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Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

ABSTRACT: Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function.

SUBMITTER: Park JW

PROVIDER: S-EPMC4314108 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

Park Ji Won JW Reed James R JR Brignac-Huber Lauren M LM Backes Wayne L WL

The Biochemical journal 20141201 2

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher le ...[more]

PMID: 25236845

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Project description:This study investigated the role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in enhancing CYP2E1 and other P450 proteins in extracellular vesicles (EVs) from alcohol-exposed rodents and human patients with alcoholism and their effects on oxidative hepatocyte injury. Female Fischer rats and wild-type or Cyp2e1-null mice were exposed to three oral doses of binge ethanol or dextrose control at 12-hour intervals. Plasma EV and hepatic proteins from alcohol-exposed rodents, patients with alcoholism, and their respective controls were isolated and characterized. The number of EVs and the amounts of EV CYP2E1, CYP2A, CYP1A1/2, and CYP4B proteins were markedly elevated in both patients with alcoholism and alcohol-exposed rats and mice. The number of EVs and EV P450 proteins were significantly reduced in ethanol-exposed rats fed a diet containing polyunsaturated fatty acids. The increased number of EVs and EV CYP2E1 and other P450 isoforms in alcohol-exposed wild types were significantly reduced in the corresponding Cyp2e1-null mice. EV CYP2E1 amounts depended on increased oxidative and endoplasmic reticulum (ER) stress because their levels were decreased by cotreatment with the antioxidant N-acetylcysteine or the CYP2E1 inhibitor chlormethiazole but increased by ER stress-inducer thapsigargin, which was blocked by 4-phenylbutyric acid. Furthermore, cell death rates were elevated when primary hepatocytes or human hepatoma cells were exposed to EVs from alcohol-exposed rodents and patients with alcoholism, demonstrating that EVs from alcohol-exposed rats and patients with alcoholism are functional and can promote cell death by activating the apoptosis signaling pathway, including phospho-c-Jun N-terminal kinase, proapoptotic Bax, and activated caspase-3. Conclusion: CYP2E1 has an important role in elevating EV CYP2E1 and other P450 isoforms through increased oxidative and ER stress. Elevated EV-CYP2E1 detected after withdrawal from alcohol or exposure to the CYP2E1 inducer pyrazole can be a potential biomarker for liver injury. (Hepatology Communications 2017;1:675-690).

Dataset Information

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

Publications

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

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