A novel Pezizomycotina-specific protein with gelsolin domains regulates contractile actin ring assembly and constriction in perforated septum formation.

Septum formation in fungi is equivalent to cytokinesis. It differs mechanistically in filamentous ascomycetes (Pezizomycotina) from that of ascomycete yeasts by the retention of a central septal pore in the former group. However, septum formation in both groups is accomplished by contractile actin ring (CAR) assembly and constriction. The specific components regulating septal pore organization during septum formation are poorly understood. In this study, a novel Pezizomycotina-specific actin regulatory protein GlpA containing gelsolin domains was identified using bioinformatics. A glpA deletion mutant exhibited increased distances between septa, abnormal septum morphology and defective regulation of septal pore closure. In glpA deletion mutant hyphae, overaccumulation of actin filament (F-actin) was observed, and the CAR was abnormal with improper assembly and failure in constriction. In wild-type cells, GlpA was found at the septum formation site similarly to the CAR. The N-terminal 329 residues of GlpA are required for its localization to the septum formation site and essential for proper septum formation, while its C-terminal gelsolin domains are required for the regular CAR dynamics during septum formation. Finally, in this study we elucidated a novel Pezizomycotina-specific actin modulating component, which participates in septum formation by regulating the CAR dynamics.

Development of a protein-ligand-binding site prediction method based on interaction energy and sequence conservation.

We present a new method for predicting protein-ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction.

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α.

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.

Co-lyophilized Aspirin with Trehalose Causes Less Injury to Human Gastric Cells and Gastric Mucosa of Rats.

BACKGROUND: Aspirin is one of the most popular NSAIDs worldwide because of its anti-inflammatory and anticoagulant effects, and however, gastrointestinal injury remains a major complication. We previously reported co-lyophilized aspirin/trehalose (Lyo A/T) decreased the aspirin-induced gastric lesions in dogs. AIM: This study investigated the mechanism of gastroprotective effects of trehalose in vitro and in vivo. METHODS: The apoptotic assays were performed in a human gastric carcinoma cell line, which was treated with aspirin, mixed aspirin/trehalose (Mix A/T) or Lyo A/T. Gastric ulcer severity was examined after oral administration of drugs in rats. In addition, the mucosal tissue apoptotic status in drug-treated rats was evaluated. Molecular dynamics simulations and laser Raman spectroscopy were performed in order to examine the molecular properties of Lyo A/T. RESULTS: DNA fragmentation was detected in AGS cells that were treated with aspirin and Mix A/T, but not in the Lyo A/T-treated cells. There were fewer apoptotic cells in the Lyo A/T-treated cells than in the other cells. Gastric injury was reduced in rats that received oral Lyo A/T compared with the others, while PGE2 synthesis was equally decreased in all groups. TUNEL assay and immunohistochemistry of cleaved caspase-3 in the mucosal tissues also revealed that Lyo A/T treatment induced less apoptosis than the others. The Lyo A/T spectrum showed clear differences in several Raman bands compared with that of Mix A/T. CONCLUSIONS: Our data showed that co-lyophilization of aspirin with trehalose reduced gastric injury, potentially through suppression of aspirin-induced mucosal cell apoptosis while retaining its anti-inflammatory effects.

Exploring the factor on sensory motor function of upper limb associated with executive function in communitydwelling older adults.

Exercise, such as cardiovascular fitness training, has been shown to have utility in improving executive function but is difficult for older adults with low mobility to perform. Accordingly, there is interest in the development of regimens other than high mobility exercises for older adults with low mobility. The aim of the present study was to evaluate the association between sensory motor function of the upper limb and executive function in community-dwelling older adults. A cross-sectional study was conducted in 57 right-handed, independent, community-dwelling older adults. Sensory motor function of upper limb, including range of motion, strength, sensation, finger dexterity, and comprehensive hand function was measured in both hands. Executive function was assessed using the Delta Trail Making Test. Multiple regression analysis indicated the finger dexterity of the non-dominant hand as independently associated with executive function (ß = -0.414, P < 0.001). The findings of the present study may facilitate the development of exercise regimens for improving executive function that are more suitable for older adults with limited physical fitness levels. As this was a cross-sectional study, further studies are required to validate the efficacy of non-dominant finger dexterity training for improving executive function in older adults.

