Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

Abstract

Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(N-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

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

“…In addition, each set of Co-IP was approximately equal in the number of proteins (116 for CYP1A2 Co-IP (Table S1) and 134 for the CYP3A Co-IP (Table S2)). Thus, our study has identified many more binding partners than in previous studies utilizing co-immunoprecipitation of P450s (Li et al, 2011;Fujiwara and Itoh, 2014;Davydov et al, 2022). Like the other studies that have utilized cross-linking and/or immunoprecipitation to identify protein interactions with P450s, both P450s were immunoprecipitated with a wide variety of microsomal proteins that are related to many cellular functions.…”

“…After immunoprecipitation from microsomes using FLAG tag antibody, co-precipitated proteins were identified by LC/MS/MS (Li et al, 2011). Finally, a recent study explored the interactome of a modified (N-truncated and C-terminal His-tagged) human CYP2E1 (Davydov et al, 2022) that was capable of light-sensitive cross linking. After the modified CYP2E1 was incorporated into human liver microsomes, the cross-linker was activated and CYP2E1 complexes were identified by mass spectrometry.…”

“…CYP3A has also been reported to form complexes with other P450 enzymes, including, CYP2C9 and CYP2E1 (Subramanian et al, 2010a;Davydov et al, 2015;Dangi et al, 2020;Davydov et al, 2022). Furthermore, both CYP1A and CYP3A have been shown to interact with phase II enzymes such as UDP-glucuronosyl transferases (UGT) and epoxide hydrolase.…”