Protein and Peptide Letters - Volume 26, Issue 5, 2019

Amyloid fibrils are a special class of self-assembled protein molecules, which exhibit various toxic effects in cells. Different physiological disorders such as Alzheimer’s, Parkinson’s, Huntington’s diseases, etc. happen due to amyloid formation and lack of proper cellular mechanism for the removal of fibrils. Therefore, inhibition of amyloid fibrillation will find immense applications to combat the diseases associated with amyloidosis. The development of therapeutics against amyloidosis is definitely challenging and numerous strategies have been followed to find out anti-amyloidogenic molecules. Inhibition of amyloid aggregation of proteins can be achieved either by stabilizing the native conformation or by decreasing the chances of assembly formation by the unfolded/misfolded structures. Various small molecules such as naturally occurring polyphenols, flavonoids, small organic molecules, surfactants, dyes, chaperones, etc. have demonstrated their capability to interrupt the amyloid fibrillation of proteins. In addition to that, in last few years, different nanomaterials were evolved as effective therapeutic inhibitors against amyloidosis. Aromatic and hydrophobic interactions between the partially unfolded protein molecules and the inhibitors had been pointed as a general mechanism for inhibition. In this review article, we are presenting an overview on the inhibition of amyloidosis by using different small molecules (both natural and synthetic origin) as well as nanomaterials for development of pharmaceutical strategies against amyloid diseases.

Antimicrobial resistance (AMR) reported to increase globally at alarming levels in the recent past. A number of potential alternative solutions discussed and implemented to control AMR in bacterial pathogens. Stringent control over the clinical application of antibiotics for a reduction in uses is a special consideration along with alternative solutions to fight against AMR. Although alternatives to conventional antibiotics like antimicrobial peptides (AMP) might warrant serious consideration to fight against AMR, there is a thriving recognition for vaccines in encountering the problem of AMR. Vaccines can reduce the prevalence of AMR by reducing the number of specific pathogens, which result in cutting down the antimicrobial need and uses. However, conventional vaccines produced using live or attenuated microorganisms while the presence of immunologically redundant biological components or impurities might cause major side effects and health related problems. Here we discussed AMPs based vaccination strategies as an emerging concept to overcome the disadvantages of traditional vaccines while boosting the AMPs to control multidrug resistant bacteria or AMR. Nevertheless, the poor immune response is a major challenge in the case of peptide vaccines as minimal antigenic epitopes used for immunization in peptide vaccines.

