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

<p>Abstract</p> <p>Background</p> <p>Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of oxidized methionine residues. Most organisms that were genetically modified to lack the <it>MsrA </it>gene have shown shortening of their life span. Methionine sulfoxide reductases B (MsrB) proteins codified by three separate genes, named <it>MsrB1</it>, <it>MsrB2</it>, and <it>MsrB3</it>, are included in the Msrs system. To date, the mechanisms responsible for the transcriptional regulation of <it>MsrB </it>genes have not been reported. The aim of this study was to investigate the regulation of MsrB1 selenoprotein levels through transcriptional regulation of the <it>MsrB1 </it>gene in MDA-MB231 and MCF-7 breast carcinoma cell lines.</p> <p>Results</p> <p>A <it>MsrB1 </it>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.</p> <p>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.</p> <p>A pivotal role for Sp1 in the constitutive expression of the <it>MsrB1 </it>gene was demonstrated through transient expression of mutant <it>MsrB1 </it>promoter-reporter gene constructs and chromatin immunoprecipitation experiments.</p> <p>Since Sp1 is ubiquitously expressed, these sites, while necessary, are not sufficient to explain the patterns of gene expression of <it>MsrB1 </it>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 <it>MsrB1 </it>promoter is controlled by epigenetic modifications.</p> <p>Conclusion</p> <p>The results of this study provide the first insights into the transcriptional regulation of the human <it>MsrB1 </it>gene, including the discovery that the Sp1 transcription factor may play a central role in its expression. We also demonstrated that the <it>MsrB1 </it>promoter activity appears to be controlled by epigenetic modifications such as methylation.</p

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

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

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