| Objective:Alzheimer’s disease（AD）,also known as senile dementia,it is a common neurodegenerative disease which is closely related to age,and also characterized by cognitive and memory decline.Until now,it affects tens of millions of patients worldwide.The aggregation of the pathogenic proteinsβ-amyloid（Aβ）and Tau in the brain of patients is the key factor to induce the occurrence of AD.The aggregation can lead to the formation of senile plaques and neurofibrillary tangles in the elderly,and then lead to neurotoxicity of brain cells,which further leads to cognitive and memory dysfunction.Although researchers have been working on the aggregation and inhibition of pathogenic proteins for a long time,no effective inhibitors or therapeutic drugs have been developed.As a non-drug therapy,physical exercise has been widely validated for the relief and delay of AD symptoms.Exercise is an activity of multiple systems and organs of the body,and the microscopic mechanism of exercise intervention in AD is still unclear.Various physiological stress factors produced during exercise can promote the binding of heat shock factors and heat shock response elements in brain,and enhance the expression of small molecular weight heat shock protein B5（HSPB5,also known asαB-crystallin）in vivo.At the same time,the upregulation of HSPB5 is associated with the improvement of symptoms in the AD model.Though the protein folding and aggregation theory and molecular dynamics simulations,this study investigated the inhibition of HSPB5 on the aggregation of two pathogenic proteins in AD and the destruction of the corresponding fibrils,and revealed the microscopic mechanism,so as to provide a theoretical basis for the potential biological mechanism of exercise delaying AD.At the same time,this study also offers a predominantly conformational basis and theoretical guidance for the design of related inhibitors to block the aggregation of pathogenic proteins.Methods:In this study,protein folding and aggregation theory,and molecular dynamics simulation were used to investigate the microscopic mechanism of HSPB5on the aggregation process of two AD pathogenic proteins（Aβand Tau）.The related system of pathogenic protein and HSPB5 was constructed using GROMACS software package.In part 1,the Aβmonomer system alone was used as the control group,and the Aβmonomer system with the addition of HSPB5 was used as the experimental group;In part 2,the Aβfibril system alone was used as the control group,and the Aβfibril system with the addition of HSPB5 was used as the experiment.group;In part 3,a single Tau monomer system was used as the control group,and the Tau monomer system with the addition of HSPB5 was used as the experimental group;In part 4,a single Tau fibril system was used as the control group,and the Tau fibril system with the addition of HSPB5 was put in as the experimental group.All systems were treated with Amber99sb-ildn force field for protein related parameters,and the simulation environment was kept consistent,and each system was independently simulated for 500ns.After the simulation,VMD software was used to check the trajectory.In addition,molecular dynamics simulation software（GROMACS package）was used for data processing and analysis of the last 200 ns of the trajectory,and self-written scripts and third-party software Origin 2021 and SPSS 25.0 were used as supplementary tools for data analysis.Results:Part 1:（1）The Root mean square deviation（RMSD）value of Aβmonomer in the control group was fluctuated at 1.21 nm,while the RMSD value of the experimental group was reduced to 1.05 nm（P<0.01）.（2）Theβ-sheet structure of Aβmonomer increased to 5.96%in the control group,while theβ-sheet structure decreased to 1.03%in the experimental group（P<0.01）,and the Helix structure increased significantly（P<0.01）.（3）The ranges of Radius of gyration（Rg）,End to end distance(d E-E),and Solvent accessible surface areas（SASA）values in the control group were small,while the ranges of Rg（P<0.05）,d E-E（P<0.05）,and SASA（P<0.01）values in the experimental group were significantly increased.Compared with the control group,the number of mainchain Hydrogen bond（H-bond）formation in the experimental group decreased to 11.73（P<0.05）.