Changes of urine metabolite profiles are induced by inactivated influenza vaccine inoculations in mice.

The safety evaluation of vaccines is critical to avoid the development of side effects in humans. To increase the sensitivity of detection for toxicity tests, it is important to capture not only pathological changes but also physiological changes. 1H nuclear magnetic resonance (NMR) spectroscopy analysis of biofluids produces profiles that show characteristic responses to changes in physiological status. In this study, mouse urine metabolomics analysis with 1H NMR was performed using different influenza vaccines of varying toxicity to assess the usefulness of 1H NMR in evaluating vaccine toxicity. Two types of influenza vaccines were used as model vaccines: a toxicity reference vaccine (RE) and a hemagglutinin split vaccine. According to the blood biochemical analyses, the plasma alanine transaminase levels were increased in RE-treated mice. Changes in metabolite levels between mice administered different types of influenza vaccines were observed in the 1H NMR spectra of urine, and a tendency toward dosage-dependent responses for some spectra was observed. Hierarchical clustering analyses and principal component analyses showed that the changes in various urine metabolite levels allowed for the classification of different types of vaccines. Among them, two liver-derived metabolites were shown to largely contribute to the formation of the cluster. These results demonstrate the possibility that urine metabolomics analysis could provide information about vaccine-induced toxicity and physiological changes.

NMR characterization of the interaction between Bcl-xL and the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 µM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 µM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

A novel neuropilin-1-binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 µM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies.

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Protein stabilizer, NDSB-195, enhances the dynamics of the ß4 -α2 loop of ubiquitin.

Non-detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB-195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB-195 enhances the microsecond-millisecond dynamics of a ß4 -α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB-195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained.

Refolding additive, dimethylbenzylammonium propane sulfonate (NDSB- 256), accelerates gly-pro cis-trans isomerization.

Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding, and many different peptide-proline cis-trans isomerases (PPIases) catalyze this reaction. The acceleration of isomerization would be beneficial for in vitro refolding of protein preparations for industrial and research purposes. So we analyzed whether low-molecular-weight compounds that have been reported to enhance protein refolding have the activity to accelerate the isomerization. To evaluate the effects of chemicals on the isomerization rate, we set up a new NMR (EXSY) method that is invulnerable to their inhibitory activity, if any, and to their large NMR signals. With this method, we found that dimethylbenzylammonium propane sulfonate (NDSB-256) increase the isomerization rate in a concentration-dependent manner for the first time. Acceleration by imidazole (suggested but not experimentally confirmed) was also demonstrated. Arginine, a most popular refolding additive, did not show any significant effects on the isomerization reaction as expected.

IDEAL in 2014 illustrates interaction networks composed of intrinsically disordered proteins and their binding partners.

IDEAL (Intrinsically Disordered proteins with Extensive Annotations and Literature, http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/) is a collection of intrinsically disordered proteins (IDPs) that cannot adopt stable globular structures under physiological conditions. Since its previous publication in 2012, the number of entries in IDEAL has almost tripled (120 to 340). In addition to the increase in quantity, the quality of IDEAL has been significantly improved. The new IDEAL incorporates the interactions of IDPs and their binding partners more explicitly, and illustrates the protein-protein interaction (PPI) networks and the structures of protein complexes. Redundant experimental data are arranged based on the clustering of Protein Data Bank entries, and similar sequences with the same binding mode are grouped. As a result, the new IDEAL presents more concise and informative experimental data. Nuclear magnetic resonance (NMR) disorder is annotated in a systematic manner, by identifying the regions with large deviations among the NMR models. The ordered/disordered and new domain predictions by DICHOT are available, as well as the domain assignments by HMMER. Some examples of the PPI networks and the highly deviated regions derived from NMR models will be described, together with other advances. These enhancements will facilitate deeper understanding of IDPs, in terms of their flexibility, plasticity and promiscuity.

IDEAL: Intrinsically Disordered proteins with Extensive Annotations and Literature.

IDEAL, Intrinsically Disordered proteins with Extensive Annotations and Literature (http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/), is a collection of knowledge on experimentally verified intrinsically disordered proteins. IDEAL contains manual annotations by curators on intrinsically disordered regions, interaction regions to other molecules, post-translational modification sites, references and structural domain assignments. In particular, IDEAL explicitly describes protean segments that can be transformed from a disordered state to an ordered state. Since in most cases they can act as molecular recognition elements upon binding of partner proteins, IDEAL provides a data resource for functional regions of intrinsically disordered proteins. The information in IDEAL is provided on a user-friendly graphical view and in a computer-friendly XML format.

