Protein anatomy: structure and function of peptide fragments corresponding to the secondary structure units of barnase

Globular proteins can be decomposed into several modules or secondary structure units. It is useful to investigate the functions of such structural units in order to understand the folding units of proteins. In our previous work, barnase was divided into six peptide fragments corresponding to modules, and some of them were shown to have RNA-binding and RNase activity [Yanagawa, et al. (1993) J. Biol. Chem. 268, 5861-5865]. Barnase mutant proteins obtained by permutation of the structural units also had RNase activity [Tsuji, T. et al. (1999) J. Mol. Biol. 286, 1581-1596]. Here we investigated the structure and function of peptide fragments corresponding to secondary structure units of barnase. The results of circular dichroism spectroscopy indicated that some of the peptide fragments form helical structures in aqueous solutions containing over 30% 2,2,2-trifluoroethanol, and the S6 (94-110) peptide fragment is induced to form a beta-sheet structure in the presence of RNA. The S6 peptide fragment forms aggregate complexes with RNA. Electron microscopic analysis showed that the aggregate complexes were comprised of filaments. These results indicate that not only modules but also secondary structure units dissected from a globular protein have functional and structure-forming capabilities.     

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.     

Axin and the Axin/Arrow-binding protein DCAP mediate glucose-glycogen metabolism

Axin was found as a negative regulator of the canonical Wnt pathway. Human LRP5 was originally found as a candidate gene of insulin dependent diabetes mellitus (IDDM), but its Drosophila homolog, Arrow, works as a co-receptor of the canonical Wnt signal. In our previous paper, we found a new Drosophila Axin (Daxin)-binding SH3 protein, DCAP, a homolog of mammalian CAV family protein. Among the subtypes, DCAPL3 shows significant homology with CAP, an essential component of glucose transport in insulin signal. Further binding assay revealed that DCAP binds to not only Axin but also Arrow, and Axin binds to not only GSK3beta but also Arrow. However, overexpression and RNAi experiments of DCAP do not affect the canonical Wnt pathway. As DCAP is expressed predominantly in insulin-target organs, and as RNAi of DCAP disrupts the pattern of endogenous glycogen accumulation in late stage embryos, we suggest that DCAP is also involved in glucose transport. Moreover, early stage embryos lacking maternal Axin show significant delay of initial glycogen decomposition, and RNAi of Axin in S2 cells revealed quite increase of endogenous glycogen level as well as GSK3beta. These results suggest that Axin and DCAP mediate glucose-glycogen metabolism in embryo. In addition, the interaction among Axin, Arrow, and DCAP implies a possible cross-talk between Wnt signal and insulin signal.