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Time (S)

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hIAPPhIAPP:rIAPP 1:1hIAPP:rIAPP 1:2hIAPP:rIAPP 1:5hIAPP:rIAPP 1:10

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nte

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hIAPPhIAPP seeded with hIAPP 1:10rat IAPP seeded with hIAPP 1:10

Wavelength (nm)

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1

hIAPPrIAPP

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hIAPPhIAPP:rIAPP 1:1hIAPP:rIAPP 1:2

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LagtimeT50

Time

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Inte

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Lag time 100%50%

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hIAPPhIAPP:rIAPP 1:1hIAPP:rIAPP 1:2hIAPP:rIAPP 1:5hIAPP:rIAPP 1:10

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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 Cao1, Fanling Meng1 and Daniel P. Raleigh1,2,3

1) Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400.

2) Graduate Program in Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794.

3) Graduate Program in Biochemistry and Structural Biology, State University on New York at Stony Brook, Stony Brook, NY 11794.

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

NH3+- KCNTATCAT QRLANFLVRS SNNLGPVLPP TNVGSNTY - C- NH2

O

Rat IAPP :

Human IAPP:

1 10 20 30 37

NH3+- KCNTATCAT QRLANFLVHS SNNFGAILSS TNVGSNTY - C- NH2

O

Humans form islet amyloid while rodents do not. The primary

sequences of the peptides are very similar aside from the 20-29

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 inhibitamyloid formation by hIAPP.

Which may explainwhy mouse models

do not form amyloid.

Prediction: rat IAPP will

inhibit amyloid formation

by human IAPP in vitro.

1 10 20 30 37

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)

T50

(S)

0 470 667

1 1180 1870

2 1530 2480

5 4590 6140

10 10410 16600

For the 1:5 and 1:10 ratios,

the lag phase increased by

a factor of approximately

10 and 20, respectively.

The effect on T50 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 FibrilGrowth Phase:

Ratio (rat/human)

Maximum rate dF/dT (S-1)

Time at max rate

(S)

Human hIAPP

10980 650

1:1 3005 1890

2:1 2570 2480

5:1 342 6060

10:1 22 16890

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).

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