4S Protein Research Articles

Structure and Function of Four Classes of the 4Fe-4S Protein, IspH.

IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate reductase, is an essential enzyme in isoprenoid biosynthesis and an important drug/herbicide target. Using X-ray crystallographic, bioinformatics, mutagenesis/kinetics/stability, and electron paramagnetic resonance (EPR) results, we show that organisms from different environments ultilize one of four main IspH classes. The classes are based on the arrangement of the aromatic residues near the 4Fe-4S cluster and the presence or absence of N- and C-terminal extensions. Class A enzymes are found primarily in anaerobic and microaerophilic bacteria. Class B enzymes are found in aerobic bacteria. Class C enzymes are found in cyanobacteria and plants. Class D enzymes are found in apicomplexan parasites. Using mutagenesis, we show that the cluster-associated aromatic groups in class A and class B IspHs enhance cluster oxidative stability. Y198A, F302A, and a C-terminal truncation mutant of the class B (Escherichia coli) IspH have catalytic activity lower than that of the wild-type protein when using methyl viologen as the electron donor, but higher activity with dithionite as the electron donor, due to ready access of the small reductant to the cluster, consistent with their increased oxygen and H2O2 sensitivity. F302A has the largest effect on the reaction rates, and EPR studies indicate this residue affects Fe-S cluster structure. Similar effects on cluster stability are seen with class A (F14A and Y98A) mutants; however, effects on ET rates are smaller, and there are no differences between the EPR spectra of mutant and wild-type proteins. Overall, the results are of general interest because they show, for the first time, that there are multiple IspH classes that have evolved to allow organisms to survive in diverse oxidative-stress environments.

Inhibition of the 4Fe-4S Proteins IspG and IspH: an EPR, ENDOR and HYSCORE Investigation.

IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e- reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as "cubane-type" metallo-biochemistry, in general.

Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

Kinetic, mutagenic, and structural homology analysis of l-serine dehydratase from Legionella pneumophila

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe–4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron–sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni–Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with Ki values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron–sulfur complex.

Flip-Flop of Fluorescently Labeled Phospholipids in Proteoliposomes Reconstituted with Saccharomyces cerevisiae Microsomal Proteins

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.

421. Liver Contains Sufficient Sulfatase Modifying Factor for 4-Sulfatase and Correction of MPS VI with Neonatal Retroviral Gene Therapy in Cats

Sulfatases are enzymes that remove a sulfate from a substrate. 4-sulfatase (4S; also known as arylsulfatase B) is a lysosomal enzyme that removes a sulfate from the 4 position of N-acetylgalactosamine, a component of the glycosomaminoglycan (GAG) dermatan sulfate. Deficient activity of 4S results in mucopolysaccharidosis VI (MPS VI), a disorder that includes bone and joint abnormalities, corneal clouding, and cardiac disease, but does not usually involve the brain. The lack of neurological involvement makes this an excellent candidate for somatic therapies. All sulfatases are modified post-translationally by enzymes designated as sulfatase modifying factors (SUMF) that convert a cysteine in the active site to a C-alpha-formylglycine, which is absolutely essential for enzyme activity. SUMF is readily saturated after over expression of a sulfatase in fibroblasts in vitro, resulting in limitations in the amount of enzyme that can be produced and a reduction in activity of other sulfatases that require the same modification. It was, therefore, unclear if hepatocytes would contain sufficient SUMF activity to modify the 4S produced by hepatocytes after liver-directed gene therapy for MPS VI. A retroviral vector (RV) designated hAAT-f4S-WPRE expressing feline 4S was generated. 4S-deficient feline fibroblasts were transduced in vitro with increasing multiplicities of infection. The 4S enzyme activity (4MU-sulfate assay) and 4S protein levels (immunoblot) were directly proportional to the DNA copy number, which leveled off at about 2 copies per cell despite using higher multiplicities of infection. This likely reflects an inhibition of transduction when large amounts of vector are used, which has been described previously, suggesting that SUMF is not limiting when relatively low copy numbers are achieved. hAAT-f4S-WPRE (4.2-3.4X10E9 TU/kg) was injected IV into 6 newborn cats with MPS VI. They achieved stable expression at 109+/-49 U/ml in serum, which is 10-fold normal and has been maintained for up to 1.5 years in the oldest animals. Analysis of liver biopsies demonstrated that the 4S enzyme activity was directly proportional to the amount of 4S protein, suggesting that all of the enzyme was functional, and that modification by SUMF was not limiting. The cats have marked improvement in the clinical skeletal manifestations. The cats also have biochemical evidence of improvement, which includes high levels of 4S and a reduction in GAG and secondary lysosomal enzyme activity in other organs. We conclude that liver contains sufficient SUMF to modify 4S, and that liver-directed gene therapy can markedly improve the clinical manifestations of this disorder.

