Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity

Abstract

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.

Fig. 1.

Comparison of the phospholipid composition of liver microsomes with that of the ordered microdomains. Rabbit liver microsomes were subjected to solubilization using 1% Brij 98 followed by discontinuous sucrose density gradient centrifugation to isolate the ordered (DRM) regions of the membrane. The phospholipid composition of the ordered region was determined and compared with that of untreated microsomes. This research was originally reported by Brignac-Huber et al. (2011).

Fig. 2.

Replacement of the NH₂-terminal region of CYP1A2 with that of CYP1A1 changes its membrane localization. The microdomain localization CYP1A2 and chimeric proteins where the NH₂ terminus was substituted with that of CYP1A1 was measured by expression of the proteins in human embryonic kidney 293T cells and partial membrane solubilization using Brij 98. After Brij treatment, the samples were subjected to centrifugation and the proteins remaining in the membranes were detected in the pellet using PAGE. (A) Schematic of the chimeric proteins generated. (B) Immunoblot of the native CYP1A proteins and the chimeras. Proteins in the pellet remain associated with the membrane, whereas those in the supernatant were solubilized. P, pellet; S, supernatant. This research was originally published in the Journal of Biological Chemistry [Park JW, Reed JR, and Backes WL. The localization of cytochrome P450s CYP1A1 and CYP1A2 into different lipid microdomains is governed by their NH₂-terminal and internal protein regions. Journal of Biological Chemistry. (2015) 290, 29449–29460. © American Society for Biochemistry and Molecular Biology].

Fig. 3.

Replacement of the NH₂-terminal region of CYP1A1 with that of CYP1A2 changes its membrane localization. The microdomain localization CYP1A1 and chimeric proteins where the NH₂ terminus was substituted with that of CYP1A2 was measured as described in the legend for Fig. 1. (A) Schematic of the chimeric proteins generated. (B) Immunoblot of the native CYP1A proteins and the chimeras. P, pellet; S, supernatant. This research was originally published in the Journal of Biological Chemistry (Park et al., 2015).

Fig. 4.

Importance of the internal regions in directing the microdomain localization of CYP1A proteins. The microdomain localization of chimeric proteins where the internal segment of CYP1A2 was substituted with the corresponding region of CYP1A1 was measured as described in Fig. 1. P, pellet; S, supernatant. This research was originally published in the Journal of Biological Chemistry (Park et al., 2015).

Fig. 5.

Illustration of P450 system protein localization in the ER. Lipid membranes are thought to be segregated into liquid-ordered (l_o) and liquid-disordered domains. The ordered domains (which also are referred to as lipid rafts) tend to be more enriched in cholesterol and SM, tend to pack more tightly, and are more resistant to solubilization by detergents (Diaz-Rohrer et al., 2014). P450 system proteins do not distribute randomly throughout the membrane, but they localize in regions of differing phospholipid composition. P450s and CPR have also been reported to be more closely associated with PE and the anionic phospholipids PS and PI, the phospholipids that surround P450 enzymes. The phospholipid regions with the predominant yellow head groups represent the l_o microdomains with the disordered regions being largely in gray. Cholesterol (aqua) and SM (black) are intercalated into the l_o regions. The anionic phospholipids are indicated by the red polar head groups. CYP1A2 and CPR localize predominantly in the l_o regions, whereas CYP1A1 and CYP2E1 are found mainly in the disordered membranes. CYP2B4 distributes between both regions. The figure also illustrates the tendency of different P450 enzymes to form P450⋅P450 complexes.