Evidence for a complex formation between CYP2J5 and mEH in living cells by FRET analysis of membrane protein interaction in the endoplasmic reticulum (FAMPIR)

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.

from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2ggRwH3
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