Transcriptional regulation of the human FPR2/ALX gene: evidence of a heritable genetic variant that impairs promoter activity.

Lipoxin (LX) A(4,) a main endogenous stop-signal of inflammation, activates the G-protein-coupled receptor FPR2/ALX, which triggers potent anti-inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immunoprecipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site-directed mutagenesis of the Sp1 cis-acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA(4) enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA-MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by ∼35-90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed ∼10- and 3-fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild-type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA(4)-FPR2/ALX axis in human disease.

Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype.

Breast cancer is one of the most frequent of human malignancies, and it is therefore fundamental to identify the underlying molecular mechanisms leading to cancer transformation. Among other causative agents in the development of breast cancers, an important role for reactive oxygen species (ROS) has emerged. However, most studies on the role of ROS in cancer have not reached specific conclusions, and many issues remain controversial. In the present study, we show that methionine sulfoxide reductase A (MsrA), which is known to protect proteins from oxidation and which acts as a ROS scavenger, is down-regulated in a number of breast cancers. Moreover, levels of MsrA correlate with advanced tumor grade. We therefore investigated the functional role of MsrA in breast cancer cells. Our data show that reduction of MsrA levels results in increased cell proliferation and extracellular matrix degradation, and consequently in a more aggressive cellular phenotype, both in vivo and in vitro. We also show that the underlying molecular mechanisms involve increased ROS levels, resulting in reduction of phosphatase and tensin homolog deleted on chromosome ten protein (PTEN), and activation of the phosphoinositide 3-kinase pathway. In addition, MsrA down-regulation results in up-regulation of VEGF, providing additional support for tumor growth in vivo.

Cysteine 10 is critical for the activity of Ochrobactrum anthropi glutathione transferase and its mutation to alanine causes the preferential binding of glutathione to the H-site.

The role of the evolutionarily conserved residue Cys10 in Ochrobactrum anthropi glutathione transferase (OaGST) has been examined by replacing it with an alanine. A double mutant C10A/S11A was also prepared. The effect of the replacements on the coniugating and thiotransferase activities, and on the thermal and chemical stability of the enzyme was analyzed. Our data support the view that in OaGST, in contrast with other beta class GSTs that display significant differences in the glutathione-binding site, Cys10 is a key residue for glutathione coniugating activity. Furthermore, analysis of the OaGST-Cys10Ala structure, crystallized in the presence of glutathione, reveals that this mutation causes a switch between the high-affinity G-site and a low-affinity H-site where hydrophobic cosubstrates bind and where we observe the presence of an unexpected glutathione.

An RPE cell line as a useful in vitro model for studying retinoic acid receptor beta: expression and affinity.

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-beta is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-beta can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-beta-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-beta and none of the other receptor subtypes. In addition, we show that the expression of RAR-beta increases with increasing passage number of the cells. Interestingly, the increase in RAR-beta expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-beta-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-beta. Using [(3)H]CD367 as the ligand, RAR-beta in HRPE cells showed an affinity of 9.6 +/- 0.6 nM and a B(max) of 780 +/- 14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-beta by investigating the ability of certain flavonoids to inhibit the binding of [(3)H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-beta was between approx. 1-30 microM, showing that the flavonoids interact with RAR-beta with low affinity.

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase.

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter.