Factors affecting the discharge destination of hip fracture patients who live alone and have been admitted to an inpatient rehabilitation unit.

[Purpose] (1) The aim of this study was to examine relations between clinical and functional assessment and discharge destination and (2) to identify the optimal cutoff point for estimating discharge to home after inpatient rehabilitation. [Subjects] The subjects were 54 hip fracture patients (15 males, 39 females; mean age 81.3 ± 7.4 years) living alone. [Methods] The patients were classified into two groups: those discharged to home and those admitted to an institution. Age, gender, side of fracture, fracture type, number of comorbidities, Functional Independence Measure motor score, and Functional Independence Measure cognitive score were compared between groups. Multiple logistic regression analysis was conducted with discharge to home as the dependent variable and age, gender, side of fracture, fracture type, number of comorbidities, Functional Independence Measure motor score, and Functional Independence Measure cognitive score as independent variables. A receiver operating characteristic curve analysis was used to identify a cutoff point for classification of the patients into the two groups. [Results] Multiple logistic regression analysis showed that the Functional Independence Measure cognitive score was a significant variable affecting the discharge destination. The receiver operating characteristic curve analysis revealed that discharge to home was predicted accurately by a Functional Independence Measure cognitive score of 23.5. [Conclusion] Information from this study is expected to be useful for determining discharge plans and for the setting of treatment goals.

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.

Structural basis of the divergent oxygenation reactions catalyzed by the rieske nonheme iron oxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.

Large-scale identification of genes involved in septal pore plugging in multicellular fungi.

Multicellular filamentous fungi have septal pores that allow cytoplasmic exchange, and thus connectivity, between neighboring cells in the filament. Hyphal wounding and other stress conditions induce septal pore closure to minimize cytoplasmic loss. However, the composition of the septal pore and the mechanisms underlying its function are not well understood. Here, we set out to identify new septal components by determining the subcellular localization of 776 uncharacterized proteins in a multicellular ascomycete, Aspergillus oryzae. The set of 776 uncharacterized proteins was selected on the basis that their genes were present in the genomes of multicellular, septal pore-bearing ascomycetes (three Aspergillus species, in subdivision Pezizomycotina) and absent/divergent in the genomes of septal pore-lacking ascomycetes (yeasts). Upon determining their subcellular localization, 62 proteins were found to localize to the septum or septal pore. Deletion of the encoding genes revealed that 23 proteins are involved in regulating septal pore plugging upon hyphal wounding. Thus, this study determines the subcellular localization of many uncharacterized proteins in A. oryzae and, in particular, identifies a set of proteins involved in septal pore function.

Prospects for tertiary structure prediction of RNA based on secondary structure information.

We developed a method, called RNA Assembler using Secondary Structure Information Effectively (RASSIE), for predicting RNA tertiary structures using known secondary structure information. We attempted a fragment assembly-based method that uses a secondary structure-based fragment library. For several typical target structures such as stem-loops, bulge-loops, and 2-way junctions, our method provided numerous good quality candidate structures in less computational time than previously proposed methods. By using a high-resolution potential energy function, we were able to select good predicted structures from candidate structures. This method of efficient conformational search and detailed structure evaluation using high-resolution potential is potentially useful for the tertiary structure prediction of RNA.

Maintenance and turnover of Sox2+ adult stem cells in the gustatory epithelium.

Continuous turnover of taste bud cells in the oral cavity underlies the homeostasis of taste tissues. Previous studies have demonstrated that Sox2+ stem cells give rise to all types of epithelial cells including taste bud cells and non-gustatory epithelial cells in the oral epithelium, and Sox2 is required for generating taste bud cells. Here, we show the dynamism of single stem cells through multicolor lineage tracing analyses in Sox2-CreERT2; Rosa26-Confetti mice. In the non-gustatory epithelium, unicolored areas populated by a cluster of cells expressing the same fluorescent protein grew over time, while epithelial cells were randomly labeled with multiple fluorescent proteins by short-term tracing. Similar phenomena were observed in gustatory epithelia. These results suggest that the Sox2+ stem cell population is maintained by balancing the increase of certain stem cells with the reduction of the others. In the gustatory epithelia, many single taste buds contained cells labeled with different fluorescent proteins, indicating that a single taste bud is composed of cells derived from multiple Sox2+ stem cells. Our results reveal the characteristics of Sox2+ stem cells underlying the turnover of taste bud cells and the homeostasis of taste tissues.

