Structural investigation of a novel rhamnoglucogalactan isolated from the fruiting bodies of the fungus Hericium erinaceus

A new heteropolysaccharide (HEP-1) was isolated from the fruiting bodies of Hericium erinaceus. It was estimated to have a molecular weight of 1.8x10(4) da and showed [alpha](D)(20) +129 (c 0.295, H(2)O). HEP-1 is composed of rhamnose, galactose, and glucose in the ratio of 1.19:3.81:1.00. Its structural features were investigated using composition analysis, methylation analysis, partial hydrolysis, and IR and NMR spectroscopy. The results showed that HEP-1 has a (1-->6)-linked alpha-d-galactopyranosyl backbone with branches that are composed of rhamnose and glucose attached to O-2.     

Axial patterning of C. elegans male sensilla identities by selector genes

The fan and rays of the C. elegans male tail constitute a compound sensory organ essential for mating. Within this organ, the individual sensilla, known as rays, have unique identities. We show that ray identities are patterned by a selector gene mechanism in a manner similar to other serially homologous axial structures. One selector gene that promotes the identities of a subset of the rays is the Hox gene egl-5. Within EGL-5-expressing rays, further patterning is provided by a Pax-6 homolog and a signal of the TGFβ family. These genes and pathway coordinately specify multiple ray properties affecting all three terminal ray cell types. These properties include complex patterns of FMRFamide-like (FaRP) neuropeptides, serotonin (5HT) and dopamine expression, and ray morphology. Differences in these differentiated characteristics give each sensillum a unique identity and potentially endow the compound ray organ with a higher-order information gathering capacity.     

Synthesis, characterization and supramolecular structures of two Ni(II) complexes with potentially heptadentate ligand N,N-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol

A potentially heptadentate ligand H₃L (N,N^′-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol) and its two Ni(II) complexes, [Ni(H₂L)H₂O](H₂O)₃ClO₄ (1) and [Ni(H₂L)(H₂O)](H₂O)Cl (2) were prepared and characterized. X-ray structural analyses indicate that complex 1 has a distorted octahedral coordination geometry, with four amine N atoms of H₂L⁻ defining the equatorial plane, one aqua O atom and one phenoxo O atom of the ligand occupying two axial positions, respectively. The Ni(II) center of 2 has coordination geometry similar to that of 1. IR and electronic spectra of 1 and 2 are in agreement with their crystal structural features. Approximately along the ab plane, 2D supramolecular structure of 1 is assembled through multiple hydrogen bonds between hydroxy groups of the ligands, coordinated and crystal lattice H₂O and π-π stacking interactions between adjacent phenyl rings of the ligands, while for that of 2, probably along the a axis, 1D chain structure is also formed by multiple hydrogen bonds, but lack of π-π stacking interactions.     

Regulation of a gamma-aminobutyric acid transporter by reciprocal tyrosine and serine phosphorylation

A feature of the rat brain gamma-aminobutyric acid transporter GAT1, and other members of the neurotransmitter transporter family, is its regulated redistribution between intracellular locations and the plasma membrane. Recent studies have focused upon defining the signaling molecules that facilitate this redistribution. Agents that promote direct tyrosine phosphorylation of GAT1 promote a relative increase in surface GAT1 levels, and this results from a slowing of the transporter internalization rate. Agents that act to increase protein kinase C (PKC) activity promote a relative decrease in surface GAT1 levels; whether this effect is caused by direct transporter phosphorylation is unknown. The opposing actions of tyrosine kinase activity and PKC activity raise the possibility that the subcellular distribution of GAT1 is associated with mutually exclusive transporter phosphorylation events. The present experiments show that GAT1 is phosphorylated on serine residues in a PKC-dependent manner, but this state is only revealed when GAT1 tyrosine phosphorylation is eliminated or greatly reduced. The relative levels of serine phosphorylation and tyrosine phosphorylation are negatively correlated. The amount of serine phosphorylation is regulated by agents that affect tyrosine phosphorylation, and vice versa. In addition, the ability of agents that affect tyrosine kinase activity to regulate GAT1 serine phosphorylation requires a change in its tyrosine phosphorylation state. These data support the ideas that GAT1 can exist in either of two mutually exclusive phosphorylation states and that the relative abundance of these states determines in part the relative subcellular distribution of the transporter.     

Protein structure preference, tRNA copy number, and mRNA stem/loop content

From statistical analyses of protein sequences for humans and Escherichia coli we found that the messenger RNA segment of m-codons (for m=2 to 6) with average high tRNA copy number (TCN) (larger than approximately 10.5 for humans or approximately 1.95 for E. coli) preferably code for the alpha helix and that with low TCN (smaller than approximately 7.5 for humans or approximately 1.7 for E. coli) preferably code for coil. Between them there is an intermediate region without correlation to structure preference. For the beta strand the preference/ avoidance tendency is not obvious. All strong preference-modes of TCN for protein secondary structures have been deduced. The mutual interaction between two factors--protein secondary structural type and codon TCN--is tested by F distribution. A phenomenological model on the relation between structure preference and translational efficiency or accuracy is proposed. It is pointed out that the structure preference of codons is related to the distribution of mRNA stem/loop content in three TCN regions.     

Antifreeze protein-induced morphological modification mechanisms linked to ice binding surface

The mechanisms by which the antifreeze protein (AFP) modifies the ice morphology are identified precisely as surface poisoning by the ice binding surface (IBS) of insect AFPs and as bridge-induced surface reconstruction by the IBS of fish AFPs and antifreeze glycoproteins. The primary surfaces of hexagonal ice have predetermined face indices. The "two-dimensional" insect type IBS has regularly spaced binding intervals in two directions. It causes surface poisoning by matching and reinforcing simultaneously intersecting strong bonding directions on the primary ice surfaces. The secondary ice surfaces have variable face indices. The "one-dimensional" and "irregular" IBS variants of fish AFPs and antifreeze glycoproteins are either linearly extended with regular ice binding intervals or have ice binding sites lacking spacing regularity. These variants can bridge transversely lattice periods or shorter oxygen-oxygen distances between parallel adjacent strong bonding directions that do not intersect. Thus, one-dimensional and irregular IBS variants induce supplementary bridges cross-wise on selected secondary surfaces by mimicking strong bonding directions that are not present in the ice structure. These proteins cause surfaces with variable face indices, which in the absence of the AFPs would not grow flat, to appear in the morphology. Whereas for the primary ice surfaces it is only the morphological importance that is determined by the experimental conditions, for the secondary ice surfaces it is the face indices themselves that become adjusted in the process of maximizing the AFP-substrate interaction through attainment of the best structural match. The growth morphology of the AFP-ice system is derived from various factors, including the face indices, surface molecular compositions, relative growth rates, and the mechanisms responsible for that morphology. The theoretical formulation agrees with experiments over a wide range and resolves these, to date, unexplained phenomena.     

Inhibition by copper(II) binding of hepatocyte growth factor (HGF) interaction with its receptor Met and blockade of HGF/Met function

Overexpression of hepatocyte growth factor (HGF) and its receptor Met often occurs in carcinoma cells, leading to establishment of an HGF/Met autocrine loop. Therefore, disruption of the HGF/Met autocrine loop may lead to down-regulation of tumorigenesis. To study the HGF/Met interaction, we have developed a cell-free system to detect HGF binding to a Met fusion protein, Met-IgG, using a modified enzyme-linked immunosorbent assay methodology. Since we previously showed that HGF can be purified by copper(II) affinity chromatography, we further explored the effect of copper(II) on the HGF/Met interaction. The divalent metal cations copper(II) and zinc(II) significantly inhibited HGF binding to immobilized Met-IgG with IC(50) values of 230-270 microM, respectively, whereas manganese(II) and magnesium(II) were less inhibitory with 20-60-fold higher IC(50) values. Incubation of 1 mM copper(II) with HGF resulted in nondenaturing and denaturing gel-mobility shifts, indicating that copper(II) binds directly to HGF. This interaction occurs at the N terminus of HGF, as incubation of 1 mM copper(II) with both HGF and the HGF derivative NK1 yielded similar results on SDS-PAGE. HGF-induced activation of Met and cell scattering were inhibited upon addition of HGF in the presence of 1 mM and 500 microM copper(II), respectively. Chemical protonation with diethyl pyrocarbonate of HGF histidine residues impeded the ability of 500 microM copper(II) to inhibit the binding of HGF to immobilized Met-IgG. Based on the NK1 domain structure, we propose that copper(II) may interact with HGF via the histidine residues in either N-terminal or kringle domains. The inhibition of HGF/Met interaction and subsequent downstream cellular functions may be through direct interference by copper(II), such as a change in charge or an induced local conformational change. This putative copper(II) binding domain may be the basis for developing potential inhibitors of HGF/Met binding and downstream functions and could lead to novel strategies for anti-cancer treatment.     

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.     

Anti-chemorepulsive effects of vascular endothelial growth factor and placental growth factor-2 in dorsal root ganglion neurons are mediated via neuropilin-1 and cyclooxygenase-derived prostanoid production

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion.