Selective protection of curcumin against carbon tetrachloride-induced inactivation of hepatic cytochrome P450 isozymes in rats

We investigated the effects of curcumin, a major antioxidant constituent of turmeric, on hepatic cytochrome P450 (CYP) activity in rats. Wistar rats received curcumin-containing diets (0.05, 0.5 and 5 g/kg diet) with or without injection of carbon tetrachloride (CCl(4)). The hepatic CYP content and activities of six CYP isozymes remained unchanged by curcumin treatment, except for the group treated with the extremely high dose (5 g/kg). This suggested that daily dose of curcumin does not cause CYP-mediated interaction with co-administered drugs. Chronic CCl(4) injection drastically decreased CYP activity, especially CYP2E1 activity, which is involved in the bioactivation of CCl(4), thereby producing reactive free radicals. Treatment with curcumin at 0.5 g/kg alleviated the CCl(4)-induced inactivation of CYPs 1A, 2B, 2C and 3A isozymes, except for CYP2E1. The lack of effect of curcumin on CYP2E1 damage might be related to suicidal radical production by CYP2E1 on the same enzyme. It is speculated that curcumin inhibited CCl(4)-induced secondary hepatic CYPs damage through its antioxidant properties. Our results demonstrated that CYP isozyme inactivation in rat liver caused by CCl(4) was inhibited by curcumin. Dietary intake of curcumin may protect against CCl(4)-induced hepatic CYP inactivation via its antioxidant properties, without inducing hepatic CYPs.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPH-dependent microsomal oxidative reactions: free radical scavenging ability of isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 > acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.     

Differential Role of CYP2E1 Binders and Isoniazid on CYP2E1 Protein Modification in NADPH-dependent Microsomal Oxidative Reactions: Free Radical Scavenging Ability of Isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl 4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole=imidazole=pyridine &#100 acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl 4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.     

The roles of ER stress and P450 2E1 in CCl(4)-induced steatosis

The role of ER stress on hepatic steatosis was investigated in a rat model. We injected CCl(4) into rats and found that CCl(4) could induce hepatic lipid accumulation, confirmed by Oil Red O staining and by measurement of triglyceride and cholesterol. The expression of ApoB, an apolipoprotein, was decreased in plasma and increased in the liver of CCl(4)-treated animals. The ER stress response was also significantly increased by CCl(4). P450 2E1 expression and activity were increased through interactions of P450 2E1 with NADPH-dependent P450 reductase (NPR) under CCl(4)-treated conditions. In HepG2 cells, intracellular lipid accumulation and its signaling were comparable to in vivo results. In order to elucidate the effect of the ER stress response itself, tunicamycin, an N-acetyl-glycosylation inhibitor, was injected into rats, followed by Oil Red O staining, lipid/triglyceride/cholesterol accumulation analysis, and examination of ApoB expression. Additionally, the ER stress response and upregulation of P450 2E1 were also confirmed in the tunicamycin-treated rats. All of the responses were similar to those seen with CCl(4). The P450 2E1 inhibitor diallyl sulphide (DAS), N-acetylcysteine (NAC), and reduced glutathione (GSH) antioxidants also regulated processes, including ApoB expression and lipid accumulation in CCl(4)-treated animals. In the presence of tunicamycin, DAS or NAC/GSH regulated all of the pathological phenomena with the exception of the ER stress response. In summary, CCl(4) induces liver steatosis, a process involving ER stress-induced P450 2E1 activation and ROS production.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum

To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention “receptor” since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.     

Mitochondria-targeted Cytochrome P450 2E1 Induces Oxidative Damage and Augments Alcohol-mediated Oxidative Stress

The ethanol-inducible cytochrome P450 2E1 (CYP2E1) is also induced under different pathological and physiological conditions. Studies including ours have shown that CYP2E1 is bimodally targeted to both the endoplasmic reticulum (microsomes) (mc CYP2E1) and mitochondria (mt CYP2E1). In this study we investigated the role of mtCYP2E1 in ethanol-mediated oxidative stress in stable cell lines expressing predominantly mt CYP2E1 or mc CYP2E1. The ER+ mutation (A2L, A9L), which increases the affinity of the nascent protein for binding to the signal recognition particle, preferentially targets CYP2E1 to the endoplasmic reticulum. The Mt+ (L17G) and Mt++ (I8R, L11R, L17R) mutant proteins, showing progressively lower affinity for signal recognition particle binding, were targeted to mitochondria at correspondingly higher levels. The rate of GSH depletion, used as a measure of oxidative stress, was higher in cells expressing Mt++ and Mt+ proteins as compared with cells expressing ER+ protein. In addition, the cellular level of F₂-isoprostanes, a direct indicator of oxidative stress, was increased markedly in Mt++ cells after ethanol treatment. Notably, expression of Mt++ CYP2E1 protein in yeast cells caused more severe mitochondrial DNA damage and respiratory deficiency than the wild type or ER+ proteins as tested by the inability of cells to grow on glycerol or ethanol. Additionally, liver mitochondria from ethanol-fed rats containing high mt CYP2E1 showed higher levels of F₂-isoprostane production. These results strongly suggest that mt CYP2E1 induces oxidative stress and augments alcohol-mediated cell/tissue injury.     