Comparison of the induction of a 4S β-naphthoflavone-binding protein, cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in β-naphthoflavone-treated rats

A profound induction of a 4S β-naphthoflavone (BNF)-binding protein, cytochrome P450 1A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase (NQO1) activities was determined in the livers of Sprague–Dawley rats following intraperitoneal administration of BNF. Time-course of this induction differed for CYP1A1 and NQO1 activities, suggesting independent regulation of the phase I and II enzymes of xenobiotic metabolism. Time-course of the induction of CYP1A1 and BNF-binding activities was similar, suggesting that regulation of a 4S BNF- binding protein is associated with that of the CYP1A1 enzyme activity. The BNF specific binding to a 4S protein was inhibited by exogenous (BNF) and endogenous (indirubin and indigo) ligands for the aryl hydrocarbon receptor.

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N-acetylgalactosamine-4-sulfatase.

Mucopolysaccharidosis type VI (MPS-VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-4-sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4-sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS-VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS-VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS-VI alleles will be difficult.

The Existence of the 4S Polycyclic Aromatic Hydrocarbon-Protein Binding in 14-Day-Old Chick Embryo Liver

Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.

Effects of carbaryl and naphthalene on rat hepatic CYP1A1/2: potential binding to Ah receptor and 4S benzo(a)pyrene-binding protein.

Activation of the CYP1A1 gene has been described to be mediated by the cytosolic Ah receptor (AhR) and a possible cooperative role of the 4S benzo(a)pyrene-binding protein (4S protein). Carbaryl (CAR) has been shown to induce human CYP1A1 gene expression without binding to the human AhR. In this study, Sprague-Dawley rats received a single i.p. dose of 20, 80, 150 micromol/kg CAR or NAPn (naphthalene, the aromatic part of CAR) and were sacrificed after 24 h. CAR increased ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase activities, the level of CYP1A1, 1A2 proteins, and CYP1A1 mRNA at the highest dose, whereas NAPn showed no effects. Moreover, CAR, naphthol (its major metabolite) and NAPn were not ligands in vitro of the TCDD binding site of AhR or the benzo(a)-pyrene binding site of 4S protein in rat, neither was CAR a ligand of these two binding sites in mice, dog, monkey or human. Molecular properties of CAR were evaluated and showed that this molecule is far from the structural characteristics of CYP 1A1 specific inducers although a planar conformation can be achieved with an energy < 5 kJ x mol(-1). The data demonstrated that CAR could also modulate the AhR-mediated responses, even though it did not meet the structural requirements to be ligand of AhR.

Mild Feline Mucopolysaccharidosis Type VI: IDENTIFICATION OF ANN-ACETYLGALACTOSAMINE-4-SULFATASE MUTATION CAUSING INSTABILITY AND INCREASED SPECIFIC ACTIVITY

The missense mutation, L476P, in the N-acetylgalactosamine 4-sulfatase (4S) gene, has previously been shown to be associated with a severe feline mucopolysaccharidosis type VI (MPS VI) phenotype. The present study describes a second mutation, D520N, in the same MPS VI cat colony, which is inherited independently of L476P and is associated with a clinically mild MPS VI phenotype in D520N/L476P compound heterozygous cats. Biochemical and clinical assessment of L476P homozygous, D520N/L476P compound heterozygous, and D520N homozygous cats demonstrated that the entire range of clinical phenotypes, from severe MPS VI, to mild MPS VI, to normal are clustered within a narrow range of residual 4S activity from 0. 5% to 4.6% of normal levels. When overexpressed in CHO-KI cells, the secreted form of D520N 4S was inactivated in neutral pH conditions. In addition, intracellular D520N 4S protein was rapidly degraded and corresponded to 37%, 14.5%, and 0.67% of normal 4S protein levels in the microsomal, endosomal, and lysosomal compartments, respectively. However, the specific activity of lysosomal D520N 4S was elevated 22. 5-fold when compared with wild-type 4S. These results suggest that the D520N mutation causes a rapid degradation of 4S protein. The effect of this is partially ameliorated as a result of a significant elevation in the specific activity of mutant D520N 4S reaching the lysosomal compartment.

