Polycyclic Aromatic Hydrocarbon Binding Protein Research Articles

ATP Depletion Affects the Phosphorylation State, Ligand Binding, and Nuclear Transport of the 4 S Polycyclic Aromatic Hydrocarbon-binding Protein in Rat Hepatoma Cells

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. Phosphorylation and dephosphorylation have been suggested to influence the function of many cytosolic receptors and transcription factors. The ATP level within H4IIE rat hepatoma cells could be depleted by treatment with sodium azide or 2,4-dinitrophenol; restoration of the original ATP levels occurred with addition of glucose to the cell culture. ATP depletion reduced the phosphate content of the 4 S protein by approximately 25-30%, which lowered the binding of benzo[a]pyrene (B[a]P) to the 4 S protein by >60%. This effect could not be reversed by the addition of ATP to the binding reaction mixtures. Alkaline phosphatase treatment of the purified 4 S protein in a cell-free system also reduced the B[a]P binding to the protein. Cells treated with a protein phosphatase inhibitor, okadaic acid, and a protein kinase inhibitor, staurosporin, affected the B[a]P binding of the 4 S protein positively and negatively, respectively. These data suggested that phosphorylation is involved in the interaction of the 4 S protein with the PAH. The nuclear translocation of the predominantly cytosolic binding protein has been investigated after ligand binding. Western blots with the immunopurified 4 S PAH-binding protein from cytosolic and nuclear lysates showed significant differences in the distribution of the 4 S receptor between cytosolic and nuclear compartments in control and ATP-depleted cells. Ligand binding stimulated the movement of the receptor into the nucleus, which was completely blocked by reducing the intracellular ATP concentration. These findings provide new information on the role of ATP and phosphorylation on the interaction of B[a]P with 4 S PAH-binding protein and its nuclear translocation.

Glycine N-Methyltransferase Is a Mediator of Cytochrome P4501A1 Gene Expression

Cytochrome P4501A1, the isozyme most closely approximating aryl hydrocarbon hydroxylase activity under conditions of induction, is thought to be regulated by several trans-acting factors, including the 4S polycyclic aromatic hydrocarbon-binding protein; this protein has recently been identified as glycine N-methyltransferase (Raha et al. (1994) J. Biol. Chem. 269, 5750-5756). Previous studies had shown that partially purified liver preparations containing the 4S binding protein interacted with 5′-flanking regions of the cytochrome P4501A1 gene. Consequently, the ability of the 4S binding protein to serve as a mediator in the regulation of the cytochrome P4501A1 gene was investigated further. Introduction of an antisense 24-mer oligonucleotide to glycine N-methyltransferase cDNA into rat hepatoma H4IIE cells by lipofectin resulted in a 60% reduction in the benzo(a)pyrene-mediated induction of ethoxyresorufin-O-deethylase activity and protein over the sense and scrambled antisense oligonucleotide controls. In addition, the antisense oligonucleotide caused a marked reduction in the steady-state level of cytochrome P4501A1 mRNA; no such effect was observed with the sense oligonucleotide. Introduction of GNMT polyclonal antibodies into H4IIE cells by a streptolysin-O permeabilization technique markedly reduced both benzo(a)pyrene-binding and benzo(a)pyrene-induced ethoxyresorufin-O-deethylase activities, but had no effect on 2,3,7,8-tetrachlorodibenzo-p-dioxin induction. Collectively, these findings suggest that, in addition to the Ah (dioxin) receptor, glycine N-methyltransferase appears to be both a polycyclic aromatic hydrocarbon-binding protein and a mediator of the induction of the cytochrome P4501A1 gene by polycyclic hydrocarbons such as benzo(a)pyrene.

Rat liver cytosolic 4 S polycyclic aromatic hydrocarbon-binding protein is glycine N-methyltransferase.

