Role of odorant binding protein C12 in the response of Tribolium castaneum to chemical agents.

Tribolium castaneum is a worldwide pest of stored grain that mainly damages flour, and not only causes serious loss of flour quality but also leads to deterioration of flour quality. Chemical detection plays a key role in insect behavior, and the role of odorant-binding proteins (OBPs) in insect chemical detection has been widely studied. OBPs can interact with small molecule compounds and thereby modulate variation in insecticide susceptibility in insects. In this study, a total of 65 small molecule compounds are selected to investigate the bound effect with TcOBP C12. The molecular docking results showed that ß-caryophyllene, (-)-catechin, butylated hydroxytoluene, diphenyl phthalate and quercetin were the top five compounds, with docking binding energies of -6.11, -5.25, -5.09, -5.05, and - 5.03 Kcal/mol, respectively. Molecular dynamics analysis indicated that odorant binding protein C12 (TcOBP C12) exhibited high binding affinity to all five tested chemical ligands, evidenced by fluorescence quenching assay in vitro. In addition, the contact toxicity assay results suggested that these chemical agents caused a dose-dependent increase in mortality rate for T. castaneum adults. The TcOBP C12 gene was upregulated >2 times after a 24-h exposure, indicating that OBP C12 may play an important role for T. castaneum in response to these chemical agents. In conclusion, our results provide a theoretical basis for future insecticide experiments and pest management.

Analysis of the effect of metal ions on the ability of Xylanase to hydrolyze wheat bran by molecular dynamics simulations.

Introduction: Wheat bran is the main by-product of wheat processing, containing about 30% pentosan and 0.4%-0.7% ferulic acid. Wheat bran is the main raw material used to prepare feruloyl oligosaccharides by hydrolysis of Xylanase, we discovered that the ability of Xylanase to hydrolyze wheat bran could be affected in the presence of different metal ions. Methods: In the present study, we have probed the effects of different metal ions on the hydrolysis activity of Xylanase on wheat bran and tried to analyze the effect of Mn2+ and Xylanase by molecular dynamic (MD) simulation. Results: Our results suggested that Mn2+ had improved the Xylanase hydrolyzing wheat bran to obtain feruloyl oligosaccharides. Particularly when the concentration of Mn2+ reached 4 mmol/L, the optimal product has been obtained 2.8 times higher to compare with no addition. Through the MD simulation analysis, our results reveal that Mn2+ can induce structural change in the active site, which enlarges the substrate binding pocket. The simulation results also revealed that the addition of Mn2+ resulted in a low RMSD value compared with the absence of Mn2+ and helped stabilize the complex. Conclusion: Mn2+ could increase the enzymatic activity of Xylanase in the hydrolysis of feruloyl oligosaccharides in wheat bran. The finding could have significant implications for the preparation of feruloyl oligosaccharides from wheat bran.

Immobilization of the extracellular recombinant Lucky9 xylanase from Bacillus subtilis enhances activity at high temperature and pH.

In the paper industry, chlorine is often used to treat the pulp for bleaching. After pulping, a large amount of xylan is present in the fiber. Xylanase can be used to degrade xylan in an eco-friendly process called biobleaching, which can help minimize the usage of chlorine in the delignification process. However, a bottleneck in the adoption of biobleaching is the cost of xylanase and the requirement that xylanase be active and stable at extreme conditions. Here, we investigated whether using sodium alginate beads to immobilize an extracellular xylanase from Bacillus subtilis (Lucky9) can reduce the potential cost of enzyme usage. The optimal pH and the activity of the immobilized enzyme were increased at optimal temperature compared with the free enzyme. In addition, immobilized xylanase was shown to be more stable than free xylanase. The results of this study suggest that the immobilized xylanase has potential applications in the biobleaching industry.

The ATPase BRG1/SMARCA4 is a protein interaction platform that recruits BAF subunits and the transcriptional repressor REST/NRSF in neural progenitor cells.

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

Characterization of a ß-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment.

A highly efficient ß-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa ß-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa ß-1,4-mannanase showed the highest activity toward locust bean gum (V max = 1,990 U/mg protein, K m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa ß-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway(®) (113 mg/g-substrate), P. adiposa ß-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa ß-1,4-mannanase a good candidate as a new commercial ß-1,4-mannanase to improve biomass pretreatment.

Characterization of an acid-tolerant ß-1,4-glucosidase from Fusarium oxysporum and its potential as an animal feed additive.

An extracellular ß-glucosidase (BGL) from Fusarium oxysporum was purified to homogeneity by a single chromatography step on a gel filtration column. The optimum activity of BGL on cellobiose was observed at pH 5.0 and 60 °C. Under the same conditions, the K(m) and V(max) values for p-nitrophenyl ß-D-glucopyranoside and cellobiose were 2.53 mM, 268 U mg protein(-1) and 20.3 mM, 193 U mg protein(-1), respectively. The F. oxysporum BGL enzyme was highly stable at acidic pH (t 1/2 = 470 min at pH 3). A commercial BGL Novo188 (Novozymes) and F. oxysporum BGL were compared in their ability to supplement Celluclast 1.5 L (Novozymes). In comparison with the commercial Novo188 (267 mg g substrate(-1)), F. oxysporum BGL supplementation released more reducing sugars (330 mg g substrate(-1)) from cellulose under simulated gastric conditions. These properties make F. oxysporum BGL a good candidate as a new commercial BGL to improve the nutrient bioavailability of animal feed.