Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.     

Evidence for cytochrome P450 2E1 catalytic activity and expression in rat blood lymphocytes

Studies initiated to characterize cytochrome P450 2E1(CYP2E1) in freshly isolated rat blood lymphocytes revealed significant mRNA of CYP2E1 in control blood lymphocytes. RT-PCR studies have shown that as observed in liver, acute treatment of ethanol (single oral dose of 0.8 ml/kg b.wt, i.p), resulted in increase in the mRNA expression of CYP2E1 in freshly isolated rat blood lymphocytes. Western blotting studies using polyclonal antibody raised against rat liver CYP2E1 demonstrated significant immunoreactivity, comigrating with the liver isoenzyme, in freshly isolated control rat blood lymphocytes. Similar to that seen in liver, pretreatment of ethanol was found to produce an increase in the CYP2E1 isoenzyme in the blood lymphocytes. Blood lymphocytes were also found to catalyze the CYP dependent N-demethylation of N-nitrosodimethylamine (NDMA), which like in liver increased 2-3 fold following pretreatment of rats with known CYP2E1 inducers. Kinetic studies have further shown significant increase in the apparent Vmax and the affinity towards the substrate in rat blood lymphocytes indicating that as observed in liver, the increase in mRNA and protein expression following exposure to CYP2E1 inducers is associated with the increased catalytic activity of CYP2E1 in freshly isolated rat blood lymphocytes. The data indicating similarities of the blood lymphocyte CYP2E1 with the liver enzyme suggest that lymphocyte CYP2E1 levels in freshly isolated rat blood lymphocytes could be used to monitor tissue enzyme levels.     

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP‐mediated microtubule anchoring

Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule‐anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule‐organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule‐organizing centers and impaired microtubule‐based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome‐targeted PACT‐SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients’ cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.     

Effect of natural phenols on the catalytic activity of cytochrome P450 2E1

The effect of protocatechuic acid, tannic acid and trans-resveratrol on the activity of p-nitrophenol hydroxylase (PNPH), an enzymatic marker of CYP2E1, was examined in liver microsomes from acetone induced mice. trans-Resveratrol was found to be the most potent inhibitor (IC(50) = 18.5 +/- 0.4 microM) of PNPH, while protocatechuic acid had no effect on the enzyme activity. Tannic acid with IC(50) = 29.6 +/- 3.3 microM showed mixed- and trans-resveratrol competitive inhibition kinetics (K(i) = 1 microM and 2.1 microM, respectively). Moreover, trans-resveratrol produced a NADPH-dependent loss of PNPH activity, suggesting mechanism-based CYP2E1 inactivation. These results indicate that trans-resveratrol and tannic acid may modulate cytochrome P450 2E1 and influence the metabolic activation of xenobiotics mediated by this P450 isoform.     

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.     

Heterologous expression of mouse cytochrome P450 2e1 in V79 cells: construction and characterisation of the cell line and comparison with V79 cell lines stably expressing rat P450 2E1 and human P450 2E1

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies.     

Lysosomal Ca2+ release channel TRPML1 regulates lysosome size by promoting mTORC1 activity

Lysosomal Ca²⁺ release channel TRPML1 has been suggested to regulate lysosome size by activating calmodulin (CaM). To further understand how TRPML1 and CaM regulate lysosome size, in this study, we report that inhibiting mTORC1 causes enlarged lysosomes, and the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. We also show that lysosome vacuolation induced by inhibiting TRPML1 is corrected by mTORC1 upregulation, and the facilitating effect of TRPML1 on the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. In the meantime, lysosome vacuolation induced by inhibiting CaM is corrected by mTORC1 upregulation, and mTORC1 overexpression corrects the inhibitory effect of CaM antagonist on the recovery of enlarged lysosomes. Conversely, the vacuolation induced by suppressing mTORC1 is not corrected by upregulating CaM. These data suggest that mTORC1 functions downstream of TRPML1 and CaM to regulate lysosome size. Together with our recent finding showing that TRPML1, CaM and mTORC1 form a macromolecular complex to control mTORC1 activity, we suggest that TRPML1 and CaM control lysosome fission through regulating mTORC1, identifying an mTORC1-dependent molecular mechanism for lysosomal membrane fission.     

