Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.     

Chelating Compound,Chrysoidine, Is More Effective in Both Antiprion Activity and Brain Endothelial Permeability Than Quinacrine

1. As an extension of our previous study of quinacrine and its derivatives, chelating chemicals were screened to obtain more effective, better brain-permeable antiprion compounds using either prion-infected neuroblastoma cells or brain capillary endothelial cells. 2. Eleven chemicals were found to have antiprion activity. Most of them shared a common structure consisting of benzene or naphthalene at either end of an azo bond. Structure–activity data suggest that chelating activity is not necessary but might contribute to the antiprion action. 3. Chrysoidine, a representative compound found here, was about 27 times more effective in the antiprion activity and five times more efficiently permeable through the brain capillary endothelial cells than quinacrine was. 4. These chemicals might be useful as compounds for development of therapeutics for prion diseases.     

Physicochemical interactions of amyloid β-peptide with lipid bilayers

The aggregation and deposition onto neuronal cells of amyloid β-peptide (Aβ) is central to the pathogenesis of Alzheimer's disease. Accumulating evidence suggests that membranes play a catalytic role in the aggregation of Aβ. This article summarizes the structures and properties of Aβ in solution and the physicochemical interaction of Aβ with lipid bilayers of various compositions. Reasons for discrepancies between results by different research groups are discussed. The importance of ganglioside clusters in the aggregation of Aβ is emphasized. Finally, a hypothetical physicochemical cascade in the pathogenesis of the disease is proposed.     

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

New oligosaccharyltransferase assay method

We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.     

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).     

C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.