（4）The binding sites of HSPB5 on Aβmonomer mainly are hydrophobic amino acids F4,Y10,Y20,I32,L34,V36,and polar amino acids H6,H13,H14,K16,G33,G37,G38.（5）β3/4 andβ8～strands of HSPB5 are the main binding regions of Aβmonomer.Part 2:（1）HSPB5 can bind to multiple sites on Aβprotofibrils,with more binding times in the elongation direction.（2）The RMSD value of Aβprotofibrils in the control group was fluctuated to 0.22 nm,while that in the experimental group was increased to0.44 nm（P<0.01）.（3）There were abundantβ-sheet contents in Aβprotofibrils in the control group,andβ-sheet structures were obviously destroyed in the experimental group P<0.01);（4）Compared with the control group,the number of mainchain H-bond（P<0.01）and side chain H-bond（P<0.05）in the experimental group was significantly decreased,while Rg（P<0.01）and SASA（P<0.01）values were significantly increased;（5）The Kink Angle and K28-A42 salt bridge in the control group were stable,while the Kink Angle（P<0.01）and K28-A42 salt bridge（P<0.01）in the experimental group were significantly destroyed.（5）There are a lot of polar and hydrophobic amino acids in the binding process.Part 3:（1）The RMSD value of Tau monomer in the control group was fluctuated to 1.55 nm,while the RMSD value of the experimental group was reduced to 1.43 nm（P<0.01）;（2）Theβ-sheet structure of Tau monomer in the control group was 10.23%,while theβ-sheet structure in the experimental group decreased to 6.67%（P<0.01）,and the Helix structure increased to 7.33%（P<0.01）.（3）The Rg,d E-E,and SASA values of the control group were small,while the Rg（P<0.05）,d E-E（P<0.05）,and SASA（P<0.05）values of the experimental group were significantly increased.Compared with the control group,the number of mainchain H-bond in the experimental group decreased to 15.83（P<0.05）,and the number of side chain H-bond formation decreased to 15.79（P<0.01）.（4）The binding sites of HSPB5 on Tau monomer are mainly polar amino acids H329,E338,V339,K340,G355,T373,and H374;（5）β5/7andβ8/9～strands of HSPB5 are the main binding regions of Tau monomer.Part 4:（1）Compared with other oligomers,the RMSD,Root mean square fluctuation（RMSF）,andβ-sheet of Tau pentamers are relatively stable;（2）Theβ-sheet structure of the control group was 49.42%,while theβ-sheet structure of the experimental group was reduced to 48.01%（P<0.05）,and the content of Coil structure was significantly increased（P<0.05）.（3）The Rg and d E-E values of the control group were small,while the Rg（P<0.05）and d E-E（P<0.01）values of the experimental group were significantly increased.Compared with the control group,the number of mainchain H-bond in the experimental group decreased to 185.80（P<0.05）,and the number of side chain H-bond formation decreased to 86.01（P<0.05）.（4）The addition of HSPB5 increased the Q351-I371 distance of Tau protofibrils（P<0.05）,decreased the Angle ofβ2/3～strand（P<0.01）and increased the Angle ofβ6/7～strand（P<0.05）.（5）Polar and Hydrogen bond interaction play a dominant role in this binding process.Conclusion:（1）After HSPB5 binds to Aβmonomer,it can inhibit the aggregation of Aβmonomer by increasing the structural stability of Aβmonomer.The interaction mode between HSPB5 and Aβmonomers is mainly polar interaction.（2）After HSPB5 is combined with Aβfibrils,it can destroy the structure of Aβfibrils by reducing the structural stability of Aβfibrils and changing their secondary structure content.The interaction mode between the two is mainly polar interaction.（3）After HSPB5 is combined with Tau monomer,it can inhibit the aggregation of Tau monomer by increasing the structural stability of Tau monomer.The interaction mode between HSPB5 and Tau monomer is mainly polar interaction.（4）After HSPB5 is combined with Tau fibrils,it can destroy the structure of Tau fibrils by reducing the structural stability of Tau fibrils and changing their secondary structure content.The interaction mode between the two is mainly polar interaction. |
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