Wayne L. Backes

This study focuses on the effect of Environmentally Persistent Free Radicals (EPFRs) on the P450 enzymes of the CYP1 family. EPFRs are a component of particulate pollutants, that are stable in the environment, but can generate free radicals, leading to oxidative stress and subsequent toxicity of the respiratory, cardiovascular, and immune systems once they enter an organism. The results show differences in the ability of EPFRs to inhibit CYP1-dependent substrate metabolism, with CYP1B1 being inhibited to the greatest extent. There also were differences in the ability of EPFRs to disrupt the POR•CYP1 complex, with CYP1B1 being the only form where EPFRs disrupted POR•CYP1B1 complex formation. Despite the inhibition of substrate metabolism, each CYP1 enzyme, when reconstituted with NADPH-cytochrome P450 reductase (POR) was able to synergistically stimulate the generation of reactive oxygen (ROS) in the presence of particulate matter. Interestingly, both POR and the CYP1 enzymes were able to stimulate ROS generation, even when in partial reconstituted systems where only one of the proteins was present. However, when both POR and CYP1 were combined in a complete reconstituted system, ROS generation was synergistically stimulated.

Environmentally persistent free radicals (EPFRs) are a recently recognized component of particulate matter that cause respiratory and cardiovascular toxicity. The mechanism of EPFR toxicity appears to be related to their ability to generate reactive oxygen species (ROS), causing oxidative damage. EPFRs were shown to affect cytochrome P450 (P450) function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs is their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230, the laboratory-generated EPFR made by heating silica 5% copper oxide, and silica [<0.2 μm in diameter] and 2-monochlorophenol at ≥230 °C) exposure. Because CYP2E1 is also known to generate ROS, it is important to understand the ability of EPFRs to influence the function of this enzyme and to identify the mechanisms involved. CYP2E1 was shown to be inhibited by EPFRs and to a lesser extent by non-EPFR particles. Because EPFR-mediated inhibition was more robust at subsaturating NADPH-P450 reductase (POR) concentrations, disruption of POR•CYP2E1 complex formation and electron transfer were examined. Surprisingly, neither complex formation nor electron transfer between POR and CYP2E1 was inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR control particle (CuO-Si). When a POR/CYP2E1-containing reconstituted system was added to the pollutant-particle systems, there was a synergistic stimulation of ROS production. The results indicate that EPFRs cause inhibition of CYP2E1-mediated substrate metabolism, yet do not alter electron transfer and actually stimulate ROS generation. Taken together, the results are consistent with EPFRs affecting CYP2E1 function by inhibiting substrate metabolism and increasing the generation of ROS. SIGNIFICANCE STATEMENT: Environmentally persistent free radicals affect CYP2E1 function by inhibition of monooxygenase activity. This inhibition is not due to disruption of the POR•CYP2E1 complex or inhibition of electron transfer but due to the uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of reactive oxygen species. These results show that environmentally persistent free radicals block the metabolism of foreign compounds and synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.

ID 25945 Poster Board 390 CYP1A1, CYP1A2, and CYP1B1 share high amino acid sequence similarity, substrate selectivity, and induction characteristics. Despite this, there are significant differences in their functional characteristics. The goal of this work was to characterize the protein-protein interactions of CYP1 enzymes and to link differences in these interactions with changes in enzyme activity. To achieve this we used bioluminescence resonance energy transfer (BRET) to study the presence and stability of CYP1 protein complexes in HEK293T cells and did kinetic analysis of CYP1 activity using recombinant protein reconstituted in liposomes with varying levels of NADPH-cytochrome P450 reductase (POR). Using BRET, we determined that both CYP1A1 and CYP1A2 (but not CYP1B1) formed homomeric complexes. When CYP1 activity was measured while varying [POR], CYP1A1 and CYP1B1 showed a hyperbolic response while the CYP1A2 response was sigmoidal. Each family 1 P450 also appeared to form a complex with heme oxygnase-1 (HO-1), though the stability of these complexes and their effect on POR binding was unique for each P450. The presence of HO-1 generally inhibited CYP1 activity, but the kinetic data were inconsistent with simple competition for POR binding. Differences in protein-protein interactions formed by CYP1A1, CYP1A2, and CYP1B1 appear to contribute to observed differences in enzyme function. The atypical kinetics of CYP1A2 can be explained by the presence of homomeric CYP1A2 complexes. Interestingly, homomeric CYP1A1 complexes did not appear to have a similar effect. Differences in HO-1/P450 complex formation and subsequent changes in POR binding and enzyme activity have the potential to affect the disposition of endogenous and xenobiotic compounds. These results demonstrate that differences in quaternary structure may contribute to the unique functional characteristics of endoplasmic reticulum membrane-resident enzymes. Our understanding of P450-mediated metabolism will be incomplete if it is limited to individual P450s interacting with POR as monomers — the participation of P450 enzymes in protein complexes must also be taken into account. Support/Funding Information: NIGMS R01 GM123253 NIEHS P42 ES013648

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, 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 to wild-type CYP1A2 (WT‑CYP1A2). On further examination, P₁‑CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared to 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 to 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.

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 K_m^POR•HO-1 without affecting the V_max^app. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the K_m^POR•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.

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.

Cytochromes P450 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 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 GFP or Renilla luciferase, and transfected into HEK-293T cells. Complexes were demonstrated by measuring energy transfer between the tags, and disruption of the complex was verified by co transfection with unlabeled P450-system proteins. CYP1A2 and CYP2B4 formed a stable complex that could not be disrupted by co-transfection 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 co transfection of either POR or CYP2B4. Both CYP1A2 and CYP2B4 activities were affected by their co-expression in a manner consistent with formation of the high affinity POR·CYP1A2·YP2B4 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.