CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

Abstract

ABSTRACT:Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b 5 (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although K m values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.Cytochromes P450 (P450s) are the primary oxidative enzymes responsible for the metabolism of xenobiotics by humans and animals (Guengerich, 2006). P450s can oxidize structurally diverse substrates ranging from small hydrocarbons to large molecules such as cyclosporine. P450s function by transferring electrons via a P450 catalytic pathway, wherein the first electron is transferred to the P450 via cytochrome NADPH P450 reductase (CPR) and the second electron is transferred via either CPR or cytochrome b 5 (b5) (Guengerich, 2002). Inefficiencies in the catalytic pathway, in part, govern the rate of transformation. P450s and CPR are bound to the cell membrane via a hydrophobic N terminus, whereas b5 is bound via its C terminus, each terminal being composed of 30 to 60 amino acids (Szczesna-Skorupa and Kemper, 2008). This hydrophobic region also contributes to protein aggregation, resulting in the formation of high molecular weight polymers in membranes and reconstituted systems (SzczesnaSkorupa and Kemper, 2008). Considering that P450s and CPR, and P450s and b5 interact within the cell membrane, P450-P450 interactions also appear conceivable. Indeed, in vitro evidence of P450-P450 interactions have been observed in several studies, including interactions of human CYP2C9-CYP2C19, rabbit CYP1A2-CYP2B4, rabbit CYP1A2-CYP2E1, human CYP3A4-CYP1A1, human CYP3A4-CYP1A2, and human CYP2C9-CYP2D6 (Cawley et al., 1995;Yamazaki et al., 1997;Backes et al., 1998;Backes and Cawley, 1999;Kelley et al., 2005Kelley et al., , 2006Subramanian et al., 2009). Substrate-dependent activation o...

CYP2C8 Exists as a Dimer in Natural Membranes

“…Experimentally, both hetero-and homodimerization have been shown to affect P450 function. Enzyme kinetic analysis of pairs of P450s, including 2B4/1A4, 2D6/2C9, 2C9/3A4, and 2C9/2C19 has provided evidence that hetero-interactions between the P450s affect the activity of one or both of the P450s in the dimer (Cawley et al, 2001;Hazai and Kupfer, 2005;Subramanian et al, 2009Subramanian et al, , 2010Reed et al, 2010). Homo-oligomerization also has functional consequences.…”

“…Solubilized P450s also have been shown to form homooligomers containing up six or eight protein molecules in some cases, which were mediated by the signal anchor sequence (Von Wachenfeldt and Johnson, 1995). Interactions among P450s may have functional significance because coexpression of a second P450 with a P450 can either inhibit or increase the activity of the first P450 (Cawley et al, 2001;Hazai and Kupfer, 2005;Subramanian et al, 2009Subramanian et al, , 2010Reed et al, 2010). The functional significance of homooligomerization is not clear, but oligomerization of CYP3A4 has been shown to decrease reduction of the P450 by dithionite (Davydov et al, 2005) or the soluble flavin domain of P450BM-3 (Davydov et al, 2010).…”

Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

“…These complexes are not only observed among P450s and their redox partners: NADPH-cytochrome P450 reductase and cytochrome b 5 (Bridges et al, 1998;Estrada et al, 2013), but also occur between multiple P450 enzymes (Davydov, 2011;. Both homomeric (Davydov et al, 1992;Szczesna-Skorupa et al, 2000;Ozalp et al, 2005;Jamakhandi et al, 2007;Davydov et al, 2010;Hu et al, 2010; and heteromeric (Yamazaki et al, 1997;Backes et al, 1998;Hazai and Kupfer, 2005;Subramanian et al, 2009;Reed et al, 2010;Subramanian et al, 2010) P450-P450 complexes have been reported among several different P450 enzymes.…”

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

“…28 Tracy and coworkers demonstrated that the hydrophobic Nterminus-binding domains of CYP2C9 and CYP3A4 are involved in heterodimer complex formation and truncation of the N-terminus of CYP2C9 led to the abolition of inhibition by CYP3A4. 29 In separate studies, it was hypothesized that the N-terminal domain might be critical for orientation of the protein into the lipid bilayer. 30 Thus, the N-terminus affects the localization on the membrane and can influence orientation and activity.…”