The 4S Benzo(a)pyrene-Binding Protein Is Not a Transcriptional Activator ofCyp1a1Gene in Ah Receptor-Deficient (AHR −/−) Transgenic Mice

In an effort to better understand the role of the 4S benzo(a)pyrene-binding protein in the induction of CYP1A1 by PAHs, we used a genetically engineered mouse line deficient in Ah receptor (AHR −/−). First, we demonstrated through binding experiments analyzed by sucrose gradient sedimentation and gel permeation chromatography that AHR −/− mice have no detectable AHR protein. In contrast, this AHR-deficient line expressed a 4S protein which efficiently binds BP as it does in hepatic cytosol from C57BL/6 mice.In vivoBP exposure in AHR-deficient mice proved the inability to sustain any CYP1A1 mRNA or CYP1A1 protein induction. These findings demonstrate the requirement of an active AHR to sustain the transactivation pathway leading to CYP1A1 induction. Surprisingly, the 4S BP-binding protein, which was previously characterized as the glycineN-methyltransferase, was completely devoid of such an enzymatic activity after purification by Sephacryl gel permeation chromatography. Moreover, sedimentation and chromatographic experiments, under nondenaturing conditions, do not support the assumption of 4S protein as a subunit of a multimeric protein (GNMT) displaying a molecular mass of 150 kDa.

The Pig as a Model for Studying AH Receptor and Other PAH-Binding Proteins In Man

In this report we compare the three major binding proteins existing in hepatic cytosols of pig and human. Many data suggest that for the AH receptor, which mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a structural as well as a functional variability exists across species. Similar conclusions can be drawn from the interspecies characterization of benzo(a)pyrene binding proteins, namely the 4S protein and the 8S protein. Using fractionation procedures such as sucrose gradient sedimentation and gel permeation chromatography we obtained enriched fractions containing each of these binding proteins. By saturation experiments and analysis of data by Scatchard and Woolf plots we determined binding characteristics and we conclude that pig and human AH receptors were closely related proteins since their Kd (18 ± 0.4nM) were found quite similar. On the other hand, 4S proteins from pig (Kd = 16.7 nM; Bmax = 5.5 pmol/mg) and from human (Kd = 14nM; Bmax= 4.5 pmol/mg) as well as 8S proteins (Kd ≍300 nM) also exhibit remarkable similarities.

Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients.

Anti-peptide antibody to the human glucocorticoid receptor (GR) was produced and used to demonstrate that a subset of the GR population resides in the plasma membrane of human leukemic cells. Characterization of the antibody with intracellular GR (iGR) showed its ability to shift [3H]triamcinolone acetonide-labeled GR (4S protein) from two human leukemic cell lines to a higher density in sucrose gradients; Western and autoradiographic analysis of affinity-labeled ([3H]dexamethasone 21-mesylate) receptor revealed an immunoreactive and competitively labeled band of 94 kDa. CCRF-CEM cell membrane GR (mGR) resolved as a > 7S protein on density gradients and immunoselected cell surface protein labeled by whole cell biotinylation or affinity-labeling with [3H]dexamethasone 21-mesylate was approximately 145 kDa, demonstrating that mGR was larger in size than iGR, as has been shown previously for the mGR of mouse lymphoma cells. Analysis of mGR in lymphocytes of leukemic patients and the CCRF-CEM cell line indicated differences in levels of expression as shown by FACS and immunocytochemical analyses. We are currently using this system to study the correlation between the quantity of membrane-resident GRs and the glucocorticoid-induced lytic response, a relationship previously shown in the murine (S-49 cell) system.

