Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies.

Alston, Kenneth; Robinson, Richard; Park, S S; Gelboin, Harry V.; Friedman, Fred K.

doi:10.1016/s0021-9258(17)35233-x

Cited by 61 publications

(14 citation statements)

References 29 publications

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“…CYP1A proteins have previously been shown to interact with CYP3A subfamily P450s (13,21,24,40). Alston et al (40) provided evidence for a physical interaction between CYP3A2 and CYP1A1 by treating liver microsomes with a bifunctional cross-linking agent to covalently link closely associated proteins. Microsomal CYP1A1 was immunopurified using a monoclonal antibody to CYP3A2.…”

Section: Discussionmentioning

confidence: 99%

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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Mixed reconstituted systems containing CYP2B4, CYP1A2 and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin-Odealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin-Odeethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.The cytochrome P450 (P450) superfamily of proteins is functionally diverse, and well distributed in nature. In addition to their roles in the metabolism of numerous endogenous compounds, P450s are important contributors to drug metabolism, primarily by catalyzing the insertion of an oxygen atom into a substrate molecule. NADPH cytochrome P450 reductase (reductase) is the major electron transfer partner, and is required for the majority of NADPHdependent oxidation reactions. Multiple forms of cytochrome P450 exist in the endoplasmic reticulum in a large excess over reductase (1), with the ratio of P450 to reductase dependent † These studies were supported by a US Public Health Service Research Grant from the National Institute of Enviromental Health Sciences ES004344 (WLB), and a predoctoral fellowship from the Stanley Scott Cancer Center (RWK). * Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, emailwbacke@lsuhsc.edu SUPPORTING INFORMATION AVAILABLE The supporting information includes ...

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Section: Discussionmentioning

confidence: 99%

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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“…Surprisingly, CYPs also exhibit protein–protein interactions with other CYPs. In the early 1990s, research by Coon, Davydov, and others demonstrated that CYPs can form homomeric complexes. − Work in the ensuing 2 decades has established that multiple CYP isoforms can interact with one another in both homomeric and heteromeric complexes and that these complexes can often have significant effects on CYP-mediated oxidation of substrates ,− (for recent reviews, please see references and ). For simplicity, we have grouped the CYP–CYP interactions into those described by homomeric (two or more molecules of the same isoform) and heteromeric (two or more molecules of different isoforms).…”

Section: Cytochrome P450 Interactions With Other Cyp Isoformsmentioning

confidence: 99%

Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

Kandel

Lampe

2014

Chem. Res. Toxicol.

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Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes.

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“…Physical and functional P450−P450 interactions have previously been described by a number of laboratories ( − ). The potential for P450 enzymes to interact raises the possibility that both homomeric and heteromeric P450−P450 interactions can influence the function of these enzymes.…”

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Effects of Ionic Strength on the Functional Interactions between CYP2B4 and CYP1A2

2005

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The presence of one P450 can influence the catalytic characteristics of a second enzyme through the formation of heteromeric P450 complexes. Such a complex has been reported for mixed reconstituted systems containing NADPH-cytochrome P450 reductase, CYP2B4 and CYP1A2, where a dramatic inhibition of 7-pentoxyresorufin-O-dealkylation (PROD) was observed when compared to simple reconstituted systems containing reductase and a single P450 enzyme. The goal of the present study was to characterize this interaction by examining the potential of the CYP1A2-CYP2B4 complex to be formed by charge-pair interactions. With ionic interactions being sensitive to the surrounding ionic environment, monooxygenase activities were measured in both simple systems and mixed reconstituted systems as a function of ionic strength. PROD was found to be decreased at high ionic strength in both simple and mixed reconstituted systems, due to disruption of reductase-P450 complexes. Additionally, the inhibition of PROD in mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of the CYP2B4-CYP1A2 complex. When ionic strength was measured as a function of CYP1A2 concentration, a shift to the right in the inflection point of the biphasic curve occurred at high ionic strength, consistent with a loss in CYP1A2 affinity for CYP2B4. When this analysis was applied to the same systems using a different substrate, 7-EFC, evidence for a high affinity complex was not observed, demonstrating that the characteristics of the CYP1A2-CYP2B4 complex are influenced by the substrates present. These results support the role for a substrate specific electrostatic interaction between these P450 enzymes.P450 catalyzes the oxidative metabolism of a wide variety of both exogenous and endogenous substrates. The enzyme system splits a dioxygen molecule, incorporating one atom of oxygen into bound substrate with the other atom of oxygen forming water. The P450 enzymes involved in foreign compound metabolism are generally found in the microsomal fraction, and receive electrons from NADPH via the flavoprotein NADPH cytochrome P450 reductase (reductase) (1;2). Electron transfer to this hemoprotein requires the formation of a 1:1 molar complex between the reductase and cytochrome P450 (3;4). The reductase sequentially transfers two electrons to P450, one before and one after the binding of molecular oxygen (5;6). In some cases cytochrome b 5 can transfer the second electron to selective P450s (7). Since the identification of the components of the P450 system (8;9), several studies have been directed towards understanding the molecular association of P450 with its redox partners. Interactions between P450 and redox partners, cytochrome b 5 and reductase, have been described as being predominately electrostatic in nature (10)(11)(12)(13)(14)(15)(16)(17)(18)). An electron transfer complex is facilitated by * Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and Stanley S. Scott Cancer Center, L...

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Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies.

Cited by 61 publications

References 29 publications

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

Effects of Ionic Strength on the Functional Interactions between CYP2B4 and CYP1A2

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