Abstract
The potential complex formation between microsomal epoxide hydrolase (mEH) and cytochrome P450-dependent monooxygenase (CYP) has been a subject of research for many decades. Such an association would enable efficient substrate channeling between CYP and mEH and as such represent an attractive strategy to prevent deleterious accumulation of harmful metabolic by-products such as CYP-generated epoxide intermediates. However, such complex formation is experimentally difficult to prove, because CYP and mEH are membrane-bound proteins that are prone to unspecific aggregation after solubilization. Here, we report the development of a FRET-based procedure to analyze the mEH–CYP interaction in living cells by fluorescence-activated cell sorting. With this non-invasive procedure, we demonstrate that CYP2J5 and mEH associate in the endoplasmic reticulum of recombinant HEK293 cells to the same extent as do CYP2J5 and its indispensible redox partner cytochrome P450 reductase. This presents final proof for a very close proximity of CYP and mEH in the endoplasmic reticulum, compatible with and indicative of their physical interaction. In addition, we provide with FAMPIR a robust and easy-to-implement general method for analyzing the interaction of ER membrane-resident proteins that share a type I topology.
Highlights
Among the many oxygenation reactions catalyzed by cytochrome P450-dependent monooxygenases (CYP), epoxygenation is a important one (Guengerich 2003; Spector 2009)
Transfection of HEK293 cells with such constructs reproducibly resulted in the expression of a single, unexpectedly large fusion protein that did not match in size with the proteins obtained when only YFP–CYP or CFP–microsomal epoxide hydrolase (mEH) alone were expressed in HEK293T cells
At least a transient physical interaction between CYP and cytochrome P450 reductase (CPR) appears essential, in view of the electron transport from CPR to CYP that initiates catalysis (Lu et al 1969). First models for this physical interaction have been developed very early (Peterson et al 1976) and a plethora of studies, nicely reviewed by Kandel and Lampe (2014), have followed, including X-ray studies that give a detailed picture of plausible modes of interaction between CPR and CYP (Hamdane et al 2009)
Summary
Among the many oxygenation reactions catalyzed by cytochrome P450-dependent monooxygenases (CYP), epoxygenation is a important one (Guengerich 2003; Spector 2009). The resulting epoxides vary substantially in their stability/reactivity While some epoxides, such as the 8,9-exo-epoxide of Aflatoxin B1, are highly electrophilically reactive and represent a major threat, due to their reactivity with DNA and proteins, other epoxides are very stable and poorly reactive (Guengerich 2003). This allows the body to use compounds of the latter type, for instance the arachidonic acid-derived epoxyeicosatrienoic acids (EETs), as signaling molecules (Spector 2009). CYP is invariably associated with cytochrome P450 reductase (CPR), the mandatory electron donor for the first step in
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