Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.     

Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts

Acid-sensing ion channel 3 (ASIC3) is a H(+)-gated cation channel primarily found in sensory neurons, where it may function as a pH sensor in response to metabolic disturbances or painful conditions. We previously found that ASIC3 interacts with the postsynaptic density protein PSD-95 through its COOH terminus, which leads to a decrease in ASIC3 cell surface expression and H(+)-gated current. PSD-95 has been implicated in recruiting proteins to lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids that organize receptor/signaling complexes. We found ASIC3 and PSD-95 coimmunoprecipitated within detergent-resistant membrane fractions. When cells were exposed to methyl-beta-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts, PSD-95 localization to lipid raft fractions was abolished and no longer inhibited ASIC3 current. Likewise, mutation of two cysteine residues in PSD-95 that undergo palmitoylation (a lipid modification that targets PSD-95 to lipid rafts) prevented its inhibition of ASIC3 current and cell surface expression. In addition, we found that cell surface ASIC3 is enriched in the lipid raft fraction. These data suggest that PSD-95 and ASIC3 interact within lipid rafts and that this raft interaction is required for PSD-95 to modulate ASIC3.     

Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis

Hevea brasiliensis Müll. Arg. is a tree that produces natural rubber, an industrially vital isoprenoid polymer. Biosynthesis of natural rubber is known to take place biochemically by a mevalonate (MVA) pathway, but molecular biological characterization of related genes has been insufficient. From H. brasiliensis, we obtained full-length cDNA of genes encoding all of the enzymes that catalyze the six steps of the MVA pathway. Alignment analysis and phylogenetic analysis revealed that in H. brasiliensis there are three acetyl-CoA acetyltransferase genes, two HMG-CoA synthase (HMGS) genes, and four HMG-CoA reductase (HMGR) genes. Gene expression analysis by type of tissue indicated that MVA pathway genes were highly expressed in latex, as compared to other types of tissue and that HMGS and HMGR, which exist in multiple copies, have different expression patterns. Moreover, these MVA pathway genes in H. brasiliensis were found to complement MVA pathway deletion mutations in yeast.     

Polymerized structure and improved physical and biochemical material properties of carboxymethyl dextran-conjugated collagen

Pepsin-solubilized collagen (PSC) was conjugated with carboxymethyl dextran (CMD) using cyanogen bromide to obtain a PSC-CMD film having improved physical properties, physiological properties, and cell affinity. The conjugation was confirmed by the loss of the alpha- and beta-subunit chains and the polymerized band on SDS-PAGE, and by a decrease in the isoelectric point to 3.2. PSC-CMD had a large polymerized structure with the 6 PSC and 228 CMD molecules. PSC-CMD was readily soluble in water, reconstructed a matrix with a less-ordered structure and a characteristic morphological shape, and lost platelet aggregation-inducing ability. The PSC-CMD film, cross-linked by ultraviolet irradiation, exhibited reduced solubility, moderate water vapor permeability, and increased flexibility. PSC-CMD coatings exhibited good cell attachment and growth for fibroblasts and vein endothrical cells.     

Potent CCR4 antagonists: synthesis, evaluation, and docking study of 2,4-diaminoquinazolines

A series of CC chemokine receptor-4 (CCR4) antagonists were examined in a previous report in an attempt to improve metabolic stability in human liver microsomes. In this study, the cycloheptylamine moiety of N-cycloheptyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine 1 was replaced with the p-chloroaniline moiety, and the resulting compound, N-(4-chlorophenyl)-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine (8c), retained its potency ([(35)S]GTPgammaS-binding inhibition and CCL22-induced chemotaxis in humans/mice). Based on the structure-activity relationships (SAR), a homology model was constructed for CCR4 to explain the binding mode of 8c. Overall, there was good agreement between the docking pose of the CCR4 homology model and the human [(35)S]GTPgammaS assay results. Administration of 8c in a murine model of acute dermatitis showed anti-inflammatory activity (oxazolone-induced contact hypersensitivity test).     

Computer based design, synthesis and biological evaluation of novel indole derivatives as HCV NS3-4A serine protease inhibitors

A series of novel indoles were designed and their molecular modeling simulation study including fitting to a 3D pharmacophore model using CATALYST program and their docking into the NS3 active site was examined as HCV NS3 protease inhibitor. Several compounds showed significant high simulation docking score and fit values. The designed compounds were synthesized and biologically evaluated in vitro using an NS3 protease binding assay, where compounds 10a-k showed significant inhibitory activity (> or =67% inhibition at 100 microg/mL). Of these, compounds 10c and 10f demonstrated potent HCV NS3 protease inhibitors with IC(50) values of 15 and 13 microM, respectively. Enantio-selective Michael addition of an indole derivative in the presence of catalytic amount of AlCl(3) and quinine at room temperature afforded the adduct 7e in excellent yield with 73% ee. The product was converted into 10l, which showed lower activity than the mixture of the corresponding diastereoisomers.     

Profiling the phenolic compounds of Artemisia pectinata by HPLC-PAD-MSn

An HPLC-PAD-MS(n) method was employed to profile the phenolic compounds of the aerial part of Artemisia pectinata (Neopallasia pectinata), a plant with no previous reports concerning its phenolic constituents. Three isomers of trans-caffeoylquinic acid accompanied by cis-5-caffeoylquinic acid, six isomers of trans-dicaffeoylquinic acid, two isomers of methyl trans-dicaffeoylquinate (including one new isomer), a trans-caffeoylferuloylquinic acid and three flavanoids were identified unambiguously by analysis of their UV and MS(n) spectra in comparison with standard compounds that were isolated from natural sources, or synthesised, or were surrogate standards (green coffee extract). Other compounds were identified by analysis of their UV and MSn data in comparison with those reported in the literature. MS(n) experiments also suggested the presence of groups of dicaffeoylquinic acid glycosides, caffeoylquinic acid diglycosides, caffeoylquinic acid glycosides and quinic acid diglycosides.     

NaCl improves siRNA delivery mediated by nanoparticles of hydroxyethylated cationic cholesterol with amido-linker

We investigated the effect of amido- (NP-OH) and carbamate linkers (NP-HAPC) in nanoparticles composed of hydroxyethylated cationic cholesterol on siRNA transfection. The presence of NaCl in forming a NP-OH nanoplex increased the suppressive effect of gene expression by increasing the size of the nanoplex and changing the cellular uptake mechanism from membrane fusion and clathrin-mediated endocytosis to clathrin- and caveolae-mediated endocytosis.