BACKGROUND: Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of oxidized methionine residues. Most organisms that were genetically modified to lack the MsrA gene have shown shortening of their life span. Methionine sulfoxide reductases B (MsrB) proteins codified by three separate genes, named MsrB1, MsrB2, and MsrB3, are included in the Msrs system. To date, the mechanisms responsible for the transcriptional regulation of MsrB genes have not been reported. The aim of this study was to investigate the regulation of MsrB1 selenoprotein levels through transcriptional regulation of the MsrB1 gene in MDA-MB231 and MCF-7 breast carcinoma cell lines. RESULTS: A MsrB1 gene promoter is located 169 base pairs upstream from the transcription start site. It contains three Sp1 binding sites which are sufficient for maximal promoter activity in transient transfection experiments. High levels of MsrB1 transcript, protein and promoter activity were detected in low metastatic MCF7 human breast cancer cells. On the contrary, very low levels of both MsrB1 transcript and promoter activity were detected in the highly metastatic counterpart MDA-MB231 cells.A pivotal role for Sp1 in the constitutive expression of the MsrB1 gene was demonstrated through transient expression of mutant MsrB1 promoter-reporter gene constructs and chromatin immunoprecipitation experiments. Since Sp1 is ubiquitously expressed, these sites, while necessary, are not sufficient to explain the patterns of gene expression of MsrB1 in various human breast cancer cells. MDA-MB231 cells can be induced to express MsrB1 by treatment with 5-Aza-2'-deoxycytidine, a demethylating agent. Therefore, the MsrB1 promoter is controlled by epigenetic modifications. CONCLUSION: The results of this study provide the first insights into the transcriptional regulation of the human MsrB1 gene, including the discovery that the Sp1 transcription factor may play a central role in its expression. We also demonstrated that the MsrB1 promoter activity appears to be controlled by epigenetic modifications such as methylation.

Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene.

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5' promoter region of the human msrA gene. Using 5'-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between -209 and -212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

Contribution of the two conserved tryptophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1.

PmGSTB1-1 (Proteus mirabilis glutathione S-transferase B1-1) has two tryptophan residues at positions 97 and 164 in each monomer. Structural data for this bacterial enzyme indicated that Trp97 is positioned in the helix a4, whereas Trp164 is located at the bottom of the helix a6 in the xenobiotic-binding site. To elucidate the role of the two tryptophan residues they were replaced by site-directed mutagenesis. Trp97 and Trp164 were mutated to either phenylalanine or alanine. A double mutant was also constructed. The effects of the replacement on the activity, structural properties and antibiotic-binding capacity of the enzymes were examined. On the basis of the results obtained, Trp97 does not seem to be involved in the enzyme active site and structural stabilization. In contrast, different results were achieved for Trp164 mutants. Conservative substitution of the Trp164 with phenylalanine enhanced enzyme activity 10-fold, whereas replacement with alanine enhanced enzyme activity 17-fold. Moreover, the catalytic efficiency for both GSH and 1-chloro-2,4-dinitrobenzene substrates improved. In particular, the catalytic efficiency for 1-chloro-2,4-dinitrobenzene improved for both W164F (Trp164-->Phe) and W164A by factors of 7- and 22-fold respectively. These results are supported by molecular graphic analysis. In fact, W164A presented a more extensive substrate-binding pocket that could allow the substrates to be better accommodated. Furthermore, both Trp164 mutants were significantly more thermolabile than wild-type, suggesting that the substitution of this residue affects the overall stability of the enzyme. Taken together, these results indicate that Trp164 is an important residue of PmGSTB1-1 in the catalytic process as well as for protein stability.

Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates.

Peptide methionine sulphoxide reductase (MsrA) and glutathione S-transferases (GSTs) are considered as detoxification enzymes. In the xenobiotics-degrading bacterium Ochrobactrum anthropi the two enzymes are co-induced by toxic concentrations of aromatic substrates such as phenol and 4-chlorophenol. In aerobic organisms, degradation of aromatic substrates by mono- and dioxygenases leads to a generation of oxidative stress that causes the occurrence of reactive oxygen species (ROS). A capillary electrophoretic method, using the intracellular conversion of dihydrorhodamine-123 into rhodamine-123, was developed to measure the content of ROS in the bacteria. The presence of toxic concentrations of the aromatic substrate 4-chlorophenol, an inducer of GST and MsrA, leads to a significant increase in the production of ROS. These results strongly suggest that GST and MsrA enzymes are part of the bacterial defence mechanism against particular oxidative stress conditions. As oxidative stress is known to be present predominantly close to the cytoplasmic membrane, we investigated the subcellular distribution of both MsrA and GST enzymes in this bacterium grown in the presence of 4-chlorophenol. By Western blotting, MsrA and GST was assayed in the cytoplasm as well as in the periplasm. Moreover, immunolocalisation by colloidal gold immunoelectron microscopy identified the two proteins associated with the cell envelope.

Liquid chromatography-electrospray mass spectrometry study of cysteine-10 S-glutathiolation in recombinant glutathione S-transferase of Ochrobactrum anthropi.

