Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.     

Regulation of the manganese-containing superoxide dismutase gene from fission yeast

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.     

Hepatitis C virus core inhibits the Fas-mediated p38 mitogen activated kinase signaling pathway in hepatocytes

The p38 mitogen activated kinase (MAPK) signaling pathway plays an essential role in regulating many cellular processes, including inflammation, cell differentiation, and cell death. Here, we report that the hepatitis C virus (HCV) core inhibits the Fas-mediated p38 signaling pathway. The Fas-mediated p38 activation is suppressed in core-expressing HepG2 cell lines, as well as in the hepatocytes of transgenic mice. In addition, core protein blocked the Fas-mediated activation of apoptosis signal-regulating kinase 1 (ASK1), a major upstream MAPKKK of p38. Treatment of a specific p38 inhibitor (SB203580) or overexpression of a kinase-defective mutant, ASK1 (K709R), promoted Fas-mediated cell death in HepG2 cells. This suggests that the p38 and ASK1 activation is required for cell survival against Fas-mediated cell death. In addition, we observed that the HCV core protein enhances Fas-mediated liver injury and lethality in transgenic mice. Collectively, our findings suggest that the HCV core inhibits the Fas-mediated p38 signaling pathway, which results in accelerated Fas-mediated cell death.     

Preliminary structure-antiangiogenic activity relationships of 4-senecioyloxymethyl-6,7-dimethoxycoumarin

Through a systematic modification of the novel angiogenesis inhibitor 4-senecioyloxymethyl-6,7-dimethoxycoumarin (1) we found that a 6,7-dimethoxy moiety is important for bioactivity of 1. Replacement of the lactone functionality in coumarin 1 by an amide decreased its activity. By substitution of the senecioyl chain with various cinnamoyl groups we discovered 6d, bearing a 4-methoxycinnamoyl instead of senecioyl side chain, with inhibitory activity in HUVEC tube formation assay enhanced by one order of magnitude compared to 1. We have also synthesized compound 12, an analogue of 6d, with equipotency and improved water solubility.     

Prodrugs of 4'-demethyl-4-deoxypodophyllotoxin: synthesis and evaluation of the antitumor activity

A series of prodrugs of 4'-demethyl-4-deoxypodophyllotoxin (DDPT) including carbamates (3-8), a carbonate (9) and water-soluble amino acid derivatives (10-17) were prepared and tested for their antitumor activity. The carbamate 6 (2-hydroxyethylcarbamoyl-DDPT), carbonate 9 (2-chloroethyloxycarbonyl-DDPT), and most of amino acid prodrugs (12-17) showed enhanced antitumor activity.     

Biochemical characterization of oligomerization of Escherichia coli GTP cyclohydrolase I

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8- dihydroneopterin triphosphate (H(2)NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.     

Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.