# The Role of the 14–20 Domain of the Islet Amyloid Polypeptide in Amyloid Formation

Authors: Sharon Gilead; Ehud Gazit
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

The molecular mechanism of amyloid formation by the islet amyloid polypeptide (IAPP) has been intensively studied since its identification in the late 1980s. The IAPP(20–29) region is considered to be the central amyloidogenic module of the polypeptide. This assumption is mainly based on the amyloidogenic properties of the region and on the large sequence diversity within this region between the human and mouse IAPP, as the mouse IAPP does not form amyloids. A few years ago, another region within IAPP was identified that seems to be at least as important as IAPP(20–29) in facilitation of molecular recognition that leads to amyloid formation. Here, we reinforce our and others' previous findings by analyzing supporting evidence from the recent literature. Moreover, we provide new proofs to our hypothesis by comparing between the amyloidogenic properties of the two regions derived from the IAPP of cats, which is also known to form amyloid fibrils.


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## Body

## 1. Introduction

The islet amyloid polypeptide (IAPP) is a 37 amino acid hormone, which is colocalized with
insulin in the pancreaticβ-cells. The two polypeptides are cosecreted in
response to β-cell stimulation [1–4]. In 1986 and 1987, Westermark
etal. and Cooper etal. identified IAPP (also designated as Amylin) as
the major component of pancreatic amyloid deposits, a characteristic pathological
feature of type II diabetes [5, 6]. As other amyloid-related
proteins and polypeptides, the IAPP identified in these deposits is the
wild-type polypeptide in most type II diabetes cases. In its physiological role,
IAPP acts as a regulator of glucose homeostasis [7–9].The formation of amyloid fibrils, which is the hallmark of a group of more than twenty diseases,
among them some of the most devastating disorders of the 20th century, is a
highly specific self-assembly process [10–14]. The accumulation of IAPP, as
well as of amyloidogenic proteins found in other amyloid-related diseases, into
amyloid fibrils proceeds via a self-recognition mechanism. The process involves a structural transition
from the protein native structure, which is considered to be random coil in the
case of IAPP, into a cross-β-pleated-sheet secondary structure conformation.The exact conditions that stimulate the aggregation of amyloidogenic proteins are yet to
be fully understood. However, comprehension of the molecular traits of the
amyloidogenic process may be highly valuable in the attempts to understand and
prevent this world affecting phenomenon. The identification of IAPP in the late
1980s paved the way for vast investigation regarding the molecular mechanism of
IAPP self-assembly process. A central issue addressed was the search for the
molecular elements that make IAPP highly prone to amyloid formation. One of the
first observations regarding IAPP amyloidogenicity was the specie specificity
of this process [15, 16]. Although more than 80% of
the IAPP sequence is conserved in mammalians, only a few species such as
humans, primates, and cats develop islet amyloid and suffer from type II
diabetes, while mice, rats, and dogs do not develop islet amyloid deposits.The most noticeable
sequence diversity is the one between human and mouse/rat IAPP. The sequence of
rodent IAPP (rIAPP) differs from that of human IAPP (hIAPP) in 6 out of 37
amino acids, 5 of them are located in a defined region between residues 20 and
29 [hIAPP(20–29) is SNNFGAILSS and rIAPP(20–29) is SNNLGPVLPP] [15–17]. Moreover, the rodent IAPP contains
within this region 3 proline residues, which are absent in the human sequence. The
proline residue is known to strongly unfavor β-sheet structures. Therefore, the hIAPP(20–29)
has been suggested to be responsible for the amyloidogenic propensities of
full-length hIAPP.The decapeptide sequence of hIAPP(20–29) was shown to be able to aggregate into
amyloids, whereas the corresponding rIAPP(20–29) did not [15–17]. Furthermore, shorter peptide
fragments derived from this region, the
penta- and hexapeptides sequences hIAPP(23–27) (FGAIL) and hIAPP(22–27)
(NFGAIL), were found to be sufficient for
the formation of amyloid-like structures [18]. Hence, the hIAPP(20–29)
has been used as a model
to study intermolecular interactions and β-sheet formation and was considered to be the
only recognition region of IAPP.In 2001, Fraser etal. identified a previously unrecognized amyloidogenic
domain of IAPP located within residues 8–20. Synthetic peptides corresponding
to this region assembled into fibrils with typical amyloid-like morphology [19]. Thereafter, we identified, using an unbiased peptide
array analysis, a domain comprising the same region that showed even higher affinity
recognition to IAPP as compared to the hIAPP(20–29) region [20]. IAPP was incubated with a
SPOT membrane containing consecutive overlapping sequences of the full-length
hIAPP. