The Assembly Regulation And High-resolution Structural Determination Of Amyloid Protein

Alzheimer's disease and type 2 diabetes caused by amyloid misfolding is increasing currently.However,the pathogenic mechanism caused by the misfolding and the structural diversity of amyloidogenic proteins aggregates remains elusive,which greatly hinders the development of the treatment methods and the related new drugs for amyloid diseases.Thus,the precise structural analysis of amyloid aggregates and the corresponding self-assembly process play a crucial role in understanding the pathogenesis of amyloid-related diseases and improving the related treatment strategies.A wide variety of characterization techniques have been developed to study the structural diversity of amyloid aggregates.However,the exploration of amyloid aggregates'structure is greatly limited by large molecular weight,poor crystallinity,and the structural uniformity of amyloid fibrils.Given that,the thesis would focus on the misfolding of human islet amyloid polypeptide(hIAPP37)and beta amyloid peptide(A?),especially the diversity and similarity of their structures and their interaction.Using the combination of high-resolution atomic force microscope,microcrystal electron diffraction(Micro ED),and theoretical simulation calculations,we proposed environmental factors such as vortex force,electric field,small chemical molecules,ion concentration,etc.,in the aggregation process of hIAPP37 and A?peptide.The usage of circular dichroism spectroscopy,transmission electron microscopy,fluorescence spectrometer,and Fourier-transform infrared spectroscopy etc.,allows us to analyze the secondary structure,the morphology,the dynamic aggregation process and the corresponding nanomechanical properties of amyloid aggregates.Finally,the cytotoxicity experiments show the change of their biological activities,and the specific pathogenic mechanisms of amyloid aggregates are explored.The main research work is as following:1.The complexity and diversity of structures and the limited knowledge about hIAPP37obstacle the exploration of its pathogenic mechanism.human islet amyloid polypeptide 20-29fragment(hIAPP20-29),which is the core fragment of hIAPP37,is vital to the study of the pathogenic mechanism of hIAPP37.The effect of vortex force induced by magnetic beads on the aggregation process of hIAPP20-29 peptide is studied.The cryo-EM method called microcrystal electron diffraction(Micro ED)captured the rare right-handed spiral structure of hIAPP20-29under the influence of magnetic beads,and revealed that the possible molecular structural conformation of hIAPP20-29,which is composed of two antiparallel?-lamellar structure composition;Atomic force microscopy shows the assembly,thin fibrils and crystal-like fibrils,the morphology of the right-handed helical structure and its twisted periodicity of hIAPP20-29,indicating the structural diversity and the self-assembly mechanism of hIAPP20-29 aggregates,which is further beneficial to the exploration of the structural diversity of hIAPP37 and the possible aggregation mechanisms.2.The structure and assembly mechanism of hIAPP37 influenced by the environmental factors are not yet clear.That could affect the exploration of the pathogenesis of type 2 diabetes.We studied the self-assembly and aggregates in morphology of hIAPP37 caused by the extended electric field.Analysis of circular dichroism results shows that the secondary structure conformation of hIAPP37 is changed under the effect of the applied electric field,resulting in the decrease in?-sheet structure and the increase in?-helical structure,which induce the morphological difference of amyloid fibrils during the aggregation process.Many thin and long fibrils appeared,improving the survival rate of INS-1 cells;The results of molecular dynamic simulation further confirmed that the effect of the electric field could change in the intramolecular interaction located in amyloid peptide and the intermolecular interaction between amyloid peptide chains.The appearance of the?-helix structure causes the conformational change of the molecular structure of the peptide.This work provides a new insight in exploring the pathogenic mechanism of human islet amyloid polypeptide.3.It is vitally important to explore the degradation mechanism of hIAPP37 aggregates for the prevention and treatment of type 2 diabetes.We choose soluble light-excited porphyrin molecule as the representative of the small chemical molecules to study the degradation process of hIAPP37 aggregates.The results of atomic force microscopy and circular dichroism showed that light-excited porphyrin caused the significant change in the morphology and the secondary structure of amyloid fibrils,that is,the content of?-sheet structure decreased and the content of?-helical structure increased,which leads to the disappearance of amyloid fibrils and the formation of aggregated particles.The result analysis of electron paramagnetic resonance spectroscopy showed that light-excited porphyrin produced superoxide radicals(O₂^?-),hydroxyl radicals(^?OH),singlet oxygen and other reactive oxygen species that may break the intermolecular force of hIAPP37 mature fibers,such as the hydrogen bonds,Van der Waals force and electrostatic force that are used to stabilize the fiber structure.At the same time,the partial energy is transferred to the amyloid fiber through the excited porphyrin during the illumination process,which destroys the stability of its?-sheet structure.As a result,the morphology of mature fiber of hIAPP37 is changed and its toxicity to INS-1 cells is reduced.The results of this chapter provide assistance in exploring the development of treatment methods for type 2diabetes.4.According to a large number of reports,hIAPP37 and A?peptide have a similarity in their structures and assembly processes.Thus,the study the assembly mechanism of A?peptide is not only benefit to the understanding of the pathogenesis of Alzheimer's disease,but also to the understanding of the assembly mechanism of hIAPP37.It is not yet clear for the pathogenic mechanism of beta amyloid peptide due to its complex morphology and structure caused by amyloid misfolding.The amyloid beta peptide 25-35 fragment(A?25-35)as the core fragment of amyloid beta protein greatly affects the pathogenic mechanism of A?peptide.The self-assembly of A?25-35 peptide incubated in solutions with different ion concentration was studied.The experimental results found that phosphate ions promoted the aggregation process of A?25-35peptide to forming a twisted fiber structure in phosphate buffer and the flat membrane structure in ultrapure water.Analysis in circular dichroism results showed that different secondary structures of A?25-35 peptide were formed in different solvents,and the secondary structure mainly with?-sheet structure was formed in the phosphoric acid solution,which led to the difference between the two nano-assembled structures and induced different degrees of cytotoxicity to SH-SY5Y cells.