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Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489–497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91–K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81–S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein–protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, ‘How are the HO-1 and P450 systems organized in the endoplasmic reticulum?’ Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

This is a small-scale qualitative study that looks at the experiences of two Japanese adult learners of English and relates their experiences in the classroom to contemporary theories on motivation, namely Dörnyei’s L2 Motivational Self System. I argue that traditional Japanese teaching practices in secondary education may inhibit the development of motivation to study English and argue that instructors at universities can play a role in ‘rescuing’ student motivation by making a clear conceptual separation between student’s prior experiences with English and a new approach or ‘fresh start’ in English oral communication classes.

Cytochromes P450s (P450s) catalyze oxygenation reactions via interactions with their redox partners. However, other proteins, particularly other P450s, also have been shown to form complexes that modulate P450 function. Previous studies showed that CYP1A2 and CYP2B4 form a complex when reconstituted into phospholipid vesicles; however, details of the interactions among the P450s and NADPH-cytochrome P450 reductase (POR) have not been fully characterized. The goal of this study was to examine P450 complex formation in living cells, using bioluminescence resonance energy transfer (BRET). Various pairs of P450 and POR constructs were tagged with either green fluorescent protein or Renilla luciferase, and transfected into human embryonic kidney 293T cells. Complexes were demonstrated by measuring energy transfer between the tags, and disruption of the complex was verified by cotransfection with unlabeled P450-system proteins. CYP1A2 and CYP2B4 formed a stable complex that could not be disrupted by cotransfection of untagged POR. Interactions of both P450s with POR were detected, with untagged CYP1A2 disrupting the POR-CYP2B4 interaction. In contrast, untagged CYP2B4 did not affect the POR-CYP1A2 interaction. These data are consistent with POR preferentially binding to the CYP1A2 moiety of CYP1A2-CYP2B4. BRET-detectable homomeric CYP1A2-CYP1A2 also was detected, and was disrupted by cotransfection of either POR or CYP2B4. Both CYP1A2 and CYP2B4 activities were affected by their coexpression in a manner consistent with formation of the high-affinity POR-CYP1A2-CYP2B4 complex. These findings demonstrate that CYP1A2 and CYP2B4 form a heteromeric POR-CYP1A2-CYP2B4 complex in living cells that has altered catalytic activities relative to the homomeric enzymes.

Nurses can prepare the public to save lives following a mass casualty event.

Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH–cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid–protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains.

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

Heme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum-bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected due to their common electron donor, they generally have been studied separately. As the expression of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function, and conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1/CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. Whereas the POR•CYP1A2 complex was readily disrupted by the addition of HO-1, the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. Whereas BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin deethylation in the presence of HO-1, its activity was actually stimulated at subsaturating POR. In contrast, HO-1-mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by simple competition for POR.

In the prehospital environment, nonmedical first responders are often the first to arrive on the scene of a traumatic event and must be prepared to provide initial care at the point of injury. In civilian communities, these nonmedical first responders often include law enforcement officers. Hemorrhage is a major cause of death in trauma, and many of these deaths occur in the prehospital environment; therefore, prehospital training efforts should be directed accordingly toward bleeding control.A bleeding control training program was implemented and evaluated in a rural police department in Pinehurst, North Carolina, from February to April 2017. A repeated measures observational study was conducted to evaluate the training program. Measured were self-efficacy (pre- and post-test), knowledge (pretest, post-test 1 [immediate], post-test 2 [at 4 weeks]), and limb-tourniquet application time (classroom, simulation exercise).The study population was composed of 28 police officers (92.9% male) whose median age was 37 (interquartile range, 22-55) years. Mean self-efficacy scores, equating to user confidence and the decision to intervene, increased from pre- to post-training (34.54 [standard deviation (SD) 4.16] versus 35.62 [SD 4.17]; p = .042). In addition, mean knowledge test scores increased from pre- to immediately post-training (75.00 [SD 16.94] versus 85.83 [SD 11.00]; p = .006), as well as from preto 4 weeks post-training (75.00 [SD 16.94] versus 84.17 [SD 11.77]; p = .018). Lower limb-tourniquet application times were more rapid in the classroom than during the simulation exercise (23.06 seconds [SD 7.68] versus 31.91 seconds [SD 9.81]; p = .005).First-responder bleeding-control programs should be initiated and integrated at the local level throughout the Nation. Implementation and sustainment of such programs in police departments can save lives and enhance existing law enforcement efforts to protect and serve communities.

