Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Enhancement of IgA production by membrane vesicles derived from Bifidobacterium longum subsp. infantis.

Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis toward host immune cells. Bifidobacterium infantis MVs consisted of a cytoplasmic membrane, and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.

Influence of mutation in the regulatory domain of α-isopropylmalate synthase from Saccharomyces cerevisiae on its activity and feedback inhibition.

Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Intrinsic nucleic acid-binding activity of Chp1 chromodomain is required for heterochromatic gene silencing.

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met.

Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75(NTR). The BDNF pro-peptide also enhances NMDA-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.

Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.

Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes.

2-Hydrazinoquinoline: a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry to detect gaseous carbonyl compounds.

The sensitivity, range of applications, and reaction mechanism of 2-hydrazinoquinoline as a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were examined. Using a reaction chamber (125L) equipped with a stirring fan and a window for moving the MALDI-MS plate and volatile samples in and out, the sensitivities of 2-hydrazinoquinoline to gaseous aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and n-butyraldehyde) and ketones (acetone, methyl ethyl ketone, and methyl isobutyl ketone) were determined to be at least parts per million (ppm) levels. On the other hand, carboxylic acids (formic acid, acetic acid, propionic acid, and butyric acid) and esters (ethyl acetate, pentyl acetate, isoamyl acetate, and methyl salicylate) could not be detected by 2-hydrazinoquinoline in MALDI-MS. In addition to 2,4-dinitrophenylhydrazine, a common derivatization reagent for analyzing carbonyl compounds quantitatively in gas chromatography and liquid chromatography, the dissolution of 2-hydrazinoquinoline in an acidic solution, such as trifluoroacetic acid, was essential for its function as a reactive matrix for MALDI- MS.

Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications.

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

Characterization of the membrane vesicle fraction from Acetobacter sp. WSS15.

A bacterium that produces membrane vesicles (MVs), strain WSS15, was isolated from a traditional vinegar in Japan called Kurozu. A phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium belongs to the genus Acetobacter. MVs and peptidoglycan-associated lipoprotein (Pal) were detected in the MV fraction of strain WSS15. In the presence of the WSS15 MV fraction, murine macrophages produced the pro-inflammatory cytokine interleukin-6 (IL-6) via the recognition by superficial Toll-like receptor 2 (TLR2). WSS15 MVs adhered to the cell surface of macrophages. The macrophages secreted IL-6 through the TLR2 recognition of an acylated N-terminal peptide of Pal. We elucidated the mode of action of WSS15 MVs on immune cells and identified the Pal peptide from strain WSS15 as an agonist of TLR2.

Crystal structure of the N-terminal domain of the yeast general corepressor Tup1p and its functional implications.

The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) → Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.

Structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii.

The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 Å. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).

Expression, refolding, and purification of active diacetylchitobiose deacetylase from Pyrococcus horikoshii.

A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GlcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GlcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80°C) Ph-Dac was obtained from 1L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GlcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GlcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac.

Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.

Characterization of extracellular vesicles from Lactiplantibacillus plantarum.

We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1ß and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.

Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus.

Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2(1), with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, ß = 95.41°.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1-22), an N-terminal region (NTR; residues 23-135) and a catalytic domain (residues 136-324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23-324) was successfully determined. The crystal diffracted to 1.9 Šresolution and belonged to space group I212121. The catalytic domain (residues 136-321) exhibited a (ß/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23-135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Mass spectrometric analysis using ruthenium (II)-labeling for identification of glycosyl hydrolase product.

Analysis of products digested by glycosyl hydrolases helps understanding of the hydrolysis mechanism and the substrate recognition in the enzymes. We developed a new universal technique, which consists of ruthenium (II) complex labeling and mass spectrometry analysis, to identify the reducing sugars released from oligosaccharides by enzymatic digestion. This method was applied to enzymatic digestion by chitinase and cellulase of the hyperthermophilic archaea Pyrococcus fusiosus and Pyrococcus horikoshii respectively.

Distinct molecular assembly of homologous peroxiredoxins from Pyrococcus horikoshii and Thermococcus kodakaraensis.

Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration.