Bartolo Favaloro

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 malignacies, 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.

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

Lipoxin (LX) A4 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 immuno‐precipitation 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. LXA4 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 LXA4‐FPR2/ALX axis in human disease —Simiele, F., Recchiuti, A., Mattoscio, D., De Luca, A., Cianci, E., Franchi, S., Gatta, V., Parolari, A., Werba, J. P., Camera, M., Favaloro, B., Romano, M. Transcriptional regulation of the human FPR2/ALX gene: Evidence of a heritable genetic variant that impairs promoter activity. FASEB J. 26, 1323‐1333 (2012). www.fasebj.org

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.

Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress

The gluthathione S-transferase gene of the atrazine-degrading bacterium Ochrobactrum anthropi (OaGST) encodes a single-subunit polypeptide of 201 amino acid residues (Favaloro et al. 1998, Biochem. J. 335, 573-579). RNA blot analysis showed that the gene is transcribed into an mRNA of about 800 nucleotides, indicating a monocistronic transcription of the OaGST gene. The modulation of OaGST in this bacterium, in the presence of different stimulants, was investigated. The level of expression of OaGST was detected both by measuring the mRNA level and by immunoblotting experiments. OaGST is a constitutive enzyme which is also inducible by several stimulants. In fact, atrazine caused an increase in the expression of OaGST even at concentrations which had no effect on growth rates of the bacteria. Moreover, the presence of other aromatic substrates of this bacterium, such as phenol and chlorophenols, leads to a marked enhancement in OaGST expression. In this case, the expression of OaGST was related to growth inhibition and membrane damage caused by these hydrophobic compounds, and to the adaptive responses of the cell membranes. On the other hand, toluene and xylene, two aromatic compounds not degradable by this bacterium, did not induce the OaGST expression. The same was observed for other stress conditions such as low pH, heat shock, hydrogen peroxide, osmotic stress, starvation, the presence of aliphatic alcohols or heavy metals. These results suggest a co-regulation of the OaGST gene by the catabolic pathways of phenols and chlorophenols in this bacterium. Therefore, OaGST could function as a detoxifying agent within the catabolism of these xenobiotics.

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.

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. Proteins 2008.

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.

An RPE cell line as a useful in vitro model for studying retinoic acid receptor β: 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.

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.

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.