In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo.

Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230 °C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50 °C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR·P450 complex.

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) are generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct affect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2- dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition.

Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450–P450 interactions.

Cytochromes P450 represent a family of enzymes that are responsible for the oxidative metabolism of a wide variety of xenobiotics. Although the mammalian P450s require interactions with their redox partners in order to function, more recently, P450 system proteins have been shown to exist as multi-protein aggregates that include the formation of P450•P450 complexes. Evidence has shown that the metabolism of some substrates by a given P450 can be influenced by the specific interaction of the enzyme with other forms of P450. Detailed kinetic analysis of these reactions in vitro has shown that the P450-P450 interactions can alter metabolism by changing the ability of a P450 to bind to its cognate redox partner, NADPH-cytochrome P450 reductase; by altering substrate binding to the affected P450; and/or by changing the rate of a catalytic step of the reaction cycle. This review summarizes the known examples of P450-P450 interactions that have been shown in vitro to influence metabolism and categorizes them according to the mechanism(s) causing the effects. P450-P450 interactions have the potential to cause major changes in the metabolism and elimination of drugs in vivo. Current research on the topic of P450-P450 interactions is focused on elucidating the extent of the functional effects of these interactions in vivo. This review also summarizes the evidence that the P450-P450 interactions influence metabolism in vivo and discusses the studies that will provide further insight into the extent of these effects in the future.

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH–POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis–Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2–CYP1A2 complexes that exhibit altered catalytic activity.

Cytochromes P450 (P450) are membrane-bound enzymes that catalyze the monooxygenation of a diverse array of xenobiotic and endogenous compounds. The P450s responsible for foreign compound metabolism generally are localized in the endoplasmic reticulum of the liver, lung and small intestine. P450 enzymes do not act alone but require an interaction with other electron transfer proteins such as NADPH-cytochrome P450 reductase (CPR) and cytochrome b(5). Because P450s are localized in the endoplasmic reticulum with these and other ER-resident proteins, there is a potential for protein-protein interactions to influence P450 function. There has been increasing evidence that P450 enzymes form complexes in the ER, with compelling support that formation of P450 · P450 complexes can significantly influence their function. Our goal is to review the research supporting the formation of P450 · P450 complexes, their specificity, and how drug metabolism may be affected. This review describes the potential mechanisms by which P450s may interact, and provides evidence to support each of the possible mechanisms. Additionally, evidence for the formation of both heteromeric and homomeric P450 complexes are reviewed. Finally, direct physical evidence for P450 complex formation in solution and in membranes is summarized, and questions directing the future research of functional P450 interactions are discussed with respect to their potential impact on drug metabolism.

Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-I (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR∙HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR-HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.

Heme oxygenase-1 (HO-1) is induced in most cell types by many forms of environmental stress and is believed to play a protective role in cells exposed to oxidative stress. Metabolism by cytochromes P450 (P450) is highly inefficient as the oxidation of substrate is associated with the production of varying proportions of hydrogen peroxide and/or superoxide. This study tests the hypothesis that heme oxygenase-1 (HO-1) plays a protective role against oxidative stress by competing with P450 for binding to the common redox partner, the NADPH P450 reductase (CPR) and in the process, diminishing P450 metabolism and the associated production of reactive oxygen species (ROS). Liver microsomes were isolated from uninduced rats and rats that were treated with cadmium and/or β-napthoflavone (BNF) to induce HO-1 and/or CYP1A2. HO-1 induction was associated with slower rates of metabolism of the CYP1A2-specific substrate, 7-ethoxyresorufin. Furthermore, HO-1 induction also was associated with slower rates of hydrogen peroxide and hydroxyl radical production by microsomes from rats induced for CYP1A2. The inhibition associated with HO-1 induction was not dependent on the addition of heme to the microsomal incubations. The effects of HO-1 induction were less dramatic in the absence of substrate for CYP1A2, suggesting that the enzyme was more effective in inhibiting the CYP1A2-related activity than the CPR-related production of superoxide (that dismutates to form hydrogen peroxide).

