The Mechanism Of Amyloid Fibrillation And The Modulation And Inhibition Of Related Processes

Amyloid fibrillation is a unique type of protein and peptide aggregation behavior.Under appropriate conditions,many proteins and peptides can self-assembly into fibrillar aggregates with cross-βstructure.Such an aggregate is called amyloid fibril.Amyloid fibrillation is relevant to many serious diseases including Alzheimer’s disease and Parkinson’s disease which are devastating to human health.These diseases can be collectively called amyloidosis.In addition,amyloid fibril is a novel type of protein and peptide based nanomaterials,having many potential applications in materials science.Therefore,investigating amyloid fibrillation is of great scientific significance.This thesis work will investigate two important topics in the field of amyloid fibrillation,namely,the mechanism of amyloid fibrillation and its modulation and the inhibition of amyloid fibrillation.The former helps us deepen our understanding of the important phenomenon of amyloid fibrillation at the level of basic research;the latter helps us discover new types of amyloid fibrillation inhibitors,thus providing useful references for the prevention and treatment of amyloidosis.This thesis includes the following parts:Lysozyme（hen egg white lysozyme）is a classic model system widely used in the field of amyloid fibrillation.Yet,the microscopic mechanism of its amyloid fibrillation is still unclear.In this work,we used atomic force microscopy（AFM）technology to study the amyloid fibril growth process of lysozyme under different conditions.Through in-depth analysis of AFM images at different stages of amyloid fibrillation,we found that the growth mechanism of lysozyme amyloid fibrillation was significantly different from that of other amyloid protein reported in the literature.In the reported mechanism,amyloid proteins first form protofilaments with a single-layered structure,and then achieve further fibril growth and thickening through protofilament-intertwining.In contrast,lysozymes first form a multi-layered protofilament structures,and then go through fibril thickening and branching under secondary nucleation mechanism and fibril growth through primary nucleation mechanism.In this work,the effects of nine Hofmeister series anions and nine Hofmeister cations on the amyloid fibrillation of lysozyme were studied using AFM and Fourier transform infrared（FTIR）spectroscopy.These anions include:SO₄^2->F^->CH₃COO^->Cl^->Br^->NO₃^->I^->Cl O₄^->SCN^-;and these cations include:NH₄⁺>K⁺>Na⁺>Cs⁺>Li⁺>Rb⁺>Mg²⁺>Ca²⁺>Ba²⁺。The AFM results indicated that under the influences of the three kosmotropic anions of SO₄^2-、F^-、COO^-,the morphology of amyloid fibril displayed branching and thickening;while under the influences of the other chaotropic anions of Cl^-、Br^-、NO₃^-、I^-、Cl O₄^-、SCN^-,the morphology of amyloid fibril displayed the common long and thin morphologies.In contrast,the nine different Hofmeister cations showed similar effects on the morphologies of lysozyme amyloid fibrils.Using FTIR spectroscopy,the correlation between the orders of anions and cations in the Hofmeister series and the orders of the contents of various secondary structures in lysozyme amyloid fibrils was revealed.This work investigated the modulation effects of the three heavy metal ions of Fe³⁺,Zn²⁺,and Cu²⁺on the amyloid fibrillation ofα-synuclein.The FTIR results indicated that the modulation effects of Fe³⁺,Zn²⁺,and Cu²⁺onα-synuclein fibrillation occurred at both secondary and quaternary structural levels,with Fe³⁺being the most effective modulator.Furthermore,a mechanistic hypothesis was proposed to interpret how metal ion could affect the morphology ofα-synuclein amyloid fibril based on the conformational plasticity property of intrinsically disordered protein.With respect to amyloid fibrillation inhibitors,the inhibitory effects of three types of carbohydrate derivatives on the amyloid fibrillation of lysozyme andα-synuclein were investigated.This includes the inhibitory effects of quinazolinone derivatives with multiple-hydroxyl linear chains and diaryl benzoimidazole derivatives on the amyloid fibrillation of lysozyme and the inhibitory effects of benzimidazole-iminosugars compounds on the amyloid fibrillation ofα-synuclein.At the same time,the inhibitory mechanisms of these compounds were explored and the differences between these compounds were compared.Quinazolinone derivatives with multiple-hydroxyl linear chains and diaryl-benzoimidazole derivatives employ non-covalent bonding and covalent bonding as their inhibitory mechanisms;benzimidazole-iminosugars compounds inhibit amyloid fibrillation by inducing the formation of amorphous aggregates.It is hoped that this work could provide useful references for the design of small molecule inhibitors for amyloid fibrillation.The thesis includes a total of eight chapters.The first chapter is the introduction;the second chapter is the study on the mechanism of the amyloid fibrillation of lysozyme;the third chapter is the effect of Hofmeister series ions on the amyloid fibrillation of lysozyme;the fourth chapter is about the modulation effects of Fe³⁺,Zn²⁺,and Cu²⁺on the amyloid fibrillation ofα-synuclein;the fifth chapter is the inhibition of the amyloid fibrillation of lysozyme by quinazolinone derivatives with multiple-hydroxyl linear chains;the sixth chapter is the inhibition of the amyloid fibrillation of lysozyme by biaryl benzoimidazole derivatives;The seventh chapter is about the inhibition of the amyloid fibrillation ofα-synuclein by benzimidazole-iminosugars compounds;the eighth chapter is conclusion and outlook.It is hoped the new findings in this thesis work can be helpful for the basic research in the field of amyloid fibrillation.