A Molecular Integrative Study on the Inhibitory Effects of WRR and ERW on Amyloid ß Peptide (1-42) Polymerization and Cell Toxicity.

Alzheimer's disease (AD) is a neurodegenerative disease and the main pathological characteristic of AD is the deposition of Aß42 in the brain. Inhibition of Aß42 polymerization is one of the important research directions. Due to the pathological complexity of Alzheimer's disease, studies on Aß42 polymerization inhibitors have not made significant progress worldwide. Using an independently constructed structure database of oligopeptides, in this study, molecular docking, umbrella sampling analysis of free energy, ThT fluorescence detection of Aß42 polymerization, transmission electron microscopy, and flow cytometry detection of reactive oxygen species (ROS) and apoptosis were performed to screen tripeptides and pentapeptides that inhibit polymerization. It was found that two tripeptides, i.e., WRR and ERW, bind stably to the core of Aß42 polymerization in the molecular dynamics analysis, and they significantly inhibited the aggregation of Aß42 and reduced their cell toxicity in vitro.

Molecular Integrative Analysis of the Inhibitory Effects of Dipeptides on Amyloid ß Peptide 1-42 Polymerization.

The major pathological feature of Alzheimer's disease (AD) is the aggregation of amyloid ß peptide (Aß) in the brain. Inhibition of Aß42 aggregation may prevent the advancement of AD. This study employed molecular dynamics, molecular docking, electron microscopy, circular dichroism, staining of aggregated Aß with ThT, cell viability, and flow cytometry for the detection of reactive oxygen species (ROS) and apoptosis. Aß42 polymerizes into fibrils due to hydrophobic interactions to minimize free energy, adopting a ß-strand structure and forming three hydrophobic areas. Eight dipeptides were screened by molecular docking from a structural database of 20 L-α-amino acids, and the docking was validated by molecular dynamics (MD) analysis of binding stability and interaction potential energy. Among the dipeptides, arginine dipeptide (RR) inhibited Aß42 aggregation the most. The ThT assay and EM revealed that RR reduced Aß42 aggregation, whereas the circular dichroism spectroscopy analysis showed a 62.8% decrease in ß-sheet conformation and a 39.3% increase in random coiling of Aß42 in the presence of RR. RR also significantly reduced the toxicity of Aß42 secreted by SH-SY5Y cells, including cell death, ROS production, and apoptosis. The formation of three hydrophobic regions and polymerization of Aß42 reduced the Gibbs free energy, and RR was the most effective dipeptide at interfering with polymerization.

Molecular integrative study on interaction domains of nuclear factor erythroid 2-related factor 2 with sirtuin 6.

Oxidative stress is one of the elements causing aging and related diseases. Inhibiting Nrf2 activity or increasing oxidative pressure can replicate the deficits of premature aging. SIRT6 is one of the few proteins that can regulate both life span and aging. Deletion of SIRT6 in human cells impairs the antioxidant capacity of cells, which results in the accumulation of intracellular reactive oxygen species and DNA oxidation products. Characterization of the binding of Nrf2 with SIRT6 is critical for understanding the modulation of Nrf2-correlated cell activities by SIRT6. The yeast two-hybrid experiments showed that the binding of Nrf2 with SIRT6 is mediated by Neh1 and Neh3 domains. The elimination of the Neh1 and Neh3 domains decreased the binding stability and free energy, according to the molecular dynamic analysis. The roles of theses domains in mediating the binding were confirmed by co-immunoprecipitation. In cells transfected with the small interfering RNA (siRNA) targeting the Nrf2 Neh1 domain and plasmids overexpressing domain-mutant Nrf2, it was discovered that Nrf2 lost its activity to stimulate the transcription of antioxidant genes in the absence of Neh1 and Neh3 domains.

Studies on HBcAg-rBlo t 5-21 Fusion Protein Vaccine That Alleviates Blomia tropicalis Airway Inflammation.

Background: In tropical and subtropical areas, allergens from the dust mite species Blomia tropicalis are common causes of allergic rhinitis and asthma. Blomia tropicalis has two main allergens: Blo t 5 and Blo t 21. Aim: To generate a chimeric virus-like particle containing HBcAg, Blo t 5 and Blo t 21 that can treat allergies caused by Blomia tropicalis. Methods: To produce allergic asthma in mice, prokaryotic expression and purification of Blomia tropicalis allergens rBlo t 5, rBlo t 21, and recombinant fusion allergen rBlo t 5-21 were utilized in the study. We created a hepatitis B core antigen (HBcAg) and rBlo t 5-21 fusion prokaryotic expression plasmid. HBcAg-rBlo t 5-21 was purified after expression and tested by transmission electron microscopy (TEM). Furthermore, the protein HBcAg-rBlo t 5-21 was employed as a protein vaccination. Results: In allergy-induced mouse model experiments, the fusion allergen rBlo t 5-21 was more effective than the individual allergens rBlo t 5 and rBlo t 21 at inducing allergy. We found that vaccinating allergic mice with the recombinant fusion protein vaccine HBcAg-rBlo t 5-21 alleviated allergy symptoms elicited by the rBlo t 5-21 allergen. Vaccination with HBcAg-rBlo t 5-21 resulted in a decrease in total serum IgE levels, suppression of anaphylaxis, and reduction of inflammatory cell infiltration into lung tissue as compared to the PBS group. Conclusion: HBcAg-rBlo t 5-21, a protein vaccine containing both the hepatitis B core antigen and the Blomia tropicalis fusion allergen rBlo t 5-21, could be a suitable vaccination for preventing allergy disorders caused by Blomia tropicalis.

Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis.

BACKGROUND AND PURPOSE: Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin. Anticoagulant effects of gene-recombinant hirudin are weaker than natural hirudin for the reason of lacking tyrosine O-sulfation at C-terminus. EXPERIMENTAL APPROACH: An integrative pharmacological study was carried out using molecular dynamic, molecular biological and in vivo and in vitro experiments to elucidate the anticoagulant effects of protein-engineered hirudins. KEY RESULTS: Molecular dynamic analysis showed that modifications of the C-termini of hirudin variant 1 of Hirudo medicinalis (HV1) and hirudin variant 2 of Hirudinaria manillensis (HM2) changed the binding energy of the C-termini to human thrombin. The study indicated that Asp61 of HM2 that corresponds to sulfated Tyr63 of HV1 is critical for inhibiting thrombin activities. Further, the anticoagulant effects of HV1 and HM2 were improved when the amino acid residues adjacent to Asp61 were mutated to Asp. These improvements were prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time of human blood, and decreased Ki and IC50 values. In the in vivo experiments, mutations at C-termini of HV1 and HM2 significantly changed partial thromboplastin time, prothrombin and thrombin time CONCLUSION AND IMPLICATIONS: The study indicated that the anticoagulant effects of gene-engineered HM2 are stronger than gene-engineered HV1 and HM2-E60D-I62D has the strongest effects and could be an antithrombotic with better therapeutic effects.

Sustained Stimulation of ß2AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway.

INTRODUCTION: This study aimed to investigate the role of ß2 adrenergic receptor (ß2AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the ß2AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression. METHODS: H9C2 cells were treated with various protein inhibitors (CGP, ß1AR inhibitor CGP20712; ICI, ß2AR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the ß2AR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot. RESULTS: Gene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of ß2AR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that ß2AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex. CONCLUSION: The sustained stimulation of ß2AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.