Cytochrome P450 is part of an electron transport chain found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with NADPH-cytochrome P450 reductase (CPR). The goals of this study were to examine how the P450 system proteins are organized in the membrane and to determine whether they are distributed in detergent-resistant lipid microdomains (DRM). Isolated liver microsomes from untreated rabbits were treated with 1% Brij 98, and DRMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that DRM fractions were enriched in cholesterol and sphingomyelin, similar to that found with plasma membrane DRMs. Approximately 73% of CYP1A2 and 68% of CPR resided in DRM fractions, compared with only 33% of total ER proteins. These DRMs were found to be cholesterol-dependent: CPR and CYP1A2 migrated to the more dense regions of the sucrose gradient after cholesterol depletion. CYP1A2 function was studied in three purified lipid vesicles consisting of 1) phosphatidylcholine (V-PC), 2) lipids with a composition similar to ER lipids (V-ER), and 3) lipids with a composition similar to the DRM fractions (V-DRM). Each system showed similar substrate binding characteristics. However, when the association between CPR and CYP1A2 was measured, V-ER and V-DRM liposomes produced lower apparent K(m) values compared with V-PC without any significant change in V(max). These findings suggest that CYP1A2 and CPR reside in ER-DRMs and that the unique lipid components of these domains enhance CYP1A2 substrate metabolism through greater efficiency in CPR-CYP1A2 binding.

Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR.

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the Km for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025–0.05 μM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

2-ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.

The pharmacokinetics, metabolism, and excretion of sitagliptin [MK-0431; (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine], a potent dipeptidyl peptidase 4 inhibitor, were evaluated in male Sprague-Dawley rats and beagle dogs. The plasma clearance and volume of distribution of sitagliptin were higher in rats (40-48 ml/min/kg, 7-9 l/kg) than in dogs ( approximately 9 ml/min/kg, approximately 3 l/kg), and its half-life was shorter in rats, approximately 2 h compared with approximately 4 h in dogs. Sitagliptin was absorbed rapidly after oral administration of a solution of the phosphate salt. The absolute oral bioavailability was high, and the pharmacokinetics were fairly dose-proportional. After administration of [(14)C]sitagliptin, parent drug was the major radioactive component in rat and dog plasma, urine, bile, and feces. Sitagliptin was eliminated primarily by renal excretion of parent drug; biliary excretion was an important pathway in rats, whereas metabolism was minimal in both species in vitro and in vivo. Approximately 10 to 16% of the radiolabeled dose was recovered in the rat and dog excreta as phase I and II metabolites, which were formed by N-sulfation, N-carbamoyl glucuronidation, hydroxylation of the triazolopiperazine ring, and oxidative desaturation of the piperazine ring followed by cyclization via the primary amine. The renal clearance of unbound drug in rats, 32 to 39 ml/min/kg, far exceeded the glomerular filtration rate, indicative of active renal elimination of parent drug.

The metabolism and excretion of [(14)C]sitagliptin, an orally active, potent and selective dipeptidyl peptidase 4 inhibitor, were investigated in humans after a single oral dose of 83 mg/193 muCi. Urine, feces, and plasma were collected at regular intervals for up to 7 days. The primary route of excretion of radioactivity was via the kidneys, with a mean value of 87% of the administered dose recovered in urine. Mean fecal excretion was 13% of the administered dose. Parent drug was the major radioactive component in plasma, urine, and feces, with only 16% of the dose excreted as metabolites (13% in urine and 3% in feces), indicating that sitagliptin was eliminated primarily by renal excretion. Approximately 74% of plasma AUC of total radioactivity was accounted for by parent drug. Six metabolites were detected at trace levels, each representing

Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2.

Cell membranes are composed of numerous lipid species that distribute heterogeneously into microdomains having unique characteristics. Ordered domains are enriched in cholesterol, sphingomyelin and...

Our lab has shown that CYP1A2 activity increased and CYP2B4 activity decreased when both proteins were incorporated into the same vesicles at subsaturating NADPH-cytochrome P450 reductase (CPR). These data, particularly the apparent activation of CYP1A2 by CYP2B4, are inconsistent with simple competition between the P450s for CPR. Previous work has also provided evidence for physical interactions between P450s. The goal of this study was to show that physical interactions can occur between CYP1A2 and CYP2B4 in the endoplasmic reticulum and to determine how these interactions might affect CPR binding and P450 activities. To achieve this we utilized bioluminescence resonance energy transfer (BRET). HEK293T cells were co-transfected with two proteins—one with a GFP tag and the other with a Renilla luciferase (Rluc) tag—and energy transfer between these proteins was measured to determine whether they formed physical complexes. A complex was formed by CYP1A2-Rluc and CYP2B4-GFP, and this complex was stable in ...

Two methods (cholate dialysis and cholate gel filtration) used to incorporate cytochromes P450 (P450s) and reductase into unilamellar phospholipid vesicles were compared with a standard reconstituted system (SRS) in which the proteins were reconstituted with preformed liposomes. Both cholate dialysis and gel filtration methods were comparable in their ability to physically incorporate reductase and either CYP2B4 or CYP1A2 into phospholipid, as determined by the elution of enzymes in the void volume using size exclusion chromatography (mol. wt. cutoff -5,000,000). Incorporation of these proteins was more efficient with both cholate methods than when reductase and P450 were mixed with preformed vesicles (SRS). Using either cholate method, more than 85% of the P450 was physically incorporated into the phospholipid vesicles, whereas less than 40% of the P450 was physically incorporated into the phospholipid vesicles using the SRS. Catalytic activities of the vesicular preparations of reductase and either CYP1A2 or CYP2B4 also were significantly higher than those resulting from the SRS using dilaurylphosphatidylcholine. Although both cholate dialysis and gel filtration methods improved protein incorporation when compared with preincubation of proteins with preformed liposomes, reductase incorporation was dependent on the relative amount of reductase used. Reductase incorporation was complete at a 0.2:1 reductase/P450 ratio; however, the efficiency of incorporation decreased to less than 50% at equimolar reductase/P450. Interestingly, reductase incorporation was higher in the presence of CYP1A2 than with CYP2B4. Both cholate methods resulted in the loss of a proportion of spectrally detectable carbon monoxyferrous P450, resulting from incubation of the proteins with detergent.

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.

Dissociation constants K d for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b 5 (b 5 ) stimulates metabolism of this substrate and dramatically decreases the substrate-related K d values. Experiments are performed to test if the b 5 -mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b 5 (Mn b 5 ) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b 5 stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b 5 is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b 5 , with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.

The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b 5 ( b 5 ) on these reactions were assessed. There did not appear to be any trends regarding the effects of b 5 when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b 5 were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b 5 effects. The rates of benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes.

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function.

Previous reports showed that CYP1A2 and CYP2B4 can affect each other’s activity when present in the same vesicles with subsaturating NADPH-P450 reductase (CPR). Specifically, systems containing both P450s exhibited increased CYP1A2 and decreased CYP2B4 activities when compared with those containing each P450 alone. These effects were consistent with the formation of CYP1A2•CYP2B4 complexes that affected the affinity of each P450 for CPR, and were corroborated in living cells using bioluminescence resonance energy transfer (BRET). Previous results also showed that CYP1A2 forms homomeric complexes whose presence was correlated with inhibition of CYP1A2 activity at subsaturating CPR. The goal of this study was to determine if homomeric P450 complexes were disrupted by the presence of another P450 using BRET. First, the potential for CYP2B4•CYP2B4 complexes was examined. Unlike with CYP1A2, we were unable to detect specific CYP2B4•CYP2B4 complexes. Next, we examined the potential for CYP2B4 to disrupt the inh...

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH–cytochrome P450 reductase, and cytochrome b5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (l(o)) domains. The goal of this study was to determine if l(o) domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in "ER-like" lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form l(o) domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model l(o)-forming systems demonstrated the coexistence of l(o)- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the l(o) regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into l(o) membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing l(o) domains.