Studies On Mechanism,Toxicity And Inhibitor Screening Of Amyloid Fibril Formation By β-Casein

The work involved the influence and mechanism of heparin sulfate（HS）and metal ions on the amyloid fibril formation by β-casein,the toxicity of the amyloid fibril formation,the screening of the inhibitors and the inhibition mechanism of the arginine on the amyloid fibril formation by β-casein.The following innovative results have been achieved.1.The influence and mechanism of HS on the amyloid fibril formation by β-caseinThe studies about the influence and mechanism of HS on the amyloid fibril formation by β-casein were carried out.（1）The results demonstrated that HS promoted β-casein amyloid fibrilization in the optimal concentration of 10 mg/m L in nucleation period,by Thioflavin T（Th T）fluorescence analysis,transmission electron microscope（TEM）,and circular dichroism.（2）β-casein-HS complexes were formed and conformation was changed during amyloid fibril formation,which were demonstrated by resonance light scattering,steady-state UV absorption spectra,fluorescence spectra and fluorescence lifetime detection.On the basis of fluorescence analysis at three temperatures,it was deduced that the fluorescence of β-casein was quenched by HS with a static quenching mechanism.There was one binding site between β-casein and HS.The thermodynamic parameters explained that a spontaneous reaction happened,and the protein-ligand complex was stabilized by hydrogen bonds and hydrophobic interaction.Furthermore,the result of fluorescence resonance energy transfer assay showed that the binding distance between HS and tryptophan residue of β-casein was 0.93 nm.Finally,it was demonstrated by synchronous fluorescence spectra that the polarity was increased and hydrophobicity was decreased around the tryptophan residue,which caused the exposure of the tryptophan residue to solvent easily and promoted the amyloid fibril formation by β-casein.2.The influence of HS on the breast amyloidosis of ratsThe influence of HS on the breast amyloidosis of rates and the further relationship between inflammation and fibrosis were investigated by mammary gland injection of HS.（1）The results of hematoxylin-eosin staining and thioflavin S staining demonstrated that HS,acted as an endogenous molecule,induced the inflammation and amyloid fibril formation at the same time;（2）There was a close relationship between breast inflammation and amyloidosis.The intense inflammation induced the breast amyloidosis,and the amyloidosis also promoted the inflammatory response.3.The influence of Ca²⁺ and Zn²⁺ on the amyloid fibril formation by β-caseinThe influence of Ca²⁺ and Zn²⁺ on the dynamics,morphology and microenvironment of amyloid fibril formation by β-casein without and with of HS was investigated.（1）The results of Th T fluorescence assay demonstrated that Ca²⁺and Zn²⁺ promoted the amyloid fibril formation by β-casein.The effect of Ca²⁺ was greater than that of Zn²⁺.In addition,the influences of Ca²⁺ and Zn²⁺ were decreased by the concentration increased.（2）TEM showed that the influence of Ca²⁺ and Zn²⁺on morphology of fibrils was different;（3）Meanwhile,it was also observed that the microenvironment of β-casein was changed by Ca²⁺ and Zn²⁺ using intrinsic fluorescence measure.（2）There was a synergistic effect of HS on the amyloid fibril formation by β-casein.4.The study on the toxicity of the amyloid fibril formation by β-caseinThe investigation about the toxicity of the amyloid fibril formation by β-casein was carried out systematically.（1）The pentamer formyl thiophene acetic acid fluorescence analysis was performed to determinate the pre-fibrils formed by β-casein.（2）The influence of pre-fibrils on HC11 cells was evaluated with MTT assay and Annexin V/PI staining.The results demonstrated that the pre-fibrils formed by β-casein were toxic to HC11 cells,which not only caused the death of HC11 cells,but also promoted the procedural apoptosis of HC11 cells.（3）The influence of pre-fibrils on the mammary gland of rats was analyzed with hematoxylin-eosin staining.The results demonstrated that the pre-fibrils formed by β-casein were toxic to the mammary gland,which not only destroyed the tissue,but also induced inflammation.5.The studies about the inhibitors of the amyloid fibril formation by β-caseinThe inhibitors of the amyloid fibril formation by β-casein were screened from the natural molecule compounds.（1）The results showed that arginine and epigallocatechin gallate inhibited the amyloid fibrils formation,but folic acid,resveratrol and procyanidins had no repressive effects.（2）The epigallocatechin gallate acted on the growth period of the fibril,while arginine inhibited amyloid fibrillations not only on the growth period,but also on the maturation period.（3）The above results were verified by the particle size measurement and TEM assay.6.The studies on the interaction mechanism between β-casein and arginineThe effects of arginine on the space structure,acting force,binding site,and energy change of β-casein were studied in order to make clear the inhibition mechanism with multi-spectroscopic approaches.（1）The results of UV absorption spectra showed that there was an interaction between β-casein and arginine.（2）Fluorescence spectra demonstrated that the conformation of β-casein was changed.（3）On the basis of fluorescence analysis at three temperatures,it was deduced that arginine quenched the fluorescence of β-casein by a static quenching mechanism.There was one binding site between β-casein and arginine.The thermodynamic results explained that a spontaneous reaction happened,and protein-ligand complex was stabilized by hydrogen bonds and Van der Waals force.（4）Furthermore,the binding distance between arginine and tryptophan residue of β-casein was 0.48 nm,showed in fluorescence resonance energy transfer assay.（5）According on the results of synchronous fluorescence experiment,the polarity was decreased around tyrosine residues,but there was no obvious change around tryptophan residue.