雌激素受体β转录激活系统的构建

目的：构建雌激素受体β（ERβ）的转录激活系统。方法：以pcDNA3-ERβ质粒为模板,利用PCR技术扩增ERβ全长及转录激活结构域1（AF1）、DNA结合结构域（DBD）、转录激活结构域2（AF2）等不同长度的基因片段,分别插入pGAL载体中,构建重组质粒,转染293T细胞,利用免疫杂交方法鉴定其表达情况,用萤光素酶报告基因（Gal4-LUC）检测转录活性。结果：构建了ERβ全长及不同功能区片段编码基因的重组质粒,转染293T细胞后检测到相应蛋白的表达;在活性实验中,雌激素（E2）诱导下pGAL-ERβ使Gal4-LUC活性升高约17倍,pGAL-ERβAF1在有无E2诱导下均能使Gal4-LUC活性升高2倍,pGAL-ERβAF2在E2诱导下使Gal4-LUC活性升高约7倍,pGAL-ERβDBD对Gal4-LUC活性没有明显作用。结论：ERβ的转录激活系统构建成功。     

Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats

Extracts of Phellinus linteus (EPB), grown on germinated brown rice, protected rats from liver injury induced by carbon tetrachloride (CCl4). Peroxidation products in the liver were decreased to 10% by EPB. Catalase and superoxide dismutase activities were significantly decreased to 55% and 39% by CCl4 administration, but EPB blocked this effect, resulting in enzyme activities at control levels. Expression of cytochromeP450 2E1 (CYP2E1) protein was significantly decreased to 88% in CCl4-treated rats but remained at control levels when EPB was also administered. EPB did not affect the altered fatty acid composition induced by CCl4. The hepatoprotective effect of EPB may be mediated by EPB's prevention of CCl4-induced CYP2E1 degradation.     

Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins

Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway.     

Multisite phosphorylation of human liver cytochrome P450 3A4 enhances Its gp78- and CHIP-mediated ubiquitination: a pivotal role of its Ser-478 residue in the gp78-catalyzed reaction

CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation.     

Co-planar 3,3',4,4',5-pentachlorinated biphenyl and non-co-planar 2,2',4,6,6'-pentachlorinated biphenyl differentially induce recruitment of oestrogen receptor alpha to aryl hydrocarbon receptor target genes

In the present study we examined the ability of 3,3',4,4',5-pentachlorinated biphenyl [PCB126 (polychlorinated biphenyl 126)], a prototypical AHR (aryl hydrocarbon receptor) agonist, and 2,2',4,6,6'-PCB (PCB104), which does not activate AHR, to induce the recruitment of ERalpha (oestrogen receptor alpha) to CYP1A1 (cytochrome P4501A1 gene) and CYP1B1 promoters in T-47D human breast cancer cells and other cell lines. PCB126 treatment strongly induced CYP1A1 and CYP1B1 mRNA expression that was unaffected by co-treatment with E2 (17beta-oestradiol). PCB104 failed to induce changes in either CYP1A1 or CYP1B1 expression levels. ChIP (chromatin immunoprecipitation) assays show that PCB126, but not PCB104, increased the promoter occupancy by ERalpha to CYP1A1 and CYP1B1 promoters. Co-treatment with PCB126+E2 significantly enhanced the promoter occupancy of ERalpha at CYP1A1, whereas co-treatment with PCB126+4-hydroxytamoxifen or ICI182,780 did not. Competitive binding studies revealed that neither PCB126 nor PCB104 bound to ERalpha. HEK-293 cells (human embryonic kidney-293 cells) stably transfected with ERalpha showed significantly higher PCB126-induced CYP1A1 expression compared with empty vector controls, whereas no increase was observed in cells stably transfected with ERalpha lacking its N-terminal AF1 (activation function-1) domain (ERalphaDeltaAF1). Despite no increase in AHR-mediated gene expression, ChIP assays revealed that ERalphaDeltaAF1 was present at CYP1A1 and CYP1B1 promoters. HC11 mouse mammary cells stably expressing shRNA (small-hairpin RNA) against ERalpha showed an 8-fold reduction in PCB126-dependent Cyp1a1 expression. Our results provide further evidence that AHR agonists induce ERalpha promoter occupancy at AHR target genes through indirect activation of ERalpha, and support a role for ERalpha in AHR transactivation.     

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.     

Regulation of cytochrome P450 2E1 by heat shock protein 90-dependent stabilization and CHIP-dependent proteasomal degradation

Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any member of this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly by various substrates, such as ethanol and CCl(4). CYP2E1 is degraded by the ubiquitin-proteasome pathway, and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balance between protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulated by the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stably expressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited by the proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressing the truncated CYP2E1(Delta3-29) yields coadsorption of CYP2E1(Delta3-29). Cotransfection of HEK cells with both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(Delta3-29) degradation, and CYP2E1(Delta3-29) co-immunoadsorbs with myc-CHIP from cytosol of cotransfected cells. Purified, bacterially expressed CYP2E1(Delta3-29) is ubiquitylated in a CHIP-dependent manner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2, and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated by the hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates, such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundly alter enzyme turnover.     

Protective effect of cornuside against carbon tetrachloride-induced acute hepatic injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl?)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl?. Sixteen h after CCl? treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl?-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl? treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl?-treated rats. Our data indicate that cornuside protects the liver from CCl?-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Caspase activation by adenovirus e4orf4 protein is cell line specific and Is mediated by the death receptor pathway

Adenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochrome c is released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1, 3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.