Characterization of the ��-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.     

Characterization of the Effect of the Mitochondrial Protein Hint2 on?Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca²⁺ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca²⁺ dynamics in this cell type. We found that in hepatocytes isolated from Hint2^−/− mice, the frequency of Ca²⁺ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca²⁺ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2^−/− mice; we found that Hint2 accelerates Ca²⁺ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca²⁺ in suspensions of mitochondria. This prediction was then confirmed experimentally.     

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ?

Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.     

Site–Site Isolation and Site–Site Interaction – Two Sides of the Same Coin

Solid-phase synthesis has contributed to a significant portion of combinatorial libraries made to date. Among many advantages of solid-phase organic synthesis, one unique property is the pseudo-dilution effect. In reality, polymer support is a dynamic system and its structural mobility is controlled by many intrinsic and external factors such as cross-linking, load, solvent, temperature and incoming reagent concentrations. Site-site interaction can often occur and affect the direction of the intended reaction.     

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes)

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.     

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.     

Characterization of the flanking regions of the S-RNase genes of Japanese pear (Pyrus serotina) and apple (Malus×domestica)

Genomic sequences of the self-incompatibility genes, the S-RNase genes, from two rosaceous species, Japanese pear and apple, were characterized. Genomic Southern blot and sequencing of a 4.5-kb genomic clone showed that the S⁴-RNase gene of Japanese pear is surrounded by repetitive sequences as in the case of the S-RNase genes of solanaceous species. The flanking regions of the S²- and S^f-RNase genes of apple were also cloned and sequenced. The 5′ flanking regions of the three alleles bore no similarity with those of the solanaceous S-RNase genes, although the position and sequence of the putative TATA box were conserved. The putative promoter regions of the Japanese pear S⁴- and apple S^f-RNase genes shared a stretch of about 200 bp with 80% sequence identity. However, this sequence was not present in the S²-RNase gene of apple, and thus it may reflect a close relationship between the S⁴- and S^f-RNase genes rather than a cis-element important in regulating gene expression. Despite the uniform pattern of expression of the rosaceous S-RNase genes, sequence motifs conserved in the 5′ flanking regions of the three alleles were not found, implying that the cis-element controlling pistil specific gene expression also locates at the intragenic region or upstream of the analyzed promoter region.     

α-Adducin translocates to the nucleus upon loss of cell-cell adhesions

The F-actin binding protein adducin plays an important role in plasma membrane stability, cell motility and cell-cell junctions. In this study, we demonstrate that α-adducin is mainly localized in the nucleus of sparsely cultured epithelial cells, whereas it is localized at cell-cell junctions when the cells are grown to confluence. Disruption of cell-cell adhesions induces a nuclear translocation of α-adducin. Conversely, α-adducin is redistributed to the cytoplasm and cell-cell junctions in the process of establishing cell-cell adhesions. We identify that α-adducin contains a bipartite nuclear localization signal (NLS) in its COOH-terminal tail domain and a nuclear export signal in its neck region. The phosphorylation of α-adducin at Ser716 that is immediately adjacent to the NLS appears to antagonize the function of the NLS. Moreover, we show that depletion of α-adducin has adverse effects on cell-cell adhesions and, to our surprise, cell proliferation. The impaired cell proliferation is associated with mitotic defects characterized by disorganized mitotic spindles, aberrant chromosomal congregation/segregation and abnormal centrosomes. Taken together, our results not only reveal the mechanism for α-adducin to shuttle between the cytoplasm and nucleus, but also highlight a potential role for α-adducin in mitosis.     

Characterization and Genetic Study of the Ovine α S2 -Casein (CSN1S2) Allele B

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine α _s2 -casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp₇₅ → Tyr₇₅ and Ile₁₀₅ → Val₁₀₅. The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp₇₅ → Tyr₇₅ substitution modifies the protein electric charge, milk properties may result affected to some extent.     

Do Postures of Distal Effectors Affect the Control of Actions of Other Distal Effectors? Evidence for a System of Interactions between Hand and Mouth

The present study aimed at determining whether, in healthy humans, postures assumed by distal effectors affect the control of the successive grasp executed with other distal effectors. In experiments 1 and 2, participants reached different objects with their head and grasped them with their mouth, after assuming different hand postures. The postures could be implicitly associated with interactions with large or small objects. The kinematics of lip shaping during grasp varied congruently with the hand posture, i.e. it was larger or smaller when it could be associated with the grasping of large or small objects, respectively. In experiments 3 and 4, participants reached and grasped different objects with their hand, after assuming the postures of mouth aperture or closure (experiment 3) and the postures of toe extension or flexion (experiment 4). The mouth postures affected the kinematics of finger shaping during grasp, that is larger finger shaping corresponded with opened mouth and smaller finger shaping with closed mouth. In contrast, the foot postures did not influence the hand grasp kinematics. Finally, in experiment 5 participants reached-grasped different objects with their hand while pronouncing opened and closed vowels, as verified by the analysis of their vocal spectra. Open and closed vowels induced larger and smaller finger shaping, respectively. In all experiments postures of the distal effectors induced no effect, or only unspecific effects on the kinematics of the reach proximal/axial component. The data from the present study support the hypothesis that there exists a system involved in establishing interactions between movements and postures of hand and mouth. This system might have been used to transfer a repertoire of hand gestures to mouth articulation postures during language evolution and, in modern humans, it may have evolved a system controlling the interactions existing between speech and gestures.     

Characterization of the interaction between Actinin-Associated LIM Protein (ALP) and the rod domain of α-actinin

Background  

The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown.     

Influence of GABA(A) Receptor α Subunit Isoforms on the Benzodiazepine Binding Site

Classical benzodiazepines, such as diazepam, interact with α_xβ₂γ₂ GABA_A receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and with receptors containing the homologous mutations in α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂. The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and homologous positions in α₂, α₃, α₅ and α₆ with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA_A receptor isoform α₁β₂γ₂, α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂.     

Characterization of the Sulfolobus host–SSV2 virus interaction

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNA^Gly (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus–host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.