Studies On The Aggregation Behaviors Of Human Islet Amyloid Polypeptide And The Inhibitors Of Amyloidosis

Highly ordered aggregation and accumulation of proteins in human body lead to a series of neurodegenerative diseases, including Alzheimer’s disease(AD), Parkinson’s disease(PD), type II diabetes(T2DM) and other age-related neurodegenerative disease. The amyloid deposit of human islet polypeptide(hIAPP) on the islet ? cells resulting from the misfolding of the peptide is believed to be a pathogenic factor of Type II diabetes. Therefore, preventing hIAPP fibrillogenesis is important not only for understanding the pathological process of type 2 diabetes, but also for the development of therapeutic drugs. In this thesis, we report the studies on the aggregation mechanism of human islet amyloid polypeptide, the inhibition effects of a D-type amino-acid inhibitor, as well as the effects of the organic solvents on the aggregation behaviors of hIAPP and on the performances of an all-D-amino-acid inhibitor in blocking fibril formation of hIAPP both in bulk solution and at phospholipid membrane surface. We also report the investigations on a serial of ultra-small sizes of CDs as inhibitors of hIAPP fibrillation and cytotoxicity. Finally, we show the ESI-MS studies on the aggregation mechanism of hIAPP8-20 segment. Overall, the work in this thesis can be summarized as following points:1. We designed an all-D-amino-acid inhibitor, namely D-NFGAIL, based on the ?-sheet core segment of hIAPP and tested the efficiency of the short peptide inhibitor in preventing the fibrillation of hIAPP using ThT assay, circular dichroism(CD), dynamic light scattering(DLS) and atomic force microscopy(AFM) measurements. We monitored the dynamic behaviors, size distribution, structure details and morphologies of hIAPP aggregates in the peptide fibrillation process and explored the mechanism underlying the inhibition of the formation of hIAPP fibrils by the inhibitor both in bulk solution and at membrane surface. The results show that the fibrillation of hIAPP can be inhibited by the all-D-amino-acid D-NFGAIL efficiently both in bulk solution and at phospholipid membrane surface. However, the mechanism of inhibition at membrane surface is different from that in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of ?-sheet oligomeric intermediates in bulk solution. This study could provide a new idea for designing the inhibitors of amyloid proteins.2. We studied the effects of two organic solvents(DMSO and HFIP) on the aggregation behaviors of hIAPP and the performances of an all-D-amino-acid inhibitor DNFGAIL in preventing hIAPP fibrillation both in bulk solution and at phospholipid membrane. Our results demonstrate that the presence of 1% v/v DMSO or HFIP decreases the rate of fibril formation of hIAPP at the lipid membrane rather than accelerates the fibril formation as what happened in bulk solution. The presence of 1% v/v DMSO or HFIP impairs the activity of the inhibitor at the lipid membrane surface dramatically, while it affects the efficiency of the inhibitor in bulk solution slightly. The inhibitor inserts into the lipid membrane more deeply or with more proportion in the presence of the organic solvents than it does in the absence of the organic solvents, which may hinder the binding of the inhibitor to hIAPP at the lipid membrane. Our results suggest that the organic solvents should be used with caution in studying membrane-induced fibrillogenesis of amyloid peptides and in testing amyloid inhibitors under membrane environments to avoid incorrect evaluation to the fibrillation process of amyloid peptides and the activity of inhibitors.3. We explored the potential role of carbon dots in regulating the aggregation behavior of hIAPP. Five kinds of CDs belonging to three categories, namely polymer dots(PDs-1 and PDs-2), carbon nanodots(CNDs and CQDs), and graphene quantum dots(GQDs), were prepared and characterized. The fibrillation behaviors of hIAPP in the presence of these CDs were monitored by the ThT assay and TEM/AFM imaging and the cytotoxicity of the systems was tested by the MTT assay. Our results showed that the polymer dots and carbon nanodots inhibit hIAPP fibrillation, while the GQDs promoted the formation of hIAPP fibrils. The PDs and GQDs that were nontoxic in INS-1 cells exerted efficiencies in decreasing the cell death induced by hIAPP through different mechanisms. The inhibitory activity and mechanism of the CDs were closely associated with their structures and surface properties. Our results shed light on a new potential application of CDs in therapy of amyloidodensis diseases.4. We chose the fragment of hIAPP8-20 as a model to study the aggregation mechanism of hIAPP further. We investigated the aggregation propensity of hIAPP8-20 and its mutants using Ion Mobility based Mass Spectrometry and Atomic Force Microscopy(AFM) to obtain the solution distributions of the peptide oligomers. This study is possibly useful for us to identify the structure of the toxic oligomers and eventually understand hIAPP pathogenesis mechanism.