Publications (50) View all
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Article: Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts.
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ABSTRACT: During bone development, osteoblast differentiation requires remodeling of the extracellular matrix. Although underlying mechanisms have not been elucidated, evidence points to the participation of the nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) system. Here, we detected increased matrix metalloproteinase (MMP)-13 mRNA, protein and activity, as well as increased inducible NO synthase (iNOS) and NO production during the differentiation of MC3T3-E1 osteoblasts. Transcriptional activity of the MMP-13 promoter was augmented by NO, 8-bromo-cGMP (8-Br-cGMP), and by a dominant-positive form of protein kinase G (PKG1-alpha). The stimulatory effect on the MMP-13 promoter was partially inhibited by mutation of the osteoblast-specific element 2 (OSE-2) binding site. Core binding factor-1 (Cbfa-1) expression peaked at 7 days of differentiation, and was phosphorylated by PKG in vitro. Cbfa-1 was localized to cell nuclei, and its translocation was inhibited by the iNOS inhibitor 1400W. Immunohistological examination revealed that MMP-13 and Cbfa-1 expression levels are both reduced in 17-day-old embryos of iNOS-deficient mice. Silencing of Cbfa-1 mRNA blocked MMP-13 expression without interfering with endogenous NO production, confirming its role in NO-induced MMP-13 expression by MC3T3-E1 cells. The results described here suggest a mechanism by which NO regulates osteogenesis.Journal of Cell Science 06/2006; 119(Pt 9):1896-902. · 6.11 Impact Factor -
Article: Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.
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ABSTRACT: Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.European Journal of Biochemistry 01/2002; 268(23):6190-6. · 3.58 Impact Factor -
Article: Mitogillin and related fungal ribotoxins.
Methods in Enzymology 02/2001; 341:324-35. · 2.04 Impact Factor -
Article: RNase U2 and alpha-sarcin: a study of relationships.
Methods in Enzymology 02/2001; 341:335-51. · 2.04 Impact Factor -
Article: Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin alpha-sarcin.
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ABSTRACT: alpha-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called alpha-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 degrees C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9 degrees C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein-vesicle interaction.Proteins Structure Function and Bioinformatics 12/2000; 41(3):350-61. · 3.39 Impact Factor