Anti-metatype peptides, a molecular tool with high sensitivity and specificity to monitor small ligands.

To develop a new immunological detection system of gibberellins (GAs), a class of phytohormones, peptides that interact with an antibody against GA4 in a GA4-dependent manner, were screened from phage display random peptide libraries. The biopanning procedure yielded peptides designated as anti-metatype peptides (AM-peps), which showed specific binding to the complex of the antibody and its ligand GA4; that is, the antibody could not be replaced with the other anti-GA4 antibody, and GA4 could not be replaced with GA1, another ligand of the antibody. Together with computational analyses such as analysis of structural propensity of the AM-peps and docking simulation of the AM-peps and the 8/E9-GA4 complex, it was suggested that AM-peps formed a helix in their central region and interacted with a part of the 8/E9-GA4 complex located in close proximity to the GA4 molecule. Based on the property of AM-peps to make a ternary complex with antibody and its ligand, a noncompetitive enzyme-linked immunosorbent assay (ELISA) system corresponding to sandwich ELISA was developed to detect GA4. GA4 as low as 30 pg, which could not be achieved by conventional competitive ELISA, could be detected by the new system, demonstrating the feasibility of this system.

Comprehensive analysis of sequence-structure relationships in the loop regions of proteins.

Local sequence-structure relationships in the loop regions of proteins were comprehensively estimated using simple prediction tools based on support vector regression (SVR). End-to-end distance was selected as a rough structural property of fragments, and the end-to-end distances of an enormous number of loop fragments from a wide variety of protein folds were directly predicted from sequence information by using SVR. We found that our method was more accurate than random prediction for predicting the structure of fragments comprising 5, 9, and 17 amino acids; moreover, the extended loop fragments could be successfully distinguished from turn structures on the basis of their sequences, which implies that the sequence-structure relationships were significant for loop fragments with a wide range of end-to-end distances. These results suggest that many loop regions as well as helices and strands restrict the conformational space of the entire tertiary structure of proteins to some extent; moreover, our findings throw light on the mechanism of protein folding and prediction of the tertiary structure of proteins without using structural templates.

Using a new GPI-anchored-protein identification system to mine the protein databases of Aspergillus fumigatus, Aspergillus nidulans, and Aspergillus oryzae.

Computational approaches provide valuable information to start experimental surveys identifying glycosylphosphatidylinositol (GPI)-anchored proteins in protein sequence databases. We developed a new sequence-based identification system that uses an optimized classifier based on a support vector machine (SVM) algorithm to recognize appropriate COOH-terminal sequences and uses a classifier implementing a simple majority voting strategy to recognize appropriate NH2-terminal sequences. The SVM classifier showed high accuracy (96%) in 5-fold cross-validation testing, and the majority voting classifier showed high recall (98.88%) when applied to a test dataset of eukaryote proteins. When applied to S. cerevisiae protein sequences, the new identification system showed good ability to classify "unseen" data. Applying our system to protein sequences of three aspergilli, we identified 115 GPI-anchored proteins in Aspergillus fumigatus, 129 in Aspergillus nidulans, and 136 in Aspergillus oryzae. Sequence-based conserved domain search found nearly half of these proteins to have conserved domains that covered a wide range of functions.

Highly accurate method for ligand-binding site prediction in unbound state (apo) protein structures.

This article describes a new method for predicting ligand-binding sites of proteins. The method involves calculating the van der Waals interaction energy between a protein and probes placed on the protein surface, and then clustering the probes with attractive interaction to find the energetically most favorable locus. In 80% (28/35) of the test cases, the ligand-binding site was successfully predicted on a ligand-bound protein structure, and in 77% (27/35) was successfully predicted on an unbound structure. Our method was used to successfully predict ligand-binding sites unaffected by induced-fit as long as its scales were not very large, and it contributed to a significant improvement in prediction with unbound state protein structures. This represents a significant advance over conventional methods in detecting ligand-binding sites on uncharacterized proteins. Moreover, our method can predict ligand-binding sites with a narrower locus than those achieved using conventional methods.

