Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin

A non-visual pigment melanopsin, which is localized in photosensitive retinal ganglion cells and is involved in the circadian photoentrainment and pupillary responses in mammals, is phylogenetically close to the visual pigments of invertebrates, such as insects and cephalopods. Recent studies suggested that melanopsin is a bistable pigment and drives a Gq-mediated signal transduction cascade, like the invertebrate visual pigments. Because detailed electrophysiological properties are somewhat different between the visual cells and the photosensitive ganglion cells, we here expressed and purified the invertebrate visual pigment and melanopsin to comparatively investigate their Gq-activation abilities. We successfully expressed and purified UV and blue light-sensitive visual pigments of the honeybee as well as the amphioxus melanopsin. Although the purified UV-sensitive pigment and the melanopsin lost their bistable nature during purification, reconstitution of the pigments in lipid vesicles resulted in return of the bistable nature. The light-dependent Gq-activation abilities among these reconstituted pigments are similar, suggesting that the electrophysiological differences do not depend on the Gq-activation step but rather on the other signal transduction steps and/or on cell properties. Our findings are also important in that this is the first report describes a heterologous large-scale expression of the Gq-coupled invertebrate visual pigments in cultured cells.     

Phosphatidic acid metabolism regulates the intracellular trafficking and retrotranslocation of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of DeltaF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of DeltaF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.     

Radical and superoxide scavenging activities of matairesinol and oxidized matairesinol

The radical and superoxide scavenging activities of oxidized matairesinols were examined. It could be assumed that the free benzylic position was important for higher radical scavenging activity. The different level of activity was observed between 7'-oxomatairesinol (Mat 2) and 7-oxomatairesinol (Mat 3). The activity of 8-hydroxymatairesinol was lower than that of matairesinol (Mat 1). The superoxide scavenging activity of the oxidized matairesinols was also demonstrated for the first time. It is assumed that the pKa value of phenol in the oxidized matairesinols affected this activity.     

Statistical Validation of Two Sample Comparison Methods for Oligonucleotide Microarray in Rat Ischemia Model

In gene expression analyses using a high-density oligonucleotide array in a rat ischemia model, two comparison methods, “pair-wise comparison” and “sample average comparison”, were evaluated based on statistical methods. The reliability of the elements screened with a 1.2 to 10-fold threshold was also evaluated. In pair-wise comparisons, most of the elements were significantly independent of the threshold value, with the percentage of significant elements remaining above 95%, when screened at 2.5-fold or higher threshold value. Pair-wise comparison structurally provided strict screening, which resulted in genes that were not selected despite significant alterations in expression. Screening by “sample average comparison” resulted in elements with low probability of significance, which suggested the necessity for increasing the reliability by additional statistical analyses after screening. When genes with altered expression were screened using an oligonucleotide array, marked differences in the numbers and reliability were proved to exist among elements screened by each sample comparison method.     

High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.     

Chondroitin sulfate characterized by the E-disaccharide unit is a potent inhibitor of herpes simplex virus infectivity and provides the virus binding sites on gro2C cells

Although cell surface chondroitin sulfate (CS) is regarded as an auxiliary receptor for binding of herpes simplex virus to cells, and purified CS chain types A, B, and C are known to interfere poorly or not at all with the virus infection of cells, we have found that CS type E (CS-E), derived from squid cartilage, exhibited potent antiviral activity. The IC(50) values ranged from 0.06 to 0.2 mug/ml and substantially exceeded the antiviral potency of heparin, the known inhibitor of virus binding to cells. Furthermore, in mutant gro2C cells that express CS but not heparan sulfate, CS-E showed unusually high anti-herpes virus activity with IC(50) values of <1 ng/ml. Enzymatic degradation of CS-E with chondroitinase ABC abolished its antiviral activity. CS-E inhibited the binding to cells of the purified virus attachment protein gC. A direct interaction of gC with immobilized CS-E and inhibition of this binding by CS-E oligosaccharide fragments greater than octasaccharide were demonstrated. Likewise, the gro2C-specific CS chains interfered with the binding of viral gC to these cells and were found to contain a considerable proportion (13%) of the E-disaccharide unit, suggesting that this unit is an essential component of the CS receptor for herpes simplex virus on gro2C cells and that the antiviral activity of CS-E was due to interference with the binding of viral gC to a CS-E-like receptor on the cell surface. Knowledge of the determinants of antiviral properties of CS-E will help in the development of inhibitors of herpes simplex virus infections in humans.     

A novel induced-fit reaction mechanism of asymmetric hot dog thioesterase PAAI

Hot dog fold proteins sharing the characteristic "hot dog" fold are known to involve certain coenzyme A binding enzymes with various oligomeric states. In order to elucidate the oligomerization-function relationship of the hot dog fold proteins, crystal structures of the phenylacetate degradation protein PaaI from Thermus thermophilus HB8 (TtPaaI), a tetrameric acyl-CoA thioesterase with the hot dog fold, have been determined and compared with those of other family members. In the liganded crystal forms with coenzyme A derivatives, only two of four intersubunit catalytic pockets of the TtPaaI tetramer are occupied by the ligands. A detailed structural comparison between several liganded and unliganded forms reveals that a subtle rigid-body rearrangement of subunits within 2 degrees upon binding of the first two ligand molecules can induce a strict negative cooperativity to prevent further binding at the remaining two pockets, indicating that the so-called "half-of-the-sites reactivity" of oligomeric enzymes is visualized for the first time. Considering kinetic and mutational analyses together, a possible reaction mechanism of TtPaaI is proposed; one tetramer binds only two acyl-CoA molecules with a novel asymmetric induced-fit mechanism and carries out the hydrolysis according to a base-catalyzed reaction through activation of a water molecule by Asp48. From a structural comparison with other family members, it is concluded that a subgroup of the hot dog fold protein family, referred to as "asymmetric hot dog thioesterases" including medium chain acyl-CoA thioesterase II from Escherichia coli and human thioesterase III, might share the same oligomerization mode and the asymmetric induced-fit mechanism as observed in TtPaaI.     

AIP1/Alix is a binding partner of Sendai virus C protein and facilitates virus budding

The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.     

Differential roles of human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases in drug metabolism

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.