In the rat, cytochrome P-450IA1 gene expression, which is most closely associated with aryl hydrocarbon hydroxylase activity, is thought to be regulated by several trans-acting factors, including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. This protein has been purified to homogeneity from rat liver using ion exchange, gel permeation, hydrophobic interaction, and affinity chromatographies. Partial sequencing of the 33-kDa band indicated its identity as glycine N-methyltransferase (GNMT). Polyclonal antibodies to GNMT immunoprecipitated PAH-binding activity from rat liver cytosol. Methyltransferase and PAH-binding activities copurified during the course of protein purification. GNMT protein and PAH binding activity were colocalized in various cytosolic fractions. These data all indicate that the 4 S PAH-binding protein and GNMT are one and the same protein or very similar proteins. Western blot analyses yielded a positive signal under denaturing (33 kDa) and nondenaturing (150 kDa, tetramer) conditions; the PAH-binding protein also was an oligomer. GNMT was detected by immunohistochemistry in nuclei from H4IIE rat hepatoma cells and rat liver. The localization of GNMT in liver nuclei is in accordance with a role in modulating cytochrome P-450IA1 gene expression.

Characterization of the 4 S polycyclic aromatic hydrocarbon-binding protein in human liver and cells

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a soluble protein that binds PAHs with high affinity in mouse, rat, and rabbit. Until now, this protein had not been detected in human placenta or human cells in culture by cytosol labeling and gradient centrifugation assay. Thanks to a preliminary fractionation of cytosol by sedimentation on sucrose gradient or/ and gel permeation chromatography, we found that PBP was present in liver, MCF-7 cell line, and hepatocytes of human. To accurately quantitate PBP binding and determine specific binding parameters, a reduction in the amount of charcoal used to adsorb nonspecifically bound benzo[ a]pyrene was required. By saturation analysis, the concentration of specific binding sites for [ 3H]BP in PBP fraction from human liver was 4.6 pmol/mg of protein compared with 14.7 ± 1.4 pmol/mg in the same fraction from DBA/2J mouse liver. Kinetic studies analyzed by Scatchard and Woolf plots indicate that human liver and MCF-7 cells contain a low-affinity PBP form: the K d derived from Woolf plot analysis were 14.2 ± 1.4 and 26.2 ± 1.8 n m, respectively. DBA/2J mouse possesses a higher-affinity PBP form, the same analysis indicating a K d of 6.1 ± 0.3 n m. These data demonstrate that, by comparison to the mouse liver, a lower-affinity form of PBP is present in reduced concentration in human liver, explaining the impossibility of detecting this protein by sedimentation of human cytosol in sucrose gradient.

The level of aryl hydrocarbon (Ah) receptor and of 4S polycyclic aromatic hydrocarbon (PAH) binding protein in diploid and polyploid hepatocytes of 2-acetylaminofluorene-treated rats.

Sequential treatment of partially hepatectomized male Wistar rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) induces the emergence of diploid hepatocyte populations. These carcinogen-induced hepatocytes are thought to include the precursor cells of liver carcinomas that arise later in this treatment protocol. The growth of the diploid hepatocytes is promoted by AAF and it has been suggested that the action of the arylamine may be receptor-mediated. AAF has been shown to bind specifically to the aryl hydrocarbon (Ah) receptor and the so-called 4S polycyclic aromatic hydrocarbon (PAH) binding protein. The present study addresses the question of whether the concentrations of the two binding proteins differ in diploid and polyploid hepatocytes from DEN/AAF-treated rats. Hepatocytes from carcinogen-treated rats were isolated and diploid, and tetraploid hepatocytes separated by means of centrifugal elutriation. Whereas Ah receptor concentrations in diploid hepatocytes were insignificantly lower (21.8 +/- 5.9 versus 29.2 +/- 6.6 fmol/mg cytosolic protein; n = 4; P = 0.1), levels of the 4S PAH binding protein in diploid hepatocytes were twice as high as in tetraploid hepatocytes (252.3 +/- 93.6 versus 124.0 +/- 18.5 fmol/mg cytosolic protein; n = 4; P = 0.04). We conclude from our results that the differences in growth control in polyploid and carcinogen-induced diploid hepatocytes are not associated with changes in the levels of the Ah receptor. The role of the 4S PAH binding protein in the process of hepatocarcinogenesis remains to be established.

Presence of the 4 S polycyclic hydrocarbon-binding protein in H4-II-E cells

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein had been implicated in regulating the expression of rat cytochrome P450IA1 which is most closely associated with aryl hydrocarbon hydroxylase (AHH). We have now investigated the presence of both the 4 S PAH-binding protein and the 8 S Ah receptor in rat hepatoma H4-II-E cells as well as the induction of P450IA1 upon their exposure to PAH's such as benzo[ a]pyrene (BP) and 3-methylcholanthrene (3MC), and halogenated dioxins such as 2,3,7,8-tetracholordibenzo- p-dioxin (TCDD) and 2,3,7,8-tetracholordibenzofuran (TCDBF). Sucrose density gradient analyses and hydroxylapatite assays indicate that, in addition to the 8 S protein, the 4 S PAH binding protein is present in these cells. This protein interacts in a saturable and high affinity manner with BP and 3MC, but not with TCDD or TCDBF. Using a P450IA1 probe, the induction of gene expression was observed by Northern blot analysis of total cellular RNA after exposure of the H4-II-E cells to BP, 3MC, or TCDBF. Since the 4 S protein was observed to interact only with BP and 3MC, these results suggest that this protein may also play a role in the PAH-induced expression of cytochrome P450IA1 gene expression in H4-II-E.

Voronoi binding site model of a polycyclic aromatic hydrocarbon binding protein

A three-dimensional Voronoi binding site model has been formulated from a series of competitors for the binding site on a recently isolated polycyclic aromatic hydrocarbon binding protein (PBP) from mouse liver. The PBP binds polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P), with high affinity and shows other characteristics associated with receptor-ligand complexes. Altogether, the in vitro binding constant of seven molecules were used to deduce the geometry and the energetics of a possible site model consisting of five regions: one tetrahedron-shaped finite central hydrophobic pocket, one infinite region representing access to the solvent, and three strongly repulsive regions representing the sterically forbidden walls of the pocket. The model then predicted the binding energies correctly for nine additional competitors and suggests that competition of monoaromatic (benzene) derivatives with B[a]P would be weak.

The 4S polycyclic aromatic hydrocarbon-binding protein: immunohistochemical localization in mice

The 4S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a cytoplasmic protein that binds PAHs with specificity and high affinity. We have used antisera for the PBP and unlabeled peroxidase anti-peroxidase immunohistochemistry to demonstrate its possible localization in cell types known to have xenobiotic metabolizing capabilities. Cellular sites of the PBP in liver, lung and kidney of C57BL/6 and DBA/2 mice were probed. The PBP was visualized in hepatocytes throughout the liver lobule and was not preferentially located in either centrilobular or periportal areas. However, cellular heterogeneity with respect to PBP content was clearly evident in the hepatocyte population. The positive reactivity correlated with substantial levels of benzo[a]pyrene (B[a]P) binding in liver cytosol. In the lung, the PBP was found in the bronchiolar epithelium and the alveolar septa, and was localized in ciliated and non-ciliated Clara and alveolar type II cells as well as in alveolar macrophages. In the kidney, the glomeruli and epithelia of proximal and distal convoluted tubules and collecting ducts were labeled. Staining for the PBP was greatest in the apical region of the pyramid and was localized in the epithelial lining of the collecting ducts. Relatively lower levels of the PBP were detected in the lung and kidney than in the liver. Staining was localized in the cytoplasmic compartment of cells in all tissues examined. Similar immunoreactivities were exhibited in the tissues of both C57BL/6 and DBA/2 mice. Treatment with beta-naphthoflavone (beta NF) altered neither the intensity nor pattern of immunostaining. Furthermore, treatment with beta NF or isosafrole has no effect on the Kd and Bmax of B[a]P binding to liver cytosolic PBP. The results of our experiments demonstrate localization of the PBP to sites of active physiological response to PAH exposure.

