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 P450⅐P450 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) CPR⅐CYP1A2, 2) CPR⅐CYP2B4, 3) a mixture of CPR⅐CYP1A2 vesicles with CPR⅐CYP2B4 vesicles, and 4) CPR⅐CYP1A2⅐CYP2B4 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 crosslinking 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.
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.
Cytochromes P450 (P450s) are membrane‐bound proteins mainly found in the endoplasmic reticulum (ER). The ER membrane is heterogeneous, containing ordered (OD) and disordered domains (DD). These OD are more tightly packed and not readily solubilized with non‐ionic detergent, and are called detergent‐resistant membranes. Recently, our lab showed that CYP1A2 and CPR localize in OD. In this study, we examined the organization of other P450s in rabbit liver microsomes by Brij98 treatment and sucrose gradient centrifugation. Interestingly, CYP2E1 localized in DD and CYP2B4 was equally distributed between the OD and DD. Cholesterol depletion disturbed the distribution of OD‐localized CYP1A2, CYP2B4 and CPR. However, CYP2E1 localization was cholesterol independent. Lastly, to test if P450 localization is a property of the P450, P450 was incorporated into reconstituted systems, consisting of 1) phosphatidylcholine (PC) and 2) lipids mimicking the ER, forming both OD and DD. In PC vesicles both CYP2B4 and CYP2E1 proteins localized in late DD fractions. However, in ER mimicking vesicles, CYP2B4 was found in both OD and DD, whereas CYP2E1 distributed exclusively into DD, consistent with microsomal data. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and the distribution is dependent on the specific P450 being examined. Supported by R01 ES004344 and P42 ES013648
P450s are membrane‐bound proteins found in the endoplasmic reticulum (ER). The ER is known to be heterogeneous, containing both ordered and disordered regions. Ordered microdomains are more tightly packed and not readily solubilized with non‐ionic detergents and are called detergent resistant membranes (DRM). The goal of this study is to determine if these microdomains affect P450 localization. The localization of P450 system proteins was examined in rabbit liver microsomes after incubation with 1% Brij 98, followed by sucrose gradient centrifugation. Unlike CYP1A2 (which localizes in the DRM), CYP2E1 proteins localized in non‐DRM and CYP2B4 was found to be equally distributed between both domains. Importantly, for P450 to function, it is necessary for this heme protein to form a complex with its electron donor CPR; however, CPR is found largely in the DRM. Consequently, for CYP2E1 to function, a mechanism for complex formation is required, such as translocation of either CYP2E1 or CPR between the lipid domains, or the interaction of CYP2E1 with the small amount of residual CPR localized in the disordered region, which would limit its activity. These results demonstrate that the components of the P450 system are organized in specific membrane regions, and not randomly distributed throughout in the ER membrane. Supported by ES004344
P450s are membrane‐bound proteins found in the endoplasmic reticulum, requiring interaction with NADPH‐cytochrome P450 reductase (CPR). Previously, using rabbit enzymes, we demonstrated that one P450 can influence the behavior of another P450 by forming P450•P450 complexes. The goal of this study was to determine (1) if similar P450•P450 complexes are found with human P450s, and (2) how these interactions are affected by membrane lipids. Human CYP1A2 and CYP3A4 were incorporated into different reconstituted systems at subsaturating CPR, and the activity of CYP1A2 and CYP3A4 was examined. Substrate metabolism by both CYP1A2 and CYP3A4 were sensitive to alterations in lipid composition, and may affect interactions among P450s. These interactions were examined by comparing simple systems, containing CPR and a single P450, and mixed systems contain multiple P450s in the same vesicles. Interestingly, when CYP1A2 and CYP3A4 were present in the same vesicles, an inhibition of CYP3A4 function was observed, consistent with studies with rabbit P450s. The result of altered function of CYP3A4 with CYP1A2 is consistent with P450•P450 complex formation. (Supported by NIH R01 ES004344 & P42 ES013648)
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