Glutathione S-transferase of Ochrobactrum anthropi (OaGST), a bacterium isolated from soils contaminated by xenobiotic pollutants, was recently purified, cloned and characterised in our laboratories. The recombinant OaGST (rOaGST), highly expressed in Escherichia coli, when purified by glutathione-affinity chromatography and then analysed by electrospray ionisation mass spectrometry (ESI-MS), has evidenced a disulphide bond with glutathione (S-glutathiolation), which was removable by reduction with 2-mercaptoethanol. Enzymatic digestion of rOaGST with endoproteinase Glu-C, followed by liquid chromatography (LC)-ESI-MS analyses of the peptide mixtures under both reducing and not reducing conditions, have shown that glutathione was covalently bound to the Cys10 residue of rOaGST. Furthermore, LC-ESI-MS analyses of overexpressed rOaGST in Escherichia coli crude extracts, with and without incubation with glutathione, have not shown any S-glutathiolation of the recombinant enzyme.

Role of apoptosis in disease.

Since the initial description of apoptosis, a number of different forms of cell death have been described. In this review we will focus on classic caspase-dependent apoptosis and its variations that contribute to diseases. Over fifty years of research have clarified molecular mechanisms involved in apoptotic signaling as well and shown that alterations of these pathways lead to human diseases. Indeed both reduced and increased apoptosis can result in pathology. More recently these findings have led to the development of therapeutic approaches based on regulation of apoptosis, some of which are in clinical trials or have entered medical practice.

Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis.

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359 Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Proteus mirabilis glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative and chemical stresses.

We investigated the effects of several xenobiotics, including antimicrobial agents and general stress factors such as starvation, heat and osmotic shock, on the modulation of expression of Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1). The level of expression of PmGST B1-1 was established by both Western- and Northern-blot experiments. Our results show that several compounds can modulate expression of PmGST B1-1. The level of PmGST B1-1 increased when bacterial cells were exposed to a variety of stresses such as 1-chloro-2,4-dinitrobenzene, H(2)O(2), fosfomycin or tetracycline. A knock-out gst B gene was also constructed using the suicide vector pKNOCKlox-Ap. Successful inactivation of the wild-type gene was confirmed by PCR, DNA sequence analysis and Western blotting. Under normal culture conditions, this mutant was viable and displayed no significant phenotypic differences compared with the wild-type. However, viability tests revealed that the null mutant was more sensitive to oxidative stress in the form of H(2)O(2) and to several antimicrobial drugs when compared with the wild-type. These results suggest that PmGST B1-1 has an active role in the protection against oxidative stress generated by H(2)O(2) and it appears to be involved in the detoxification of antimicrobial agents.

A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site.

Screening of a liver tumour cDNA library from Xenopus laevis resulted in the isolation of a full-length cDNA clone encoding a novel Pi-class amphibian glutathione transferase (GST) isoenzyme (designated as XlGSTP1-1). The gene encodes a protein of 212 amino acids with a calculated molecular mass of 24428 Da. The product of the gene has been overexpressed in Escherichia coli and characterized. XlGSTP1-1 has one of the highest specific activities towards 1-chloro-2,4-dinitrobenzene (1310 micromol/min per mg of protein) obtained with any GST. A notable feature of XlGSTP1-1 is the presence in the H-site of Phe(111) and Pro(208) in place of tyrosine and glycine residues respectively, present in other mammalian Pi-class GSTs. Site-directed mutagenesis indicate that Phe(111) is involved in substrate specificity of XlGSTP1-1. We provide evidence showing that XlGSTP1-1 is present only in the embryo and its expression might be associated with cellular proliferation.

Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1.

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu(65), Ser(103) and Glu(104) are in hydrogen-bonding distance of the N-terminal amino group of the gamma-glutamyl moiety of the co-substrate, GSH. Glu(65) was mutated to either aspartic acid or leucine, and Ser(103) and Glu(104) were both mutated to alanine. Glu(65) mutants (Glu(65)-->Asp and Glu(65)-->Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser(103)-->Ala and Glu(104)-->Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser(103) and Glu(104) are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu(65) is crucial for catalysis.