TEMPLATE DESIGN © T 50 Lag time 100%50% Human IAPP Rat IAPP Rodent Islet Amyloid Polypeptide Inhibits Amyloid Formation by Human Islet Amyloid Polypeptide: Implications For the Design of Inhibitors and For Animal Models of Diabetic Amyloid Ping Cao 1, Fanling Meng 1 and Daniel P. Raleigh 1,2,3 1) Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY ) Graduate Program in Biophysics, State University of New York at Stony Brook, Stony Brook, NY ) Graduate Program in Biochemistry and Structural Biology, State University on New York at Stony Brook, Stony Brook, NY General Characteristics of Amyloid Fibrils Insulin, Human Islet Amyloid Polypeptide and Type 2 Diabetes Rat IAPP Does Not Form Amyloid Fibrils Rat IAPP Inhibits Amyloid Formation by Human IAPP Rat IAPP Lengthens the Lag Phase Electron Micrographs of Human Rat IAPP Mixture (ratio: hIAPP/rIAPP) Comparison of the Maximum Growth Rate of Amyloid Formation CD Spectra Demonstrate that Rat and Human IAPP Interact with Each Other Seeding Experiments Show that Human Fibrils Can Not Seed the Rat Peptide The Sequence of Rodent and Human IAPP Are Different 1:2 1:5 1:10 Rat IAPP Human IAPP 1:1 Scale bar represents 100 nm Conclusions Rat IAPP : Human IAPP :  Humans form islet amyloid while rodents do not. The primary sequences of the peptides are very similar aside from the region. Rat IAPP contains three proline residues in this region.  We have proposed that helical intermediates are involved in amyloid formation by IAPP and rat IAPP is predicted to bind to the helical intermediates. Hypothesis: Rat IAPP might inhibit amyloid formation by hIAPP. Which may explain why mouse models do not form amyloid. Prediction: rat IAPP will inhibit amyloid formation by human IAPP in vitro Rat IAPP  The red curve shows a typical human IAPP fibrillization reaction.  The black curve shows the result for rat IAPP, no significant change in thioflavin-T fluorescence is observed.  TEM images of human IAPP display the classic features of amyloid fibrils.  TEM images of rat IAPP reveal that no fibrils were formed. Human IAPP  Kinetic assays of mixtures of human and rat IAPP at ratios of 1:1, 1:2, 1:5 and 1:10, i.e. at equal amounts of human IAPP and with rat IAPP in excess.  The final fluorescence intensity of the human and rat IAPP 1:5 mixture decreased by 67% relative to the intensity of human IAPP and the 1:10 mixture decreased by 85%. Ratio (rIAPP/hIAPP) Lag time (S) T 50 (S)  For the 1:5 and 1:10 ratios, the lag phase increased by a factor of approximately 10 and 20, respectively.  The effect on T 50 is similar to the effect on the lag phase time.  TEM images were recorded where the final fluorescence reaches the steady-state value for all ratios at pH 7.4 in 2% HFIP.  Fewer fibrils were formed at higher ratios of rat to human IAPP, and they are thinner. + Fibril Growth Phase: Ratio (rat/human) Maximum rate dF/dT (S -1 ) Time at max rate (S) Human hIAPP : : : : numerical sum of hIAPP and rIAPP (1+2) hIAPP:rIAPP 1:2 experimental result numerical sum of hIAPP and rIAPP (1+1) hIAPP:rIAPP 1:1 experimental result  Hypothesis: Rat IAPP can bind to Human IAPP but the prolines in the rat peptide likely prevent formation of the  -sheet structure.  Rat IAPP is monomeric and is largely unstructured in aqueous solution.  Rat IAPP is a moderate inhibitor of amyloid formation by human IAPP. It lengthens the lag phase and decreases the amount of amyloid.  Our data strongly suggest that transgenic mice that express both rat and human IAPP are not good models for amyloid formation in type-2 diabetes.  Islet Amyloid is a common pathological feature of type 2 diabetes (Figure 1). The deposition of islet amyloid contributes to beta- cell failure and the decline in insulin secretion.  Islet Amyloid Polypeptide (IAPP) is responsible for islet amyloid.  Consist of 3-9 protofilaments which pack with a left handed coil.  Dyes such as thiofalvin-T and Congo Red bind to amyloid. Figure 2. Model of thioflavin-T binding to amyloid β-sheet structure. (a)Structure of thioflavin-T. (b)Thioflavin-T binds to channels on the surface of the fibril. (c)A protofilament composed of three β–sheets. When thiofalvin-T binds to the beta sheet in amyloid oligomers, the dye undergoes a characteristic red shift and an increase in the fluorescence signal at 482nm. Figure 1. IAPP amyloid fibrils contribute to cell death. Nucleation-Dependent Pathway of Amyloid Fibril Assembly Two-step process:  Initial lag phase: form nuclei.  Polymerization phase: early protofibrils grow and assemble to render mature amyloid fibrils.  Seeding: Addition of pre-formed fibrils to reduce the length of lag phase. Figure 3. Model of the nucleation-polymerization pathway of amyloid formation. Inhibition of Amyloid Formation by IAPP Figure 4. Kinetic plots of possible effect of different inhibitors on amyloid formation. Types of Inhibitors:  Inhibitors both lengthen the lag phase and reduce the amount of amyloid (green curve).  Inhibitors only lengthen the lag phase (black curve).  Inhibitors only decrease the ultimate amount of amyloid fibrils (red curve) best target