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

An integrated metabolomics approach for the research of new cerebrospinal fluid biomarkers of multiple sclerosis

(1) Lipid profiling in MuS and OND patients. (2) Search of alterations associated with MuS. (3) Characterization of differences

LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

BACKGROUND: Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances. RESULTS: LIMPIC preprocessing proves to be superior than other classical preprocessing techniques, allowing for a reliable decomposition of the background noise and the baseline drift from the MALDI-TOF mass spectra. It provides lower coefficient of variation associated with the peak intensity, improving the reliability of the information that can be extracted from single spectra. Our results show that LIMPIC peak-picking is effective even in low protein concentration regimes. The analytical comparison with commercial and freeware peak-picking algorithms demonstrates its superior performances in terms of sensitivity and specificity, both on in-vitro purified protein samples and human plasma samples. CONCLUSION: The quantitative information on the peak intensity extracted with LIMPIC could be used for the recognition of significant protein profiles by means of advanced statistic tools: LIMPIC might be valuable in the perspective of biomarker discovery

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

Advances in Lipidomics for Cancer Biomarkers Discovery

Lipids play critical functions in cellular survival, proliferation, interaction and death, since they are involved in chemical-energy storage, cellular signaling, cell membranes, and cell–cell interactions. These cellular processes are strongly related to carcinogenesis pathways, particularly to transformation, progression, and metastasis, suggesting the bioactive lipids are mediators of a number of oncogenic processes. The current review gives a synopsis of a lipidomic approach in tumor characterization; we provide an overview on potential lipid biomarkers in the oncology field and on the principal lipidomic methodologies applied. The novel lipidomic biomarkers are reviewed in an effort to underline their role in diagnosis, in prognostic characterization and in prediction of therapeutic outcomes. A lipidomic investigation through mass spectrometry highlights new insights on molecular mechanisms underlying cancer disease. This new understanding will promote clinical applications in drug discovery and personalized therapy