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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.
Rat IAPP Lengthens the Lag Phase
Insulin, Human Islet Amyloid Polypeptide and Type
2 Diabetes
CD Spectra Demonstrate that Rat and Human IAPP
Interact with Each Other
The Sequence of Rodent and Human IAPP Are
Different

• 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.

Human IAPP

• For the 15 and 110 ratios, the lag phase
  increased by a factor of approximately 10 and 20,
  respectively.

1 10
20
30 37
T50
Lag time
100
50
Rat IAPP
1 10
20
30 37
Ratio (rIAPP/hIAPP) Lag time (S) T50 (S)
0 470 667
1 1180 1870
2 1530 2480
5 4590 6140
10 10410 16600

• 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.

Figure 1. IAPP amyloid fibrils contribute to cell
death.

• The effect on T50 is similar to the effect on the
  lag phase time.

General Characteristics of Amyloid Fibrils
numerical sum of hIAPP and rIAPP (11)

• Consist of 3-9 protofilaments which pack with a
  left handed coil.
• Dyes such as thiofalvin-T and Congo Red bind to
  amyloid.

Electron Micrographs of Human Rat IAPP Mixture
(ratio hIAPP/rIAPP)
Scale bar represents 100 nm
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.
hIAPPrIAPP 11 experimental result
Rat IAPP
Human IAPP 11
Which may explain why mouse models do not form
amyloid.
Hypothesis Rat IAPP might inhibit amyloid
formation by hIAPP.
Prediction rat IAPP will inhibit amyloid
formation by human IAPP in vitro.
numerical sum of hIAPP and rIAPP (12)
Rat IAPP Does Not Form Amyloid Fibrils

• 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.

• Figure 2. Model of thioflavin-T binding to
  amyloid ß-sheet structure.
• Structure of thioflavin-T.
• Thioflavin-T binds to channels on the surface of
  the fibril.
• A protofilament composed of three ßsheets.

Human IAPP
12
15
110
hIAPPrIAPP 12 experimental result
Nucleation-Dependent Pathway of Amyloid Fibril
Assembly
Rat IAPP
Seeding Experiments Show that Human Fibrils Can
Not Seed the Rat Peptide

• 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.

Rat IAPP
Human IAPP

• TEM images of human IAPP display the classic
  features of amyloid fibrils.
• TEM images of rat IAPP reveal that no fibrils
  were formed.

• 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.

Comparison of the Maximum Growth Rate of Amyloid
Formation

Fibril
Growth Phase
Fibril
Rat IAPP Inhibits Amyloid Formation by Human IAPP

• Kinetic assays of mixtures of human and rat IAPP
  at ratios of 11, 12, 15 and 110, i.e. at
  equal amounts of human IAPP and with rat IAPP in
  excess.

Ratio (rat/human) Maximum rate dF/dT (S-1) Time at max rate (S)
Human hIAPP 10980 650
11 3005 1890
21 2570 2480
51 342 6060
101 22 16890
Figure 3. Model of the nucleation-polymerization
pathway of amyloid formation.

• Hypothesis Rat IAPP can bind to Human IAPP but
  the prolines in the rat peptide likely prevent
  formation of the ?-sheet structure.

Inhibition of Amyloid Formation by IAPP
Types of Inhibitors

• Inhibitors only decrease the ultimate amount of
  amyloid fibrils (red curve).

Conclusions

• Rat IAPP is monomeric and is largely
  unstructured in aqueous solution.

• The final fluorescence intensity of the human and
  rat IAPP 15 mixture decreased by 67 relative to
  the intensity of human IAPP and the 110 mixture
  decreased by 85.

• Inhibitors only lengthen the lag phase (black
  curve).

• Rat IAPP is a moderate inhibitor of amyloid
  formation by human IAPP.
• It lengthens the lag phase and decreases the
  amount of amyloid.

• Inhibitors both lengthen the lag phase and reduce
  the amount of amyloid (green curve).

• 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.

Figure 4. Kinetic plots of possible effect of
different inhibitors on amyloid formation.
----- best target