Robert Fuchs

The cytochromes P450 are a set of enzymes in the liver critical for metabolism of drugs, toxins, and hormones. These proteins interact extensively with the endoplasmic reticulum (ER), a membranous organelle composed of heterogeneously organized lipids. Most of the ER surface area is composed of anionic phospholipids; such regions are termed disordered microdomains (ld). Conversely, select parts of the membrane contain cholesterol, sphingomyelin, and other specialized lipids; these areas are called ordered microdomains (lo, “lipid rafts”). Many membrane-related proteins show strong preference for ordered or disordered regions. It is currently unclear what factors drive enzymes into their preferred microdomains and whether microdomain localization plays a role in maintaining enzyme activity. This project will study whether specific cytochrome P450 enzymes preferentially sort into specific ER membrane regions. It is hypothesized that a specific amino acid motif targets P450 proteins into ordered or 44 disordered regions, and that disruption of normal microdomain localization influences enzyme activity. To test this hypothesis, chimeric fusion proteins will be generated for two specific P450s, CYP1A1 and CYP1A2, which respectively localize to disordered and ordered regions. Microdomain localization for each chimera will be determined using detergent solubilization. It is expected that chimeric CYP1A1 containing a critical amino acid motif from CYP1A2 will show microdomain localization pattern characteristic of CYP1A2, and vice versa. For chimeric proteins that mobilize outside their native microdomain, enzyme activity on CYP1A1- and CYP1A2-specific substrates will be measured, and it is predicted that constructs that do not localize to their native microdomains will exhibit altered enzyme activity. Results from this project will reveal a basic mechanism for regulation of P450 activity in live cells, and will guide future research on variability in P450 enzyme activity.