Evaluation of the 4S polycyclic aromatic hydrocarbon binding protein in the Harlan Sprague-Dawley rat

The polycyclic aromatic hydrocarbon (PAH)-induced expression of the rat Cyp1A1 gene is a complex process which appears to be regulated by several trans-acting factors including the 8S (Ah receptor) and 4S PAH binding proteins. This gene is closely associated with aryl hydrocarbon hydroxylase enzyme activity (AHH), which is known to bioactivate PAHs. The current study was undertaken to examine the hepatic 4S PAH binding protein in male Harlan Sprague-Dawley (HSD) rats using sucrose gradient analysis of 100,000 g supernatant solutions incubated with 10 nM [3H]benzo[a]pyrene (B[a]P) and a 200-fold excess of competitive ligands. Our results indicate that the 4S PAH binding protein is present in HSD rats and exhibits specific and saturable binding to B[a]P, but not to the dioxin congener 2,3,7,8-tetrachlorodibenzo-p-furan. The detection of the 4S protein in Harlan rats with B[a]P was found to be dependent on ligand and protein concentration. However, under standard assay conditions (10 nM [3H]B[a]P and 4.0 mg/ml cytosolic protein), HSD rats contained equivalent amounts of the 4S PAH binding protein compared to standard SD rats. Under standard conditions, no specific binding was observed to the 8S protein in HSD rats when B[a]P was used as the radioligand. Our data suggest that the role of the 4S protein in Cyp1A1 gene regulation in Harlan rats requires further evaluation.

"4S" polycyclic aromatic hydrocarbon binding protein. Its role as a benzo(a) pyrene cytosolic carrier to the microsomes of DBA/2N mouse liver.

Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.

Purification to homogeneity of the major "4S" PAH binding protein from "non responsive" DBA/2N mouse liver by affinity chromatography.

DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [ 3H]benzo[ a]pyrene and other PAHs (W. H. Houser et al. (1985) Biochemistry 24, 7839–7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5′ half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either λ exonuclease or exonuclease III, tentatively identified two protected regions at −225 and −455 bp 5′ from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5′-upstream sequences (—882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (−95 to −724 or −240 to −720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.

Implication of the "4S" polycyclic aromatic hydrocarbon binding protein in the transregulation of rat cytochrome P-450c expression.

A protein which specifically binds [3H]benzo[a]pyrene and other polycyclic aromatic hydrocarbons has been purified over 6000-fold from rat hepatic cytosol by using ion-exchange, gel permeation, and hydrophobic interaction chromatography. The binding protein differs from the 9S binding protein characterized in other laboratories. A Stokes radius of 2.75 nm was determined by gel filtration on Sephadex G-100. A sedimentation coefficient of 3.3 S was determined by using sucrose gradient analysis. The ability of this protein to bind total rat liver DNA as well as subclones containing portions of the rat cytochrome P-450c gene was investigated. Under high stringency conditions, this binding protein was found to interact in a specific and saturable manner with several subclones of the rat cytochrome P-450c gene containing 5'-upstream sequences, as well as portions of intron 1. Binding was not observed to the coding portions of the gene. These data implicate the "4S" binding protein in the transregulation of rat cytochrome P-450c expression.

The binding of benzo(a)pyrene to subcellular structures of rat liver

1. 1. The development of the polycyclic aromatic hydrocarbon binding protein in cytosol of rat liver has been studied. The highest amount was observed in the cytosol of the 11 day old rat (60 fmoles/mg protein). Adult levels (20 fmoles/mg protein) were reached at the age of 25 days. The fetal rat liver contains only minute amounts of the binding protein. 2. 2. Benzo(a)pyrene (BP) proved to be an inducer of the binding protein. This induction was age dependent, the highest induction (about 10-fold) being observed in liver of rats older than 30 days. No induction could be observed in the fetal liver, while during the period of high endogenous content of the binding protein, the content of the binding protein was lower after treatment of the rat with BP. A high endogenous amount of binding protein was found to parallel a high degree of induction of cytosolic NAD(P)H dehydrogenase (DT-diaphorase) by BP. 3. 3. The induction of the binding protein proved to be sensitive to SKF-525A. Since SKF-525A does not induce the binding protein, it is concluded that metabolic conversion of BP is required for the induction. 4. 4. BP bound to the 100,000 g supernatant of rat liver was bound to nuclei, nuclear envelopes, and microsomes. The dissociation constant for this binding was found to be 0.3 nM. The highest amount of binding was observed in nuclei isolated from the adult rat liver. Fetal rat liver nuclei demonstrated about 40 × less binding of BP bound to 100,000 g rat liver supernatant than adult rat liver nuclei.