QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online April 1, 2009
doi: 10.1242/10.1242/jcs.038430

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kim, H.-R. Articles by Chae, H.-J. Search for Related Content PubMed PubMed Citation Articles by Kim, H.-R. Articles by Chae, H.-J. Social Bookmarking                         What's this?

Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1

Hyung-Ryong Kim^1,*, Geum-Hwa Lee^2,*, Eun Yi Cho³, Soo-Wan Chae^2,4, Taeho Ahn^5, and Han-Jung Chae^2,4,

¹ Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk, 570-749, Republic of Korea
² Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, 560-182, Republic of Korea
³ School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
⁴ Clinical Trial Center, Chonbuk Hospital, Jeonju, 561-712, Republic of Korea
⁵ Department of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effect of BI1 on ER stress-initiated ROS accumulation. (A) Neo-(neomycin-resistant) and BI1-overexpressing HepG2 cells (namely Neo and BI1) were lysed, and the lysates were then immunoblotted with anti-HA (inset). The Neo and BI1 cells were treated with 5 µM thapsigargin or 5 µg/ml tunicamycin for 20 hours. DCFDA was loaded into the cells, and the fluorescence was measured. (B) Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin for 20 hours. The microsomal fractions were then isolated from the Neo and BI1 cells, and the ER lipid peroxidation was measured. The data indicate the mean±s.e.m. (n=4). *P<0.05, significantly different from control-Neo cells; #P<0.05, significantly different from 5 µM thapsigargin-treated Neo cells; $P<0.05, significantly different from 5 µg/ml tunicamycin-treated Neo cells. CNTL, control; THAP, thapsigargin; TUN, tunicamycin.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Effect of BI1 on the expression of P450 2E1. (A) Neo and BI1 cells were immunoblotted with anti-P450 2E1 and β-actin antibodies. (B) Neo and BI1 cells were treated with 5 µM thapsigargin or 5 µg/ml tunicamycin for 0, 2, 4, 6, 8 and 12 hours. Cell lysates were immunoblotted with anti-P450 2E1 and β-actin antibodies. The expression of P450 2E1 was normalized to that of β-actin as a relative ratio. The data indicate the mean±s.e.m. (n=7). *P<0.05, significantly different from the amount of P450 2E1 expressed in the Neo cells without any treatment; #P<0.05, significantly different from the amount of P450 2E1 expressed in the Neo cells under each of the indicated conditions.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of a P450 2E1 inhibitor on ER stress-induced ROS accumulation. (A) Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin in the presence or absence of 1 mM 4-MP for 20 hours. The microsomal fractions were then isolated from the Neo and BI1 cells, and the ER lipid peroxidation was measured. *P<0.05, significantly different from the amount of malondialdehyde in the Neo cells; #P<0.05, significantly different from the amount of malondialdehyde in thapsigargin-treated Neo cells (left); #P<0.05, significantly different from the amount of malondialdehyde in tunicamycin-treated Neo cells (right). (B) Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin in the presence or absence of 1 mM 4-MP for 20 hours. DCFDA was loaded into the cells, and the fluorescence was measured. (C) Neo and BI1 cells were transfected with non-specific (NS) and P450 2E1 siRNA. Sixteen hours later, the expression of P450 2E1, BI1 and β-actin was analyzed by western blot (top). NS or P450 2E1 siRNA-transfected Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin for 20 hours. DCFDA was loaded into the cells, and the fluorescence was measured (bottom). MDA, malondialdehyde; CNTL, control; THAP, thapsigargin; TUN, tunicamycin; 4MP, 4-methylpyrazole.