The Influence of Lipid Microdomain Heterogeneity on Protein-Protein Interactions: Proteomic Analysis of Co-Immunoprecipitated Binding Partners of P450 1A2 and P450 3A in Rat Liver Microsomes.

Liver cytochrome P450s (CYPs) of the endoplasmic reticulum (ER) are involved in the metabolism of exogenous and endogenous chemicals. The ER is not uniform, but possesses ordered lipid microdomains containing higher levels of saturated fatty acids, sphingomyelin, and cholesterol and disordered regions containing higher levels of polyunsaturated fatty acid chains. The various forms of drug-metabolizing P450s partition to either the ordered or disordered lipid microdomains with different degrees of specificity. P450s readily form complexes with ER-resident proteins, including other forms of P450. This study aims to ascertain whether lipid microdomain localization influences protein-P450 interactions in rat liver microsomes. Thus, liver microsomes were co-immunoprecipitated with CYP1A2-specific and CYP3A-specific antibodies, and the co-immunoprecipitating proteins were identified by liquid chromatography mass spectrometry proteomic analysis. These two P450s preferentially partition to ordered and disordered microdomains, respectively. More than 100 proteins were co-immunoprecipitated with each P450. Segregation of proteins into different microdomains did not preclude their interaction. However, CYP3A interacted broadly with proteins from ordered microdomains, whereas CYP1A2 reacted with a limited subset of these proteins. This is consistent with the concept of lipid raft heterogeneity and may indicate that CYP1A2 is targeted to a specific type of lipid raft. Although many of the interacting proteins for both P450s were other-drug metabolizing enzymes, other interactions were also evident. The consistent CYP3A binding partners were predominantly involved in phase I/II drug metabolism; however, CYP1A2 interacted not only with xenobiotic metabolizing enzymes, but also with enzymes involved in diverse cellular responses such as ER stress and protein folding. SIGNIFICANCE STATEMENT: This work describes the protein interactomes in rat liver microsomes of two important cytochromes P450s (CYP1A2 and CYP3A) in drug metabolism and describes the relationship of the interacting proteins to lipid microdomain distribution.