The Effect of Ethanol on Disassembly of Amyloid-β1-42 Pentamer Revealed by Atomic Force Microscopy and Gel Electrophoresis

The most common type of dementia, Alzheimer’s disease, is associated with senile plaques formed by the filamentous aggregation of hydrophobic amyloid-β (Aβ) in the brains of patients. Small oligomeric assemblies also occur and drugs and chemical compounds that can interact with such assemblies have attracted much attention. However, these compounds need to be solubilized in appropriate solvents, such as ethanol, which may also destabilize their protein structures. As the impact of ethanol on oligomeric Aβ assembly is unknown, we investigated the effect of various concentrations of ethanol (0 to 7.2 M) on Aβ pentameric assemblies (Aβp) by combining blue native-PAGE (BN-PAGE) and ambient air atomic force microscopy (AFM). This approach was proven to be very convenient and reliable for the quantitative analysis of Aβ assembly. The Gaussian analysis of the height histogram obtained from the AFM images was correlated with band intensity on BN-PAGE for the quantitative estimation of Aβp. Our observations indicated up to 1.4 M (8.3%) of added ethanol can be used as a solvent/vehicle without quantitatively affecting Aβ pentamer stability. Higher concentration induced significant destabilization of Aβp and eventually resulted in the complete disassembly of Aβp.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

Characterisation of a highly diverged mitochondrial ATP synthase peripheral stalk subunit b in Trypanosoma brucei

The mitochondrial F1Fo ATP synthase of Trypanosoma brucei has been studied in detail. Whereas its F1 moiety is relatively highly conserved in structure and composition, the same is not the case for the Fo part and the peripheral stalk. A core subunit of the latter, the normally conserved subunit b, could not be identified in trypanosomes suggesting that it might be absent. Here we have identified a 17 kDa mitochondrial protein of the inner membrane that is essential for normal growth, efficient oxidative phosphorylation and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicate that the protein is associated with the F1Fo ATP synthase. Its ablation reduces the levels of Fo subunits, but not those of F1, and disturbs the cell cycle. HHpred analysis showed that the protein has structural similarities to subunit b of other species, indicating that the Fo part of the trypanosomal ATP synthase does contain a highly diverged subunit b. Thus, the Fo part of the trypanosomal ATPase synthase may be more widely conserved than initially thought.

Deciphering the Amendment-Induced Secretion of Ligninolytic Enzymes by Pleurotus pulmonarius

Pleurotus pulmonarius belonging to the white-rot fungal basidiomycetes group secretes extracellular ligninolytic enzymes for the degradation of agroresidues. The present study was carried out to evaluate the efficacy of different agro-residues for the enhanced production of ligninolytic enzymes and to authenticate their ability by protein analysis. The morphological and molecular sequences of white-rot fungi were characterised. Besides, the efficacy of organic and inorganic amendments in the secretion of ligninolytic enzymes by P. pulmonarius was characterised using SDS-PAGE and native PAGE analysis. The characterised strain of P. pulmonarius secreted enhanced laccase enzyme levels in the liquid medium through supplementation with organic and inorganic amendments. Wheat bran and groundnut cake each @5% enhanced secretions of Laccase, LiP and MnP. Copper sulphate at 150 μM enhanced the laccase enzyme and at 100 μM enhanced the LiP enzyme level by P. pulmonarius. Similarly, supplementation with manganese sulphate at 150 μM enhanced laccase, LiP and MnP enzyme levels compared to control. SDS-PAGE results showed protein banding patterns in the range of 50–85 kDa for the Lac enzyme in samples drawn from wheat bran and groundnut cake-supplemented substrates. Native PAGE results of laccase enzymes also showed that wheat bran (5%) + groundnut cake (5%) + CuSO4 (150 M) + MnSO4 (150 M) induced four laccase isozymes. Supplementing organic and inorganic amendments to the substrates would enhance the secretion of laccase enzyme that would aid in better breakdown of lignin.

Domain Organization of the UBX Domain Containing Protein 9 and Analysis of Its Interactions With the Homohexameric AAA + ATPase p97 (Valosin-Containing Protein)

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α–1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1’ intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

TTYH family members form tetrameric complexes within the cell membrane

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.

Purification and Characterization of Polyphenol Oxidase Enzyme from Damson Plum (Prunus insititia) by Spectrophotometrically

Polyphenol oxidase enzyme, performing browning reactions in fruits and vegetables, was purificated from damson plum (Prunus insititia) which has a high antioxidant activity. Firstly, partially purified polyphenol oxidase was treated by 0-80% ammonium sulfate precipitation and dialysis, respectively. Characterization studies were carried out by using catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05M/ pH:7.2/ 25°C; 0.2M/ pH:4.5/ 10°C; 0.01M/ pH:6.8/ 5°C and 0.2M/ pH:8.5/ 10°C, respectively. The kinetic constants of Vmax and KM were calculated for the same substrates as 17219.97 U/(mL*min) and 11.67mM; 7309.72 U/(mL*min) and 5mM; 12580.12 U/(mL*min) and 3.74mM; 12100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest Vmax value when compared to others. In the second step, purification was performed by using Sepharose 4B-L-Tyrosine-p-amino benzoic acid and Sepharose 6B-L-Tyrosine-p-amino benzoic acid affinity gels. A single band of approximately as 50-55 kDa was observed in SDS-PAGE and Native-PAGE. 90 and 10.2 purification folds were obtained for Prunus insititia PPO by the reference Sepharose-4B-L-Tyrosine-p-aminobenzoic acid and original Sepharose-6B-L-Tyrosine-p-aminobenzoic acid gels, respectively. PPO enzyme purification from Prunus insititia by affinity chromatography has not been investigated in literature yet.

Recombinant Production of IL-1Ra in Fusion to Albumin Binding Domain for Its Extended Half-Life

Background: Anakinra, a FDA approved biological drug for Rheumatoid Arthritis, must be injected daily due to its short Half-life, leads to the lower patient compliance. So, the aim of this study was to produce IL-1Ra in fusing to albumin binding domain to extend its half-life and evaluate its biological effects.Methods and Results: The expression of IL-1Ra-ABD was performed in E. coli in fusing to intein1 of pTWIN1 in soluble and purified. The affinity of IL-1Ra-ABD to HSA was determined on Native-PAGE and its release percent toward time was determined. Finally, MTT assay was used to determine the antagonizing properties of recombinant IL-1Ra-ABD against IL-1β, on A375 cells. the expression induction of intein1-IL-1Ra-ABD using 0.1mM of IPTG at 15°C, and its cleavage represented a band approximately in 50 and 23 kDa respectively. Native-PAGE results showed that about 78% of IL-1Ra-ABD attached to the HSA after 2 hours of incubation, and MTT assay results showed no significant differences between the effects of our recombinant protein and native IL-1Ra.Conclusion: the production of soluble IL-1Ra-ABD with similar antagonizing effects to IL-1Ra was successfully performed. IL-1Ra-ABD showed suitable interaction with HSA and release over the time. However, pharmacokinetics and furthur biological evaluations are required.