The possible mechanisms of CYP2E1 interactions with HSP90 and the influence of ethanol on them
© Kitam et al.; licensee BioMed Central Ltd. 2012
Received: 10 September 2012
Accepted: 14 December 2012
Published: 17 December 2012
Microsomal CYP2E1 metabolizes about 160 hydrophobic exogens, many of which are environmental pollutants. While metabolising xenobiotics CYP2E1 on one hand facilitates in their excretion and on the other hand activates them into the cytotoxins, which may damage the cell. Thus the CYP2E1 activity level significantly affects the processes in cell. Posttranslational stabilization of CYP2E1 seems to be the main mechanism of its regulation in living cell. It is known that degradation of CYP2El takes part in cytoplasmic proteasome system. The efficiency of such degradation depends on the presence of molecular chaperones (HSP90) as was shown from in vitro experiments. But the processes that involve HSP90 in the degradation of CYP2E1 and the mechanisms of transfer of microsomal CYP2E1 to the proteasome system remain unknown. This paper investigates HSP90-dependent processes in mechanisms of CYP2El degradation and the possible role of ethanol in them.
With the help of computational methods we have shown that CYP2E1 can interact with HSP90 resulting in dissociation of CYP2E1 from membrane and formation of the CYP2E1-HSP90 complex for its further transfer to the proteasome for degradation. The twofold increase of both CYP2E1 and HSP90 in the mouse liver under the constant alcohol administration was shown using WB methods. Also, as was shown in silico, ethanol molecule, while binding to the CYP2E1 active site, prevents its interaction with HSP90, thus resulting in accumulation of CYP2E1 in cell.
Cytoplasmic HSP90 and membrane-bound CYP2E1 may directly interact with each other as partner proteins, leading to the dissociation of the CYP2E1 from the membrane. This makes it possible to transfer microsomal CYP2E1 in complex with HSP90 to the proteasome for proteolysis. The ethanol molecule inhibits the interaction of HSP90 with CYP2E1 leading to the suppression of its proteasome degradation, thus increasing level of this protein in the cell. Other substrates of CYP2E1 should increase level of this protein in the same way. This may be one of the mechanisms of substrate-dependent regulation of the CYP2E1 expression in the cell.
As the main role of microsomal CYP2E1 is detoxification of xenobiotics (exogenous low weight compounds) the highest level of its constitutive expression is found in liver and kidneys, organs that mostly utilize and excrete harmful substances off the organism[1, 2]. There are 160 hydrophobic substances of exogenous origin known as substrates for CYP2E1 (http://cpd.ibmh.msk.su/). Most of them are environmental pollutants (industrial wastes, fertilizers and solvents), components of food additives, drugs and cosmetics. CYP2E1 on one hand contributes in withdrawal of xenobiotics by metabolizing them in liver (thus taking active part in adaptation of organism to the adverse environmental factors). On the other hand CYP2E1, while metabolizing, may activate its substrates into cytotoxins, which causes different cell damages. Substrates of CYP2E1 induce its protein expression level but the mechanisms of such induction steel need to be investigated. Such increase in CYP2E1 protein level causes homeostasis misbalance in cell. Thus it is shown that introduction of ethanol (one of the most widely used CYP2E1 substrate) into the animals causes intensification of peroxidation processes and depletion of hepatocyte antioxidant system. Activation of peroxidation processes may be caused by the ethanol-dependent induction of CYP2E1, which can generate oxygen radicals during its catalytic cycle. As a result an oxidation stress usually develops in cell. Herewith ethanol does not only stimulate CYP2E1-dependent peroxidation, but serves as a source of free radicals itself (during the CYP2E1-dependent oxidation of ethanol a 1-hydroxyethyl radical is being formed[3, 5]). Posttranslational stabilization of protein molecule with substrates is one of the main mechanisms of regulation of CYP2E1 expression level in cell. It is believed that substrates while in the active site of enzyme, change and stabilize protein structure, thus preventing its fast degradation with proteasomes[6–8]. The mechanisms of such substrates-dependent CYP2E1 protein stabilization mostly are staying uninvestigated. The processes of transfer of microsomal CYP2E1 to the proteasome (where its degradation takes place) also need to be investigated. It is thought that cytoplasm heat shock proteins, in particular HSP90, actively participate in these processes[9–11]. This work is devoted to investigating the role of HSP90 in the degradation of microsomal CYP2E1 and studying the influence of ethanol on these processes.
Results and discussion
The main characteristics of HSP90 complexes with CYP2E1 and CYP2E1-ethanol
Binding energy (−ΔGGibbs, kJ/mole)
Сontact area (Å2)
Resides of CYP2E1 involved in contact with HSP90
L32, F37, P38, P40, I41, Y71, S74, Q75, R76, D102, P104, H107, A108, H109, R110, D111, R112, G119, P120, T121, R198, Y218, P222, S223, L225, H226, I236, H232, R233, K237, A240, E241, K243, E244, Y245, S247, E248, K251, A280, E281, M286, D287, T290, V291, R374, D375, L382, K385, G386 (49)
N135, Y136, G139, K140, Q141, G142, E144, S145, Q148, R149, H152, F153, E156, R159, K160, Q162, K187, S336, R337, I338, A340, P491 (22)
The main domains and functionally active sites in the structure of CYP2E1
Localization (residue number from N-term)
Active site and its channel
41-57, 70–79, 113–119, 202–218, 298–305, 387–396, 467-471
Comparative characteristics of the CYP2E1 and CYP2E1-ethanol spatial structures
Gyration radius (Å)
Polar surface area (Å2)
Surface area (Å2)
RMSD – Cα (Å)
RMSD – all atoms (Å)
Cytoplasmic chaperone HSP90 and membrane-bound CYP2E1, as partner proteins may directly interact with each other, leading to the dissociation of the CYP2E1 from the membrane. This makes it possible to transfer microsomal CYP2E1 to the proteasome for proteolysis. The ethanol molecule inhibits the interaction of HSP90 with CYP2E1 leading to the increased content of this protein in the cell. We assume that other substrates of CYP2E1 should increase its content in the same way. This may be one of the mechanisms of substrate-dependent regulation of the CYP2E1 level in the cell.
The Institute of Molecular Biology and Genetics Bioethics Committee (head Prof. Dr. Lukash LL) according to the "Recommendations of the ethics committees that carry out the examination of biomedical research" (WHO, 2000), the order of the AMS of Ukraine № 50 from 06.07.2001 "On establishment of committees of medical ethics in research institutions of Academy of Sciences of Ukraine" and the "General ethical principles of animal experiments" (1 National Congress on Bioethics, Kyiv, 2001) approved procedures involved in the breeding and handling of animals (protocol № 10 dated 24.09.2008).
We used BALB/c mouse males, 3.5 months old, with the average weight of 30 g from vivarium of the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine (Kyiv) in our work. Mice were kept at standard conditions with inverted diurnal light regime (8 night hours), at temperature 18-20°C and on standard diet. Animals were divided into two groups: experimental and control (intact). The experimental mice were fed with 10% ethanol in water. On day 35 mice of both groups were decapitated under the light ether anaesthesia.
Western blotting and protein measurement
The relative level of CYP2E1 and HSP90 protein in the liver was determined by Western blot analysis. The liver tissues were homogenized in ice-cold RIPA-buffer (1:3) containing 20 mM TrisHCl, pH 7.5; 0.15 M NaCl; 1 mM EDTA; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS and 1 mM protease inhibitor PMSF was added to liver tissue frozen in liquid nitrogen. The extraction of proteins was carried out on ice for 45 minutes. Centrifugation was then carried out at 11 000 g. for 20 min. at + 4°C. The protein concentration in the supernatant was determined. Proteins from the liver of each mouse (50 μg per line) were separated using 12% polyacrylamide gel with 0.1% SDS. The semi-dry electrotransfer of proteins to the nitrocellulose membranes was held at 200 mA for 40 minutes. Western blot analysis was held in the following way: nitrocellulose membranes were pre-incubated in 4% nonfat milk (Sigma, USA) in PBST-buffer, and then treated with polyclonal anti-CYP2E1 or anti-HSP90 antibodies (obtained in Rabbit in our institute earlier) diluted in 4% nonfat milk for 1 hour at room temperature. Membranes were incubated with peroxidase-conjugated secondary anti-Rabbit antibodies (Sigma, USA) for 1 hour. The GAPD (used as control) was identified using anti-GAPD antibodies (obtained in Rabbit in our institute earlier). The treatment of membranes with secondary antibodies was followed by chemiluminescence detection according to manufacturers’ instructions (Pierce). Membranes were exposed to autoradiography film (Agfa, Belgium) for 0,5 to 1 minutes. Digital images of immunoblots were analyzed using densitometric scanning analysis program Scion image 3.53.346.0 (http://www.scioncorp.com/). The level of CYP2E1 and HSP90 protein was calculated as the ratio of protein values to GAPD and presented as relative units.
Statistical analysis was performed using STATISTICA 7.0 (StatSoft, Inc. 2004, USA). Results are presented as mean ± standard deviation (SD). Differences between groups were identified using an unpaired two-tailed distribution of Student’s T test. P values < 0.05 were considered to be statistically significant.
The authors thank to the head of the laboratory of molecular mechanisms of autoimmune processes of cell signalling systems in the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, LL Sidorik for kindly providing anti-HSP90 and anti-GAPD antibodies.
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