Mechanism of antimutagenicity of wheat sprout extracts

In this paper we have demonstrated that wheat sprout extract, which has been shown to be antimutagenic towards benzo[ a]pyrene (BP), reduced formation of BP metabolites by hepatic microsomes of either benzo[ a]pyrene- or phenobarbital-treated rats as analyzed in high-pressure liquid chromatography (HPLC). Comparing the time dependence of profiles and values of BP metabolites, formed in experiments in which the same dose of wheat sprout extract was added to the incubation medium, it has been observed that the later this extract was added the higher the percent of BP that was metabolized. In a bacterial test (cytochrome P450 induction assay) high inhibition of mutagenic activity of cyclophosphamide and ethidium bromide, in the presence of wheat sprout extract, reflected decreased levels of cytochromes P4502B1 and P450A1 respectively. Decreased levels of both cytochromes P4501A1 and P4502B1 were also observed in either wheat sprout extract- or wheat sprout extract plus benzo[ a]pyrene-treated rats. In all of these studies it has been observed that wheat sprout extract displays much more affinity for cytochrome P450A1 than for the P450B1 form. On the other hand the wheat sprout extract had higher affinity for carcinogen binding protein (4S protein) than for the aryl hydrocarbon receptor. The strong inhibition of BP mutagenicity and BP metabolism with non-chlorophyllic wheat sprout extract suggests that chlorophyll is not the main compound responsible for the antimutagenic activity of wheat sprout extract. The similar chromatographic behavior of both the main inhibitory fraction, obtained from wheat sprout extract, and two pure glycosides of apigenin — shaftoside, purified from wheat sprout extract and synthetic swertisine — suggest that antimutagenic compound(s) contained in the wheat sprout extract belong(s) to this family of flavonoids.

Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.

Binding characteristics of 4s PAH‐binding protein andAhreceptor from rats and mice

Cytochrome P-450IA1 (Cyto-P450IA1) is the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH). At least two distinct high-affinity binding proteins may regulate its expression, the 4S protein that primarily binds polycyclic aromatic hydrocarbons (PAHs), and the 8S Ah receptor that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to investigate ligand binding characteristics of the 4S and 8S binding proteins before and after separation from liver cytosol in the presence and absence of sodium molybdate. Liver cytosol and 4S and 8S receptor-enriched fractions from livers of male Sprague-Dawley rats (AHH-responsive), and from C57BL/6N (AHH-responsive) and DBA/2N and AKR/N mice (AHH-nonresponsive) served as sources of these proteins. Competitive binding studies were performed using 10 nM [3H]benzo[a]pyrene (BaP) or [3H]-TCDD in the presence and absence of a 200-fold excess of BaP, 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed by using either sucrose density gradient analysis or a hydroxylapatite assay. Our results indicate that before and after the separation of liver cytosol into 4S and 8S fractions, ligand binding characteristics were relatively unaltered for the 4S protein in comparison to that for the Ah receptor, particularly in the presence of molybdate. The 4S protein had high affinity for BaP and 3-MC but very low affinity for TCDBF; the 8S protein had high affinity for TCDBF, lesser affinity for 3-MC, and low affinity for BaP. In the presence of sodium molybdate, the Ah receptor fractions were significantly stabilized, whereas the 4S protein was relatively unaffected. After the separation of Ah receptor fraction from liver cytosol in the presence of molybdate, 3-MC consistently bound to a greater extent. These results affirm the existence of two distinct PAH-binding proteins.

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.

Intracellular lipoproteins as carriers for 2,3,7,8-tetrachlorodibenzo- p-dioxin and benzo( a)pyrene in rat and mouse liver

The possible role of hepatic lipoproteins as intracellular carriers in the transport of 2,3,7,8-tetrachlorodibenzo- p-dioxin and benzo( a)pyrene was assessed by in vitro and in vitro studies. Following administration of [ 3H]2,3,7,8-tetrachlorodibenzo- p-dioxin or unlabelled 2,3,7,8-tetrachlorodibenzofuran to C57 BL/6 mice or Sprague-Dawley rats these compounds were bound to lipoproteins which subsequently underwent rapid and pronounced degrActative processing, possibly catalysed by lipoprotein lipase, to heavier entities. At the highest doses of xenobiotics administered, an almost complete disappearance of lipoprotein particles was observed. The in vitroincubation of [ 3H]2,3,7,8-tetra-chlorodibenzo- p-dioxin-lipoprotein and [ 3H]benzo( a)pyrene-lipoprotein complexes with separated Ah receptor and 4S protein, respectively, demonstrated that a passive transfer occurred; the latter was likely dependent on both the relative affinities of the ligands towards the different cellular binding components as well as on their quantitative binding capacity. Taken together, these findings support the idea of a carrier-role for lipoproteins in the intracellular transport of hydrophobic xenobiotics and it may be asked whether the widespread modulators of lipoprotein level such as fibrates or others affect drug transfer or action.