Binary classification of protein molecules into intrinsically disordered and ordered segments.

BACKGROUND: Although structural domains in proteins (SDs) are important, half of the regions in the human proteome are currently left with no SD assignments. These unassigned regions consist not only of novel SDs, but also of intrinsically disordered (ID) regions since proteins, especially those in eukaryotes, generally contain a significant fraction of ID regions. As ID regions can be inferred from amino acid sequences, a method that combines SD and ID region assignments can determine the fractions of SDs and ID regions in any proteome. RESULTS: In contrast to other available ID prediction programs that merely identify likely ID regions, the DICHOT system we previously developed classifies the entire protein sequence into SDs and ID regions. Application of DICHOT to the human proteome revealed that residue-wise ID regions constitute 35%, SDs with similarity to PDB structures comprise 52%, while SDs with no similarity to PDB structures account for the remaining 13%. The last group consists of novel structural domains, termed cryptic domains, which serve as good targets of structural genomics. The DICHOT method applied to the proteomes of other model organisms indicated that eukaryotes generally have high ID contents, while prokaryotes do not. In human proteins, ID contents differ among subcellular localizations: nuclear proteins had the highest residue-wise ID fraction (47%), while mitochondrial proteins exhibited the lowest (13%). Phosphorylation and O-linked glycosylation sites were found to be located preferentially in ID regions. As O-linked glycans are attached to residues in the extracellular regions of proteins, the modification is likely to protect the ID regions from proteolytic cleavage in the extracellular environment. Alternative splicing events tend to occur more frequently in ID regions. We interpret this as evidence that natural selection is operating at the protein level in alternative splicing. CONCLUSIONS: We classified entire regions of proteins into the two categories, SDs and ID regions and thereby obtained various kinds of complete genome-wide statistics. The results of the present study are important basic information for understanding protein structural architectures and have been made publicly available at http://spock.genes.nig.ac.jp/~genome/DICHOT.

Interaction of anti-aggregation agent dimethylethylammonium propane sulfonate with acidic fibroblast growth factor.

Prevention of aggregation is critical for analyzing protein structure. Non-detergent sulfobetaines (NDSBs) are known to prevent protein aggregation, but the molecular mechanisms of their anti-aggregation effect are poorly understood. To elucidate the underlying mechanisms, we analyzed the effects of dimethylethylammonium propane sulfonate (NDSB-195) on acidic fibroblast growth factor (aFGF). NDSB-195 (0.5M) increased both aggregation and denaturation temperatures of aFGF by 4 degrees C. Chemical shift perturbation analyses indicated that many affected residues were located at the junction between a beta-strand (or 3(10)-helix) and a loop, irrespective of the chemical properties of the residue. The apparent dissociation constants of the interaction ranged from 0.04 to 3M, indicating weak interactions between NDSB and protein molecules.

Microwave-assisted dechlorination of polychlorobenzenes by hypophosphite anions in aqueous alkaline media in the presence of Pd-loaded active carbon.

Microwave-assisted dechlorination of chlorobenzene and the three dichlorobenzenes takes place in the presence of the hypophosphite (NaH(2)PO(2)) reductant and Pd-loaded activated carbon (Pd/C) in alkaline media at relatively low temperatures. The extent of loss/dechlorination at 90 degrees C followed the order: o-DCB approximately m-DCB>CB>p-DCB. Detected final products were mostly benzene and phenol. Dechlorination of pentachlorobenzene (PeCB) through reduction was slight even when both NaH(2)PO(2) and Pd/C were simultaneously employed in the absence of NaOH, nor when NaH(2)PO(2) alone was present in excess. The generated HCl proved to be an inhibitor, thus the need for the presence of NaOH to enhance dechlorination. Conventional heating of the reacting mixture above 90 degrees C to a reaction temperature of 180 degrees C led to no further dechlorination of the PeCB. Intermediate products of dechlorination of PeCP were the tetrachlorobenzenes with final products being benzene and phenol (GC-FID spectral analyses). Both salicylic acid (a constituent of humic acid) and l(+)-ascorbic acid used as possible promoters proved to be rather ineffective. The simultaneous presence of NaH(2)PO(2), Pd-loaded activated carbon and NaOH was crucial in the dechlorination of PeCB by microwave dielectric heating with maximal reduction of PeCB being ca. 75% under these conditions.

Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators.

The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.