Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry
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)....
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MDPI AG
2022-01-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/12/2/185 |
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| author | Dmitri R. Davydov Bikash Dangi Guihua Yue Deepak S. Ahire Bhagwat Prasad Victor G. Zgoda |
| author_facet | Dmitri R. Davydov Bikash Dangi Guihua Yue Deepak S. Ahire Bhagwat Prasad Victor G. Zgoda |
| author_sort | Dmitri R. Davydov |
| collection | DOAJ |
| description | 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-(<i>N</i>-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. |
| first_indexed | 2024-03-09T22:30:53Z |
| format | Article |
| id | doaj.art-6444d483dcc44f8dbd13dcbf1a4ad328 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-09T22:30:53Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-6444d483dcc44f8dbd13dcbf1a4ad3282023-11-23T18:57:51ZengMDPI AGBiomolecules2218-273X2022-01-0112218510.3390/biom12020185Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass SpectrometryDmitri R. Davydov0Bikash Dangi1Guihua Yue2Deepak S. Ahire3Bhagwat Prasad4Victor G. Zgoda5Department of Chemistry, Washington State University, Pullman, WA 99164, USADepartment of Chemistry, Washington State University, Pullman, WA 99164, USADepartment of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USADepartment of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USADepartment of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USAOrekhovich Institute of Biomedical Chemistry, Pogodinskaya 10, 119121 Moscow, RussiaAiming 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-(<i>N</i>-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.https://www.mdpi.com/2218-273X/12/2/185chemical crosslinking mass spectrometry (CXMS)alcohol exposurealcohol-drug interactionsdrug metabolismprotein-protein interactionscytochrome P450 |
| spellingShingle | Dmitri R. Davydov Bikash Dangi Guihua Yue Deepak S. Ahire Bhagwat Prasad Victor G. Zgoda Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry Biomolecules chemical crosslinking mass spectrometry (CXMS) alcohol exposure alcohol-drug interactions drug metabolism protein-protein interactions cytochrome P450 |
| title | Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry |
| title_full | Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry |
| title_fullStr | Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry |
| title_full_unstemmed | Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry |
| title_short | Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry |
| title_sort | exploring the interactome of cytochrome p450 2e1 in human liver microsomes with chemical crosslinking mass spectrometry |
| topic | chemical crosslinking mass spectrometry (CXMS) alcohol exposure alcohol-drug interactions drug metabolism protein-protein interactions cytochrome P450 |
| url | https://www.mdpi.com/2218-273X/12/2/185 |
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