Aggregation Mechanisms Of Human Islet Amyloid 11-20 And The Study Of Peptide Inhibitors

It is known that conformational diseases are induced by the misfolding of protein,which can be characterized that proteins are unable to maintain their normal conformation,misfolding into β-sheet structures and becoming amyloid deposits.Extensive studies indicate that these deposites may damage the cytoskeleton and trigger cellular apoptosis.With the increasing prevalence rate yearly,Type 2 diabetes mellitus（T2DM）,a kind of conformational disease,has become an epidemic disease,which seriously endangers the quality of life and health of human beings.The deposition of islet amyloid polypeptide has been considered as one of the major pathological features of T2 DM.Though,oral hypoglycemic agents and insulin injection can be directly used for the therapy of T2 DM,these intervention treatments only can improve and maintain the normal blood glucose level at initial period.In the later stage of treatment,with the gradual occurrence of complications,these therapeutic methods present some limitations.The formation of amyloid deposits of human islet amyloid peptide（hIAPP）may damage the memebrane of pancreatic β cells,trigger cellular apoptosis,resulting in aggravating patients’ conditions.Inhibition of the accumulation of hIAPP could decrease the incidence rate of T2 DM,which develop a new method for preventing the aggregation of h IAPP.To block the π-stacking interactions in the amyloid formation of hIAPP self-assembly is an effective way to decrease the amyloid fibrils.As different fragments of hIAPP shows different properties,h IAPP_11-20 is considered as the target peptide in inhibiting events.In this paper we have studied the aggregation behavior of h IAPP_11-20 and designed a series of peptide inhibitors.First of all,the studies on molecular interaction between an organic dye fluorescein isothiocyanate（FITC）and bovine serum albumin（BSA）were investigated by a promising method named microscale thermophoresis to establish a method to study the self-aggregation behavior of protein or peptide.Furthermore,based on the established method,the aggregation behaviors of hIAPP_11-20 in different solutions were investigated.Finally,a serial of potential peptide inhibitors have been designed and synthesized,possessing different length,composition and conformation.The mechanisms of these peptides on inhibiting amyloid formation have been investigated by microscale thermophoresis and ThT assays,summarizing main factors which may have some influence on inhibiting amyloid formation.The specific studies are described as follows: 1.A novel method for the study for molecular interaction and aggregation behavior of proteins by using microscale thermophoresisThe fundamental investigating of the binding events between protein and its partner are important in drug development.In this study,molecular interaction between an organic dye fluorescein isothiocyanate（FITC）and bovine serum albumin（BSA）was investigated by a promising method named microscale thermophoresis（MST）and convential fluorescence spectroscopy.The results from MST experiments demonstrated that the interaction between BSA and FITC was a biphasic binding pattern in a short interaction time.By using competitive strategies in which warfarin and ibuprofen acted as the site markers of BSA,FITC was demonstrated to mainly bind to the hydrophobic pocket of site II of BSA compared to site I of BSA.Depending on the competitive strategies,it is proven that the interaction between FITC and BSA in this study is not a labeling process but weak interaction.Except for the binding affinity,MST also offered additional information with respect to the aggregation of BSA and the binding of FITC to BSA aggregates,which is unobtainable by fluorescence spectroscopy.This work suggests that MST is not only as a new approach for investigation of protein-small molecule interaction,but also as a powerful and reliable method for the studies on aggregation behavior of proteins.2.New insights into side effect of solvents on the aggregation of human islet amyloid polypeptide _11-20The formation of highly ordered fibrils for the human islet amyloid polypeptide（hIAPP）is considered as one of the precipitating factors of type 2 diabetes mellitus.In this study,MST and conventional ThT fluorescence assays were utilized to investigate the aggregation behavior of hIAPP_11-20,giving a new insight of the solvent effect on the aggregation of hIAPP_11-20.h IAPP_11-20 displayed different aggregation behaviors in various buffers,revealing that hIAPP_11-20 not only self-aggregates but also binds to solvent components.hIAPP_11-20 had a higher binding affinity for Tris than other selected buffers because multiple hydrogen bonds form,resulting in weaker self-aggregation of h IAPP_11-20 at the early stage of aggregation and prolonging the fibril formation process.hIAPP_11-20 displayed similar self-aggregation in both HEPES and pure water.Negatively charged phosphate ions in the PBS solution ‘neutralize’ the charges carried by h IAPP_11-20 itself to some extent,causing rapid aggregation of h IAPP_11-20,and leading to a shorter fibrillation process of h IAPP_11-20.These results revealed that solvents contribute to the aggregation of hIAPP_11-20 and demonstrated the affect of solvents on the activity of biomolecules,implying that buffer is more than buffering agent.Hence,the side effects of solutions should be taken into account in the study of h IAPP_11-20 self-aggregation.Additionally,as a new technique,microscale thermophoresis offers a powerful and promising approach to study the early stages of aggregation of peptides or proteins.3.New peptide inhibitors modulate the self-assembly of islet amyloid polypeptide residues _11-20 in vitroThe structural transition and misfolding of human islet amyloid polypeptide may cause a common metabolic disease Type 2 diabetes mellitus.Seventeen peptides have been engineered and synthesized,possessing different length,composition and peptide conformation.In this study,using model h IAPP_11-20,these peptides were assessed for their ability to decrease amyloid formation by the conventional ThT fluorescence assay and microscale thermophoresis.The final amyloid fibrils of h IAPP_11-20 were characterized by transmission electron microscopy（TEM）.The results showed that short peptides AT,SA,RF,KS,KT and KN,and cyclic peptides cyclic-KS,cyclic-KT and cyclic-KN displayed considerable inhibitory effect on h IAPP_11-20 fibril formation and strong affinity for hIAPP_11-20.The detailed investigation indicated that phenylalanine residue and some special peptide composition are significant in inhibiting amyloid formation.The peptide conformation may also play an important role on the inhibitory effect of the designed peptide inhibitors.Microscale thermophoresis quantified the binding affinities between h IAPP_11-20 and peptides and displayed that high affinity more likely leads to inhibiting fibril formation of hIAPP_11-20 and vice versa,in accordance with the results from ThT assays and molecular docking results.These findings suggest a feasible model of peptide inhibitor design for inhibiting the amyloid formation and extended the field on exploring other inhibitors of conformational diseases.In addition,microscale thermophoresis was proven as a promising rapid method in preliminarily screening inhibitors of h IAPP_11-20.4.Design and study of lipopeptide inhibitors on preventing aggregation of human islet amyloid polypeptide residues _11-20As lipopeptides have some hydrophobic groups,which are similar to the reported aggregation inhibitors,and some lipopeptides could prevent cells from depositing of amyloid fibrils,several potential lipopeptide inhibitors have been engineered and synthetized,which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of h IAPP_11-20 by using the conventional ThT fluorescence assay and microscale thermophoresis.The final amyloid fibrils of h IAPP_11-20 were characterized by transmission electron microscopy（TEM）.Results suggested that with the increasing length of carbon chain,the antiaggregation efficiency of lipopeptide inhibitors towards hIAPP_11-20 increased gradually.Meanwhile,the amount of arginines may also have some influence on inhibiting.As the binding events shows that the inhibitory efficiency of these lipopeptide inhibitors were enhanced with the increase of affinities,MST could be regarded as a appropriate and rapid method to screen potential inhibitors of h IAPP_11-20 or other amyloid protein.This study also broadens the types of inhibitors on inhibiting amyloid formation of hIAPP.In this study,an approach to study aggregation behavior of proteins was established by MST.The self-aggregation behaviors of h IAPP_11-20 in different solutions were invstigated by this method and other conventional methods.Discussing the inhibiting effects by different types of inhibitors,some impact factors on amyloid fibril formation of h IAPP_11-20 were also summarized,providing some experimental formation for the design of peptide inhibitors,broadening the types of inhibitos,and establishing a simple and rapid method to screening potential aggregation inhibitors.