Cytochrome P450 2E1 and hyperglycemia-induced liver injury

Cytochrome P450 2E1 (CYP2E1), a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury. The review deals with the changes in various cellular pathways in liver linked with the changes in regulation of CYP2E1 under hyperglycemic conditions. Some of the hepatic abnormalities associated with hyperglycemia-mediated induction of CYP2E1 include increased oxidative stress, changes in mitochondrial structure and function, apoptosis, nitrosative stress, and increased ketone body accumulation. Thus, changes in regulation of CYP2E1 are associated with the injurious effects of hyperglycemia in liver.     

Cytochromes P450 2C1/2 and P450 2E1 are retained in the endoplasmic reticulum membrane by different mechanisms

Cytochrome P450 (P450) 2C1/2 contains redundant endoplasmic reticulum (ER) retention signals and is excluded from the recycling pathway. Other P450s, such as P450 2E1, have been detected in the plasma membrane and Golgi apparatus. To examine whether the mechanisms of ER retention might differ for P450 2C1/2 and P450 2E1, chimeras of green flourescent protein and the full-length proteins, N-terminal signal/anchor sequences, or the cytoplasmic catalytic domains from these proteins have been expressed in COS1 cells. Chimeras with either the N-terminal signal/anchor sequence or the cytoplasmic domain of P450 2C1/2 were retained in the ER and the distribution was not altered by treatment with nocodazole. A chimera with full-length P450 2E1 was located in the ER, but in contrast to P450 2C1/2, treatment with nocodazole resulted in redistribution to a vesicular pattern, which suggested that this protein was retained in the ER by a retrieval mechanism. In support of this possibility, the P450 2E1 chimera, but not the P450 2C1/2 chimera, was included in transport vesicles generated in an in vitro budding assay. A chimera with only the N-terminal signal/anchor sequence of P450 2E1 fused to green fluorescent protein was located in the ER and nocodazole treatment altered its distribution, whereas a chimera with only the cytoplasmic domain of P450 2E1 was not efficiently retained in the ER and accumulated primarily in the Golgi region. These results demonstrate that the mechanisms for retention in the ER of two closely related members of the P450 superfamily are different and that the N-terminal signal/anchor sequence contains the dominant retention signal.     

Computer modeling of cytochrome P450 2E1 three-dimensional structure

A computer model of human cytochrome P450 2E1 (CYP2E1) three-dimensional structure and active site was constructed based on homology with crystallographic coordinates of CYP2C5 and CYP2C9. A high degree of secondary structure homology for human, mouse, rat and rabbit CYP2E1 was demonstrated. The location of heme and the supporting alpha-helices was established. CYP2E1, CYP2C5 and CYP2C9 active sites are distinguished by pocket size and their amino acid residues composition. Key amino acid residues forming the active site channel and substrate-binding cavity are presented. Active site surface area and volume for CYP2E1, CYP2C5 and CYP2C9 were calculated.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

Characterization of cytochrome P450 2E1 activity by the [14C]nitrosodimethylamine breath test

The objective of this study was to measure the rate of demethylation of nitrosodimethylamine in vivo in the rat and determine its value to assess CYP2E1 activity in intact animals. Nitrosodimethylamine labeled with 14C on both methyl groups was administered to rats and exhaled 14CO2 was collected during 2-3 h. The nitrosodimethylamine breath test was increased by inducers of CYP2E1, such as ethanol (+139%) and 4-methylpyrazole (+115%), and decreased by the inhibitor diallyl sulfide (-53%). In addition, the nitrosodimethylamine breath test was not changed significantly by inducers specific for other cytochrome P450 such as beta-naphthoflavone, dexamethasone, and phenobarbital. The specificity of the induction by 4-methylpyrazole and of the inhibition by diallyl sulfide for CYP2E1 was determined using the [14C]caffeine (CYP1A2), [14C]aminopyrine (CYP2C11), and [14C]erythromycin (CYP3A2) breath tests. 4-Methylpyrazole treatment caused a small increase of the caffeine (+33%) and aminopyrine (+9%) breath tests and no change of the erythromycin breath test. Diallyl sulfide treatment led to a small decrease of the caffeine breath test (-33%) and of the aminopyrine breath test (-13%) but a 23% increase of the erythromycin breath test. It is concluded that the [14C]nitrosodimethylamine breath test is useful to assess CYP2E1 activity in vivo in the rat.     

Selection of human cytochrome P450 1A2 mutants with enhanced catalytic activity for heterocyclic amine N-hydroxylation

Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens. Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products. Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation. Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with >10-fold increased MeIQ activation based on the E. coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k(cat)/K(m)) in steady-state N-hydroxylation assays done with isolated membrane fractions. SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines. The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H(2)O(2) formation. Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate. In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein.