Screening Of Small Molecule Inhibitors Of Amyloid Protein And Simulation Of Intracellular Microenvironment Of Amyloidogenic Proteins

In recent years,an increasing number of Amyloid deposition diseases such as type II diabetes,Parkinson's syndrome,Alzheimer's disease have been widely reported,and these Amyloidosis diseases have become the major diseases that plague older people one.Due to the low activity of the body of the elderly in the body,in some of the protein metabolism in the process of misfolding and wrong cutting,and the elderly immune system is gradually aging,mistakenly folded with the wrong cut the unwanted protein can not be cleared in time A large number of accumulation,the formation of Amyloid fiber,and then lead to Amyloid deposition disease.There is no effective treatment for these Amyloidosis.The availability of a large number of high purity,representative Amyloid precursor proteins is a prerequisite and guarantee for further study of protein folding and aggregation mechanisms.Combined with in vitro protein fiber culture experiments and in vivo protein aggregation detection to build anti-protein aggregation inhibitor screening platform,to find a protein aggregation,fibrosis inhibition effect of small molecule inhibitors is the current study of Amyloid deposition The focus of effective disease treatment strategies.In order to solve the problem of effective inhibition of different stages in the process of Amyloid fibroblasts,we have selected typical Amyloid chicken lysozyme and human ?-Amyloid protein as model proteins for anti-Amyloid aggregation small molecule compounds,And the yeast yeast-based simulation of the incorrect folding protein was constructed and the yeast-based PDI gene was constructed.Human lysozyme is a classical model protein for the study of Amyloid deposition disease,and chicken lysozyme has high homology with human lysozyme.In the extreme environment,the structure of chicken lysozyme is destroyed,the hydrophobic core is exposed,And the new protein monomer is added in turn to form an unbranched Amyloid fiber.Lignans and cyclic peptides are widely found in flax plants.Studies have shown that they have a wide range of anti-cancer,anti-inflammatory and anti-oxidative effects as polyphenols.However,studies on fibroblast inhibition as anti-Amyloid are rarely reported.In this study,five representative lignans and two cyclic peptides were selected as candidate compounds for screening anti-Amyloid fibrils.Chicken lysozyme was selected as fibrin.It has been found that lignans have a certain degree of ability to inhibit Amyloid fibroblasts,but the inhibitory effect is much lower than that of mulin in comparison with the positive control of polyphenols.It was hypothesized that molecular weight studies were related to the specific sites in the groove of chicken lysozyme,which prevented the extension of protein fibers and the molecular weight of lignans was too large to enter the surface of the lysozyme.With the increase of the concentration,it is very likely to attach to the surface of the protein,thereby reducing the protein and water molecules,through the hydrophobic interaction of protein molecules to maintain the stability of the protein structure.In this study,the use of animal-derived Amyloid protein for fibroblast inhibition experiments at the same time,try to Pichia pastoris expression system,a large number of expression of human secretion Amyloid-? protein.Amyloid-beta protein is a typical Amyloid and is the major pathogen of Alzheimer's disease.Amyloid-? has the characteristics of short fiber-forming period and high cytotoxicity of oligomers.It is widely used in the study of human Amyloid protein.However,Amyloid-? protein is derived from chemical synthesis,with higher cost and lower purity.This study attempts to express the Amyloid-? protein efficiently by using the Pichia pastoris eukaryotic expression system to provide cheap,high-purity feedstock proteins for the screening of Amyloid fibroblast inhibitors.In the intracellular experiment,we used the PDI gene with molecular chaperone and disulfide isomerase as the core to try to simulate the microenvironment of the aged cells.The CRISPR / Cas9 gene editing system was used to knock out the Pichia pastoris protein Disulfide isomerase(PDI)gene,and to construct the quality management system to isolate the strain,lay the foundation for the study of intracellular aggregation of Amyloid protein.On the other hand,the use of Pichia pastoris secretion system a large number of disulfide deletion and improper folding of Amyloid protein,for the screening of small molecule inhibitors to provide a more convenient conditions.