generated a two-dimensional data set where both P450 and CPR were simultaneously varied over a wide range of concentrations.…”

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confidence: 99%

Global Analysis of Protein−Protein Interactions Reveals Multiple CYP2E1−Reductase Complexes

et al. 2007

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Although a single binary functional complex between cytochrome P450 and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain experimentally observed catalytic activity of recombinant P450 2E1 in dependence on the total concentration of added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450:CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450•CPR, the quaternary complex (P450) 2 • (CPR) 2 , and the homodimer (P450) 2 . The second plausible model additionally involves a weakly bound ternary complex (P450) 2 •CPR. Importantly, only the binary complex P450•CPR seems catalytically active in either of the two most probable mechanisms.Protein-ligand and protein-protein interactions are fundamental to biological processes such as signal transduction, chemical transformations, and electron transport. An understanding of the role of these processes in biological function requires the identification of the detailed interaction mechanisms. These details provide a framework in which to understand how the process of molecular recognition maintains proper homeostasis, or leads to deleterious conditions. In this study, we characterize the details of protein-protein interactions involving cytochrome P450 1 (P450) and the cytochrome P450 reductase (CPR). Traditional experimental protocols and data-analytic methods applied to this two-component system failed to provide a clear answer regarding the binding stoichiometry. We describe an alternative data-analytic approach applicable to any multi-component system. The method is based on a general numerical analysis of simultaneous biochemical equilibria, without any restriction on the number of component molecular species or the number of complexes they form (1). Our results Microsomal P450 enzymes are major catalysts in the oxidative transformation of a structurally diverse class of compounds including steroids, fatty acids, hormones, antibiotics, and a wide variety of artificially produced chemicals (xenobiotics), such as drugs, food additives, and environmental contaminants (2). P450s convert lipid-soluble molecules t...

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Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

2019

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Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane-bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2-CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2-CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid-solubilized CYP2J2-CPR system by increasing the K m and decreasing the V max , and effect that is size-dependent. Crowding also affects the CYP2J2-CPR-ND system by decreasing both the K m and V max for Dextran-based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2-CPR membrane protein system. Additional Supporting Information may be found in the online version of this article. Statement of importance:This work is of interest to researchers studying macromolecular crowding, protein-lipid interactions, and cytochrome P450s. This work provides key insights into the kinetic effects of lipid composition as well as macromolecular crowding, a constant force within any cell. We also determine the effects of a crowded environment on membrane protein stability. As CYPs are important drug metabolizers, such data are pertinent to current and future studies.

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Association of Cytochromes P450 1A2 and 2B4: are the Interactions between Different p450 Species Involved in the Control of the Monooxygenase Activity and Coupling?

Cited by 27 publications

References 14 publications

Interactions among Cytochromes P450 in Microsomal Membranes

Interactions among Cytochromes P450 in Microsomal Membranes

Global Analysis of Protein−Protein Interactions Reveals Multiple CYP2E1−Reductase Complexes

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

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