Pentapeptide Inhibitor FLPNF Attenuates HIAPP Amyloid Aggregation And Its Molecular Mechanisms

Background: In vivo amyloid deposition due to misfolded proteins can lead to a series of protein conformation disease,such as type 2 diabetes mellitus,Alzheimer’s disease,Parkinson’s disease,and some other neurodegenerative diseases.In patients with type 2 diabetes mellitus and islet transplantation,human islet amyloid polypeptide(hIAPP)is the main components of the amyloid deposits in the islets.It is considered to be one of the important reasons for type 2 diabetes and loss function of islets implants.Therefore,it is becoming more and more important to study the aggregation and toxicity of hIAPP and to explore how to reduce the aggregation of hIAPP in the islet.Polypeptide hIAPP consist of 37 amino acids and is stored in the islet cell secretion vesicle after synthesis.It is secreted together with insulin by glucose stimulation.HIAPP could regulate and maintain blood glucose balance together with glucagon and insulin.But in pathologic condition,the oligomers and amyloid deposits formed by aggregation process cause functional damage to beta cells and result in beta cell death,due to the strong self-aggregation tendency of hIAPP.It has been suggest that the π-π interaction of the aromatic residues result in the interaction of hIAPP to form amyloid aggregation.The regions containing more aromatic amino acid are critical areas for hIAPP aggregation.The methods to reduce hIAPP aggregation include mainly the following 3 aspects: reduce the expression of hIAPP,improve the misfolding hIAPP clearance,and increase the stability of hIAPP and inhibit the aggregation.Many inhibitors have been reported including certain small molecule compounds,metal ions,autophagy activators,molecular chaperone and short peptide inhibitors.In which that short peptide inhibitors are homologous to the critical regions of the protein which are prone to form amyloid aggregation.These short peptides can specifically and readily bind to the critical regions of the target protein to stabilize the structure of the target protein conformation.They have no cytotoxicity and are easy to synthesize,are paid more and more attentions.At present,there are few studies on short peptide inhibitors that inhibit hIAPP aggregation.This study optimizes the design of 8 new short peptides,and verifies the effect to inhibit hIAPP aggregation of them in buffer solution.We screen out one pentapeptide inhibitor FLPNF with inhibiting effect.Then we test the effects of FLPNF on inhibiting hIAPP aggregation in cells,the effects on cell apoptosis and insulin secretion function,and its possible molecular mechanisms.We carried out the investigation using hIAPP-INS1 cells which secreting hIAPP.The study has certain significance for clinical development of medicine for treating type 2 diabetes and protecting the function of islet implants.Methods: 1.According to the critical areas of hIAPP aggregation,8 short peptides are designed and synthesised optimally.In buffer environment,the effect of short peptides on inhibition of hIAPP aggregation are detected by ThT staining to screen out effective short peptide inhibitors and the optimum inhibitory concentration of the short peptide inhibitors in buffer is detected by ThT staining.The inhibitory ability is also observed by transmission electron microscopy.2.A ThT staining method is used to verify the hIAPP aggregation in hIAPP-INS1 cells and membrane.Apoptosis of hIAPP-INS1 cells is detected by Annexin V-PI staining.The glucose-stimulated insulin secretion function of hIAPP-INS1 cells is detected by Elisa.FITC-labeled short peptides at N-terminal is constructed,and their ability to enter cells is detected by confocal microscopy and flow cytometry.In cellular environment by ThT staining,we test the short peptides inhibiting hIAPP aggregation in hIAPP-INS1 cell and the cell membrane.Influence of short peptides on cell apoptosis of hIAPP-INS1 cells is detect by Annexin V-PI staining.Influence of short peptides on glucose stimulation of insulin secrete function of hIAPP-INS1 cells is detected by Elisa.3.Bio-Layer Interferometry is used to verify the combination of FLPNF and hIAPP.Influence of FLPNF on the enzymatic activity of Neprilysin is examined by enzyme activity assay in hIAPP-INS1 cells.ThT staining is used to determine whether hIAPP is degradated by affecting the enzymatic activity of Neprilysin.Results: 1.Design,synthesis and infuluence of short peptide inhibitors on hIAPP aggregation in buffer solution.According to the critical regions of hIAPP,8 short peptides are optimal designed and synthesized.They have achieved good physicochemical properties by software predicts.In buffer environments,FLPNF can significantly inhibit amyloid aggregation formation of hIAPP.The optimum inhibitory concentration of FLPNF in buffer is 10:1 to the hIAPP on molar ratio.2.Influence on inhibition hIAPP amyloid aggregation,cell apoptosis and insulin secrete function by pentapeptide inhibitor FLPNF in hIAPP-INS1 cells.Compared with the control cell line CON-INS1 and rIAPP-INS1,hIAPP are significantly increased in hIAPP-INS1 cells.The apoptosis rate is increased significantly and insulin secretion is decreased with high glucose stimulation.FLPNF can assemble in hIAPP-INS1 cells,which means good ability of FLPNF to enter cells.In cellular environment,FLPNF can significantly inhibit hIAPP aggregation in hIAPP-INS1 cells and the optimal concentration is 200μM.At that concentration,FLPNF can reduce apoptosis rate significantly and improve the insulin secrete function of hIAPP-INS1 cells.3.Experimental study on molecular mechanisms of pentapeptide inhibitor FLPNF to reduce hIAPP aggregation.FLPNF inhibits hIAPP aggregation by binding to hIAPP.In hIAPP-INS1 cells,FLPNF may also increase the enzymatic activity of Neprilysin to promote hIAPP degradation.Conclusion: Pentapeptide inhibitor FLPNF can be observed in hIAPP-INS1 cells.It can inhibit amyloid aggregation of hIAPP in both buffer environment and cellular environment.By reducing amyloid aggregation,FLPNF can reduce apoptosis rate and improve the insulin secrete function of hIAPP-INS1 cells.FLPNF inhibit hIAPP aggregation formation by directly binding with polypeptide hIAPP.It also can enhance the enzymatic activity of Neprilysin of hIAPP-INS1 cells,thus promote the degradation of hIAPP.FLPNF can be used as a new effective short peptide inhibitor for inhibiting hIAPP amyloid aggregation.