Alteration of the substrate specificity of the angular dioxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.

Significance of NS5B Substitutions in Genotype 1b Hepatitis C Virus Evaluated by Bioinformatics Analysis.

To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.0%, respectively. Genotype 1b HCV strains with NS5B-C316N mutation were located in the leaves different from those in which HCV strains without such substitutions were present on the phylogenetic tree. Structural modeling revealed that amino acid 218 was located on the surface of the NTP tunnel. Free energy analysis based on molecular dynamics simulations demonstrated that the free energy required to pass through the tunnel was larger for triphosphate SOF than for UTP in NS5B polymerase carrying A218S, but not in wild-type. However, no susceptibility change was observed for these substitutions to SOF in replicon assay. Furthermore, the SVR rate was 100% in patients enrolled the Phase-3 trial. In conclusion, NS5B A218S and C316N were detected in all patients who relapsed following LDV/SOF in real-world practice. These substitutions did not impact the overall SVR rate after LDV/SOF, however, further studies are needed to elucidate the impact of these substitutions.

Crystal structure of the 2-iminoglutarate-bound complex of glutamate dehydrogenase from Corynebacterium glutamicum.

The NADP+ -dependent glutamate dehydrogenase from Corynebacterium glutamicum (CgGDH) is considered to be one of the key enzymes in the industrial fermentation of glutamate due to its high glutamate-producing activity. We determined the crystal structure of CgGDH complexed with NADP+ and 2-iminoglutarate. Among six subunits of hexameric CgGDH-binding NADP+ , only four subunits bind 2-iminoglutarate in a closed form, while the other two are in an open form. In the closed form, 2-iminoglutarate is bound to the substrate-binding site with the 2-imino group stacked by the nicotinamide ring of the coenzyme, suggesting a prehydride transfer state in a hypothesized reaction scheme with the imino intermediate. We also conducted MD simulations and provide insights into the extreme preference for the glutamate-producing reaction of CgGDH. DATABASE: The atomic coordinate and structure factors have been deposited in the RCSB PDB database under the accession number 5GUD.

Development of a sugar-binding residue prediction system from protein sequences using support vector machine.

Several methods have been proposed for protein-sugar binding site prediction using machine learning algorithms. However, they are not effective to learn various properties of binding site residues caused by various interactions between proteins and sugars. In this study, we classified sugars into acidic and nonacidic sugars and showed that their binding sites have different amino acid occurrence frequencies. By using this result, we developed sugar-binding residue predictors dedicated to the two classes of sugars: an acid sugar binding predictor and a nonacidic sugar binding predictor. We also developed a combination predictor which combines the results of the two predictors. We showed that when a sugar is known to be an acidic sugar, the acidic sugar binding predictor achieves the best performance, and showed that when a sugar is known to be a nonacidic sugar or is not known to be either of the two classes, the combination predictor achieves the best performance. Our method uses only amino acid sequences for prediction. Support vector machine was used as a machine learning algorithm and the position-specific scoring matrix created by the position-specific iterative basic local alignment search tool was used as the feature vector. We evaluated the performance of the predictors using five-fold cross-validation. We have launched our system, as an open source freeware tool on the GitHub repository (https://doi.org/10.5281/zenodo.61513).

Upper-limb motor and sensory function in patients with hip fracture: Comparison with community-dwelling older adults.

BACKGROUND: Upper-limb function is important in patients with hip fracture so they can perform activities of daily living and participate in leisure activities. Upper-limb function of these patients, however, has not been thoroughly investigated. OBJECTIVE: The aim of this study was to evaluate the upper-limb motor and sensory functions in patients with hip fracture by comparing these functions with those of community-dwelling older adults (control group). METHODS: We compared the results of motor and sensory function tests of upper-limb function - range of motion, strength, sensibility, finger dexterity, comprehensive hand function - between patients with hip fracture (n= 32) and the control group (n= 32). RESULTS: Patients with hip fracture had significantly reduced grip strength, pinch strength, finger dexterity, and comprehensive hand function compared with the control group. CONCLUSIONS: Most upper-limb functions are impaired in the patients with hip fracture. Thus, upper-limb function of patients with hip fracture should be considered during treatment.