Biology subjects

Dangi, B.

Publications and source records attributed to Dangi, B..

3 recordsLinked to original sources

bioRxiv2021

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

Aiming to elucidate the grounds of 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 (proteome) 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 (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS). 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. Of particular interest are the interactions of CYP2E1 with the latter two enzymes, which are involved in the synthesis and disposal of vasoactive and pro-inflammatory eicosanoids. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1 (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.

biochemistry↗

bioRxiv2020

Multifold Activation of CYP3A4 Caused by Alcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes

Here we investigate the effect of alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. In these studies we used a model that implements enrichment of HLM samples with CYP2E1 through membrane incorporation of the purified protein. Enrichment of HLM with CYP2E1 considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. Incorporation of CYP2E1 also eliminates the activating effect of -Naphthoflavone (ANF) on BQ metabolism seen in some untreated HLM samples. To probe the physiological relevance of these effects we compared three pooled preparations of HLM from normal donors (HLM-N) with a preparation obtained from heavy alcohol consumers (HLM-A). The composition of the P450 pool in all four samples was characterized with mass-spectrometric determination of 11 cytochrome P450 species. The molar content of CYP2E1 in HLM-A was from 2.5 to 3.3 times higher than that found in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all four HLM samples. Despite of that, HLM-A exhibited much higher rate of metabolism and lower degree of homotropic cooperativity with BQ, similar to that observed in CYP2E1-enriched HLM-N. In order to substantiate the hypothesis on the involvement of physical interactions between CYP2E1 and CYP3A4 in the observed effects we probed hetero-association of these proteins in Supersomes containing recombinant CYP3A4 with a technique based on homo-FRET and employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments demonstrated high affinity interactions between the two enzymes and revealed an inhibitory effect of ANF on their hetero-association. Our results demonstrate that the catalytic activity and allosteric properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and imply a profound impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

biochemistry↗

bioRxiv2019

Non-additive effects of changing the cytochrome P450 ensemble: incorporation of CYP2E1 into human liver microsomes and its impact on CYP1A2

The aim of this study is to investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system we used CYP2E1-enriched microsomes obtained by incorporation of purified CYP2E1 protein into HLM. Using the method based on homo-FRET in homo-oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with microsomes result in dissociation of the protein homo-oligomers. The finding that this effect is much better pronounced in HLM as compared to the microsomes containing no P450 proteins indicates the formation of mixed oligomers of CYP2E1 with other P450 species that takes place in expense of dissociation of the homo-oligomers.\n\nIncorporation of CYP2E1 into HLM results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol (pNP) and Chlorzaxozone (CLZ). The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or about 50% CYP2E1 in the P450 pool). These results demonstrate that the incorporated CYP2E1 becomes a fully-functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1 in HLM.\n\nEnrichment of HLM with CYP2E1 results in a pronounced alteration of the metabolism of 7-etoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2, that suggests an important augmentation of the involvement of CYP1A2 in its metabolism. This effect goes together with a remarkable increase in the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin by CYP2E1-enriched HLM. Furthermore, probing the interactions of CYP2E1 with model microsomes (Supersomes) containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

biochemistry↗