Background: Arabinoxylan (AX) is the main non-starch polysaccharide in wheat. Wheat malts are traditional raw materials for beer brewing. AX is divided into water-soluble arabinoxylan (WEAX) and waterinsoluble arabinoxylan (WUAX). In the mashing stage of beer production, WUAX in malt is degraded by arabinoxylanase to WEAX, which is further degraded to smaller molecules and retained in the final beer. The viscosity of WEAX is related to its molecular weight. WEAX with higher molecular weight and viscosity can increase viscosity and turbidity and reduce filtration speed of wort and beer; WEAX with moderate molecular weight and viscosity contributes to the foaming characteristics and foam stability, and promotes the taste and texture of a beer; WEAX with small molecular weight has the functions of anti-tumor and lowering blood pressure and is regarded as a prebiotic. Because WEAXs with different molecular weight and properties have different impacts on the beer brewing process and qualities of the final beer, it becomes more important to control the degradation of AX during the brewing process of a beer. Endo-1,4-β-xylanase (EC 3.2.1.8) is the most important AX degrading enzyme, which cleaves the β -xylosidic bond between two d-xylopyranosyl residues linked in β-(1,4). The study of enzymatic properties of endo-1,4-β-xylanase from wheat malt is very important for the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Objective: In this article, our motivation is to study the enzymatic properties (including optimum pH and temperature, pH and temperature stability, the effect of inhibitors) of wheat malt endo-1,4-β-xylanase. Methods: In this article, we prepared crude enzyme according to the method of Guo with minor modifications. The endo-1,4-β-xylanase activity was determined according to the method of Biely in the previous report with minor modifications. The 0.5 mL crude enzyme sample was mixed with 0.5 mL 1 mg/mL 4-O-methyl-dglucurono- d-xylan dyed with Remazol Brilliant Blue R (RBBR-Xylan) solution, intensively mixed, and incubated at 40 °C for exactly 90 min. The reaction was stopped by precipitation using 2 mL absolute ethanol, and the reaction mixture was stirred acutely and placed at room temperature for 30 min. Then, the mixture was mixed again and centrifuged at 6000 g for 10 min. The supernatant was collected and the absorbance was measured at 590 nm. Absolute ethanol and RBBR-Xylan were added to the control tubes first, and after the reaction was completed, the crude enzyme sample was added. One unit of endo-1,4-β-xylanase was defined as at pH 5.5 and 40 °C liberate 1 μmol xylose equivalents in 1 min per g dry wheat malt. Results: The results showed that the optimal activity of endo-1,4-β-xylanase was achieved at pH 5.5-6.0, and the enzyme was extremely stable at pH 4.5, 5.5 and 6.5 after incubation for 30, 50 and 60 min, respectively. The optimal temperature was 40-45 °C and the deactivation temperature was 75 °C. Endo-1,4-β-xylanase was stable at 20 °C and 40 °C; the stability was slightly decreased at 50 °C and rapidly decreased at 55 °C. The enzyme activity was mildly inhibited by K+, Na+, and Pb2+, moderately inhibited by Ca2+, Mg2+ and Mn2+ and severely inhibited by Cu2+, Ag+ and EDTA. Conclusion: We have got the enzymatic properties of endo-1,4-β-xylanase from wheat malt, so during wort mashing, we could apply this research result to carry out the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Expected to solve the technical problems such as high viscosity, slow filtration speed and so on, but also highlight the typical flavors of WEAX such as rich and persistent foam and mellow texture during the brewing process of a beer.

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Background: The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. Objective: In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. Methods: In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. Results: Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. Conclusion: After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for the vaccine improvement.

Background: The house dust mite Suidasia pontifica (Sp) is an important source of allergens in tropical regions that trigger IgE-mediated allergic reactions such as allergic asthma, atopic dermatitis and allergic rhinitis. Detection of Sp-specific proteins are important in the management and prevention of allergic diseases. Objective: The study aimed to provide a proof of concept for a gold nanoparticle-labeled sandwich format Lateral Flow Immunoassay (LFIA) kit for the detection of Sp-specific proteins. Methods: Protein A chromatography-purified rabbit anti-Sp polyclonal antibodies were labeled with gold nanoparticles (AuNP) synthesized from chloroauric acid using the citrate reduction method, then dispensed on a glass fiber pad. Unlabeled antibodies and anti-rabbit IgG were immobilized onto nitrocellulose membrane as test line and control line respectively. Cellulose fiber pad, glass fiber, and the nitrocellulose membrane pad were then assembled as LFIA kit. Results: Protein-A affinity chromatography purification with pre-concentration yielded 1.45 mg/mL of anti-Sp polyclonal antibodies. Synthesized AuNPs with ~20 nm sizes observed under transmission electron microscope were used for antibody conjugation at an optimal pH of 8.5 (borate buffer) and an optimal ratio of 10 μ L 50μg/mL antibody:100 μ L AuNP. Optimal color intensity and fastest migration time were observed with the treatment of 0.05% Tween20 and 10% sucrose in the conjugate pads; 5% BSA and 0.05% Tween20 in the sample pads, and 1% BSA in the test pads. The limit of detection of the LFIA Sp-specific proteins is 0.076 μg/mL. The sensitivity of the Sp LFIA kit is 83% while the specificity is 100%. Conclusion: This is the first report of a prototype for a cost-effective, rapid, and equipment-free detection of the house dust mite Suidasia pontifica.

Background: Natural products are produced via primary and secondary metabolism in different organisms. The compounds obtained via secondary metabolism are not essential for the survival of the organism, but they can have a different value for humans. Objective: The objective of this study was to examine inhibitory effects of Usnic Acid (UA), a well-known lichen secondary metabolite, and Carnosic Acid (CA), the primary antioxidant compound of Rosmarinus officinalis L., on purified Human Paraoxonase, (PON1), Glutathione Reductase (GR) and Glutathione S-Transferase (GST). These enzymes have antioxidant properties and a protective effect on the oxidation of free radicals. Hence, deficiencies of such enzymes inside cells can result in a buildup of toxic substances and cause some metabolic disorders. Methods: UA and CA were tested in various concentrations against human GST, PON1, and GR activity in vitro and they reduced human GST, PON1, and GR activity. Results: UA Ki constants were calculated as 0.012±0.0019, 0.107±0.06 and 0.21±0.1 mM for GST, PON1, and GR enzymes. CA Ki constants were determined as 0.028±0.009, 0.094±0.03 and 0.79±0.33 mM, for GST, PON1, and GR enzymes. UA and CA showed competitive inhibition for GR and GST enzymes, while they exhibited non-competitive inhibition for PON1. Conclusion: These findings indicate that UA and CA could be useful in drug development studies.

Background: Leishmaniasis is caused by a protozoan parasite, Leishmania. It is common in more than 98 countries throughout the world. Due to insufficient availability of antileishmanial chemotherapeutics, it is an urgent need to search for new molecules which have better efficacy, low toxicity and are available at low cost. Objectives: There is a high rate of diabetic cases throughout the world that is why we planned to test the antileishmanial activity of glyburide, an effective sugar lowering drug used for the treatment of diabetes. In this study, glyburide showed a significant decrease in the parasite growth and survival in vitro in a dose-dependent manner. Methods: Anti-leishmanial activity of glyburide was checked by culturing Leishmania donovani promastigotes in the presence of glyburide in a dose and time dependent manner. Docking study against Leishmania donovani-Trypanothione synthetase (LdTrySyn) protein was performed using Autodock Vina tool. Results: Growth reversibility assay shows that growth of treated parasite was not reversed when transferred to fresh culture media after 7 days. Moreover, docking studies show efficient interactions of glyburide with key residues in the catalytic site of Leishmania donovani- Trypanothione synthetase (LdTrySyn), a very important leishmanial enzyme involved in parasite’s survival by detoxification of Nitric Oxide (NO) species, generated by the mammalian host as a defense molecule. Thus this study proves that the drug-repurposing is a beneficial strategy for identification of new and potent antileishmanial molecules. Conclusion: The results suggest that glyburide binds to LdTrySyn and inhibits its activity which further leads to the altered parasite morphology and inhibition of parasite growth. Glyburide may also be used in combination with other anti-leishmanial drugs to potentiate the response of the chemotherapy. Overall this study provides information about combination therapy as well as a single drug treatment for the infected patients suffering from diabetes. This study also provides raw information for further in vivo disease model studies to confirm the hypothesis.

Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. Objective: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. Methods: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. Results: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively. Conclusion: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.

Background: The structural transition of aggregating Abeta peptides is the key event in the progression of Alzheimer’s Disease (AD). Objective: In the present work, the structural modifications of toxic Aβ25-35 and the scrambled Aβ35-25 were studied in Trifluoroethanol (TFE) and in aqueous SDS micelles. Methods: Using CD spectroscopic investigations, the conformational transition of Aβ25-35 and Aβ35-25 peptides were determined in different membrane mimicking environments such as TFE and SDS. An interval scan CD of the peptides on evaporation of TFE was performed. TFE titrations were carried out to investigate the intrinsic ability of the structural conformations of peptides. Results: We show by spectroscopic evidence that Aβ25-35 prefers beta sheet structures upon increasing TFE concentrations. On the other hand, the non-toxic scrambled Aβ35-25 peptide only undergoes a transition from random coil to α-helix conformation with increasing TFE. In the interval scan studies, Aβ25-35 did not show any structural transitions, whereas Aβ35-25 showed transition from α-helix to β-sheet conformation. In membrane simulating aqueous SDS micelles, Aβ25-35 showed a transition from random coil to α-helix while Aβ35-25 underwent transition from random coil to β-sheet conformation. Conclusion: Overall, the current results seek new insights into the structural properties of amyloidogenic and the truncated sequence in membrane mimicking solvents.