Studies On The Aggregation And Inhibitors Of Polypeptides Related To Diabetes And Other Diseases

Protein misfolding diseases(PMDs)refer to proteins that have normal physiological functions,which have been shown to misfold,aggregate and deposit in tissues and organs,triggering a series of diseases.At present,more than 30 diseases have been found to be associated with protein misfolding,such as neurodegenerative diseases Alzheimer's disease(AD),Parkinson's disease(PD),and metabolic related type 2 diabetes(T2D).Although the proteins associated with PMDs have different amino acid sequences and conformational structures,they can form insoluble amyloid deposits with ?-sheet structures from soluble proteins,suggesting that there may be a similar pathogenic mechanism among different PMDs.A typical pathological feature of type 2 diabetes is amyloid deposition in pancreatic islets.Human islet amyloid polypeptide(hIAPP),composed of 37 amino acid residues,is the major component of amyloid deposition in islets.hIAPP is synthesized in pancreatic ?-cells and co-secreted with insulin,which can regulate blood glucose more precisely with insulin and other blood glucose regulating hormones.However,hIAPP is also one of the peptides with the strongest aggregation tendency.The aggregates formed during the fibrillation of hIAPP can induce apoptosis of islet ?-cells by destroying the integrity of cell membranes,causing mitochondrial damage,inducing intracellular oxidative stress and endoplasmic reticulum stress.The loss of ?-cell function and ?-cell mass make the islet unable to maintain normal physiological function,which triggers to or aggravates the pathological development of T2D.The aggregation behavior of hIAPP is closely related to its own amino acid fragments,but also affected by the interaction of cell membrane,metal ions in the surrounding environment and pH value.In addition,hIAPP may be associated with Alzheimer's disease by crossing the blood-brain barrier and forming hIAPP deposition in the brain.Therefore,understanding the aggregation process of hIAPP and developing potential inhibitors can provide a new method for the treatment of T2D and other PMDs.In this thesis,electrospray ionization coupled with ion mobility-mass spectrometry(ESI-IM-MS)was used as the major research method combined with fluorescence spectroscopy and other analytical approach,to study the factors affecting the hIAPP aggregation and the inhibition of several natural products.Firstly,the interaction between A? fragments and hIAPP was investigated to understand the influence of the coexistence of A? and hIAPP on the self-aggregation of both peptides and whether there was co-assembly behavior in the process of fibrillation.The results suggested that the aggregation of hIAPP and A?25-35 were two-separate process in the binary mixtures.The first aggregation stage was mainly generated by the fibrillation of hIAPP and the second stage corresponded to the aggregation of A?25-35.hIAPP and A?25-35 could form a series of hetero-oligomers formed in the mixtures of hIAPP and A?25-35 in the early stage.The aggregation of A?25-35 was accelerated through the cross-reaction between hIAPP monomers and A?25-35 monomers as well as a little of oligomers during primary nucleation stage.However,due to the obvious difference in aggregation ability between hIAPP and A?25-35,this cross-interaction had a little impact on the self-assembly of hIAPP.The interaction between the hydrophilic region 1-16 of A? peptide and hIAPP was also studied.It was proved that although hIAPP has high binding affinity with hydrophilic and hydrophobic regions of A? peptide,A?25-35may play a more important role in the interaction between the full-length A?and hIAPP.Subsequently,we investigated the effects of Cu2+on the conformation and fibrillation of hIAPP,and the oxidation of hIAPP by Cu2+and H2O2.The results of ESI-IM-MS analysis showed that Cu2+may interfere with the normal aggregation pathway of hIAPP by binding with the extended conformers of hIAPP,and finally affect the amyloid formation.It was also found that the synergistic effect of H2O2 and Cu2+ could oxidize hIAPP to form a dityrosine cross-linked dimer.The results of fluorescence and CD assays showed that the cross-linked dimer could not aggregate to form amyloid fibers with ?-sheet structure,indicating that the aggregation behavior or aggregation pathway of hIAPP might be altered after the cross-linking.Finally,a screening method was established to screen the inhibitors of hIAPP aggregation in vitro.The inhibitory effects of lithospermic acid(LA)and flavonols on hIAPP aggregation were studied by multiple analytical methods from various aspects such as the conformational changes,the aggregation kinetics and the exposure of surface hydrophobicity of hIAPP.The results of ESI-IM-MS analysis showed that the relative abundance of the compact and extended conformers was altered by the formation of hIAPP-LA complexes,which promoted the transition of more extended structures to the compact conformers.It was proved that LA can effectively inhibit the aggregation of hIAPP and reduce the fiber formation.Moreover,LA exhibited potential protective effects against hIAPP-induced cell toxicity.In addition,the structure-activity relationships of the interaction between flavonols and hIAPP were analyzed.The results showed that the glycosyl groups and hydroxyl groups of aglycones in flavonols could improve their binding affinity with hIAPP,and enhance the gas phase stability of the complexes formed by hIAPP and ligands.However,the effects of multiple hydroxyl groups of aglycones on IAPP aggregation was more obvious,followed by the number of glycosyl groups and different types of glycosyl groups.Among the five flavonols,rutin had the strong inhibitory action on the fibrillation of hIAPP,which could make more hIAPP monomers in stable conformation.These above results could help us better understand the aggregation mechanism of hIAPP,and provide a new basis for screening potential natural small molecule inhibitors for the treatment of T2D and other PMDs.