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of P450 2E1 inhibitor on ER stress-induced cell death. (A) Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin in the presence or absence of the indicated concentrations of 4-MP for 48 hours, and cell death was measured as described in the Materials and Methods. The data shown indicate the mean±s.e.m. (n=6). (B) Non-specific or P450 2E1 siRNA-transfected Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin for 48 hours, and cell death was measured as described in the Materials and Methods. The data shown indicate the mean±s.e.m. (n=9). *P<0.05, significantly different from the viability in thapsigargin-treated Neo cells (NSsiRNA-transfected); #P<0.05, in tunicamycin-treated Neo cells (NSsiRNA-transfected). THAP, thapsigargin; TUN, tunicamycin; 2E1 siRNA, P450 2E1 siRNA; NS (siRNA), non-specific siRNA. (C) Neo and BI1 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin in the presence or absence of 1 mM NAC or GSH for 48 hours, and cell death was measured as described in the Materials and Methods. The data shown indicate the mean±s.e.m. (n=4).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of BI1 on the production of H2O2 by P450 2E1. The amount of H2O2 produced was measured spectrophotometrically, with increasing ratios of BI1/NPR (A) or with increasing ratios of BI1/total amount of proteins in microsomes (w/w) (B). (C) Immuno-inhibition of BI1 induced H2O2 production was also measured with increasing ratios of antibodies (against the C-terminal region of BI1) to BI1 (w/w) at a BI1/NPR molar ratio of 2.0. During all experiments, the concentration of NPR was fixed at 0.8 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Emission spectra of Amplex Red reagent. The amount of H2O2 generated from a reconstitution system containing P450 2E1 was measured using an Amplex Red assay kit according to the instructions of the manufacturer. (A) The emission spectra were recorded in the wavelength range of 560-650 nm, under an excitation wavelength of 545 nm. Lines a, b, c and d represent each of the emission spectra, which had BI1/NPR ratios of 0, 0.5, 1.0 and 1.5, respectively. (B) The max (fluorescence intensity at 590 nm) was plotted with increasing BI1 (or BI1 deletion mutants)/NPR ratios. WT, MT (8) and MT (16) represent native BI1 and deletion mutants missing 8 and 16 amino acids at the C terminus, respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 7. The binding affinity between BI1 and NPR. (A) HEK293T cells were co-transfected with HA-BI1 and GFP-NPR or with HA-C-BI1 (C-terminal deleted mutant) and GFP-NPR. (B) Cells were independently co-transfected with HA-BI1 and GFP-P4502E1 or with HA-C-BI1 and GFP-P450 2E1. Cell lysates were immunoprecipitated with anti-HA-antibody and immunoblotted with anti-GFP antibody. Using the reverse process, cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-HA antibody. In parallel, the cell lysates were immunoprecipitated with IgG. (C) The lysates were then immunoblotted with either anti-HA or anti-GFP antibody. IAEDANS-labeled BI1 and NRP were incorporated into liposomes, and the emission intensity at 490 nm was measured under an excitation wavelength of 290 nm to activate Trp fluorescence with increasing NPR/BI1 or BI1/NPR ratios. (D) IAEDANS-labeled BI1 and P450 2E1 FRET assay was performed as for the assay of BI1 and NPR. In the normalized figure (inset), the fluorescence intensity at the protein ratio of 2.0 (in the case of FRET assay between BI1 and NPR) or 8.0 (between BI1 and P450 2E1) was set to 100%, and other data points were plotted relative to this point. Data shown indicate the mean±s.e.m. of five independent samples.

View larger version (33K): [in this window] [in a new window]   Fig. 8. The effect of BI1 on the association of NPR and P450 2E1. Neo, BI1 and C-BI1 cells were immunoprecipitated with anti-NPR or anti-P450 2E1 antibodies or IgG. The lysates were then immunoblotted with either anti-NPR or anti-P450 2E1 antibodies.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Catalytic activities of P450 2E1. (A) The activities were measured with decreasing amounts of NPR incorporated into assay samples. Control represents the normalized catalytic activities of P450 2E1 at each indicated ratio of NPR/P450 in the absence of BI1. Indicated numbers represent the ratio of NPR/P450 in the presence of equal molar concentrations of BI1 and NPR. (B) The enzyme assays were repeated with increasing amounts of BI1 in the presence of fixed concentrations of P450 2E1 and NPR (the ratio of NPR/P450 2E1 was 0.5). The y-axes represent the relative ratios of BI1 to P450 2E1. CNTL, control.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter