Peptide ligand screening of α-synuclein aggregation modulators by in silico panning

<p>Abstract</p> <p>Background</p> <p>α-Synuclein is a Parkinson's-disease-related protein. It forms aggregates <it>in vivo</it>, and these aggregates cause cell cytotoxicity. Aggregation inhibitors are expected to reduce α-synuclein cytotoxicity, and an aggregation accelerator has recently been reported to reduce α-synuclein cytotoxicity. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.</p> <p>Results</p> <p>We screened peptide ligands against α-synuclein by <it>in silico </it>panning, a method which we have proposed previously. In this study, we selected as the target a very hydrophobic region known as the amyloid-core-forming region. Since this region cannot be dissolved in water, it is difficult to carry out the <it>in vitro </it>screening of its peptide ligand. We carried out 6 rounds of <it>in silico </it>panning using a genetic algorithm and a docking simulation. After the <it>in silico </it>panning, we evaluated the top peptides screened <it>in silico </it>by <it>in vitro </it>assay. These peptides were capable of binding to α-synuclein.</p> <p>Conclusion</p> <p>We demonstrated that it is possible to screen α-synuclein-binding peptides by <it>in silico </it>panning. The screened peptides bind to α-synuclein, thus affecting the aggregation of α-synuclein.</p

Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface

BACKGROUND: Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. RESULTS: Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3–4°C higher over wild type. All mutants showed 2-fold higher thermal stability at 55°C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. CONCLUSIONS: We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity

In silico panning for a non-competitive peptide inhibitor

BACKGROUND: Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH). RESULTS: The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs), which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with K(i )value of 20 μM. PQQGDH activity, in terms of the V(max )value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (K(D)) value was calculated as 60 μM by surface plasmon resonance (SPR) analysis. CONCLUSION: We demonstrate an effective methodology of in silico panning for the selection of a non-competitive peptide inhibitor from small virtual peptide library. This study is the first to demonstrate the usefulness of in silico evolution using experimental data. Our study highlights the usefulness of this strategy for structure-based screening of enzyme inhibitors

Development of engineered chromatic acclimation sensor with strict and reverse response to light signal, and application to optogenetic control in cyanobacteria

Genetic regulation and metabolic engineering enabled cyanobacteria to produce renewable chemical compounds from carbon dioxide via photosynthesis. Optogenetic control enables to precisely regulate the timing and level of gene expression without chemical inducer which is environment-hazardous. We recently developed a green-light regulated gene expression system in a model cyanobacterial strain Synechocystis sp. PCC6803 (hereafter PCC6803) [1] and a fast-growing marine cyanobacterial strain Synechococcus sp. NKBG15041c (hereafter NKBG15041c) [2] using a PCC6803-derived chromatic acclimation sensor, CcaS/CcaR two-component system [3]. However, the regulation of gene expression by CcaS is not strictly controllable and the background expression level under non-inductive condition is not negligible. Furthermore, altering the direction of gene expression, that is induction under red-light and repression under green-light, may expand its flexibility as one of the genetic tools. To obtain stricter and versatile system, we fabricated engineered CcaSs focusing on its domain structure using Escherichia coli expression system. One of the engineered CcaSs, CcaS#11, showed reverse response to light signal, i.e. inducible under red-light and strictly repressible under green-light [4]. To investigate the potential application and versatility of CcaS#11 as the red-light regulated gene expression system in cyanobacteria, we next introduced CcaS#11/CcaR two-component system and GFPuv as a probe of gene expression into PCC6803 after knocking out genomic CcaS/CcaR two-component system to exclude the interference. In this strain, the gene expression was induced under red-light and strictly repressed under green-light as we expected. Then, we applied this system to NKBG15041c. Similarly, red-light inducible gene expression with 2-fold higher ON/OFF ratio compared with the original system was successfully observed in NKBG15041c. Remarkably, there was no leaky expression under green-light, indicating that this system enables strict regulation of gene expression by light signal. In conclusion, we successfully constructed the engineered CcaS, CcaS#11, with strict and reverse response to light signal. Then we also confirmed its versatility and applicability as the red-light regulated gene expression system with strict regulation in cyanobacteria. Further development of the light regulated bioprocess will be expected using cyanobacterial hosts with this system, as a cell factory for the renewable chemical compounds production. [1] K. Abe et al., ‘Engineering of a Green-light Inducible Gene Expression System in Synechocystis sp. PCC 6803’, Microb. Biotechnol. 7 (2014) 177-183 [2] A. Badary et al., ‘The Development and Characterization of an Exogenous Green-light-regulated Gene Expression System in Marine Cyanobacteria’, Mar. Biotechnol. 17 (2015) 245-251 [3] Y. Hirose et al., ‘Cyanobacteriochrome CcaS is the Green Light Receptor That Induces the Expression of Phycobilisome Linker Protein’, Proc. Natl. Acad. Sci. USA 105 (2008) 9528-9533 [4] M. Nakajima et al., ‘Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal’, Sci. Rep. 6 (2016) 3759

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring

Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1–100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy

Migration of foreign body from mouth to nose

A man appeared in the Emergency Department complaining of discomfort in his neck because he had swallowed a toothpick while taking a nap. The examining physician could find no foreign body in the patient\u27s mouth or pharynx. An additional examination using a fiberscope disclosed the existence of a foreign body in the nose. The toothpick was thought to have migrated to the nose from the pharynx after it was swallowed. Foreign bodies of various sizes may migrate to the nose from other parts of the body. Therefore, protocols must be designed for additional examination of the nose

An Amine-Reactive Phenazine Ethosulfate (arPES)—A Novel Redox Probe for Electrochemical Aptamer-Based Sensor

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method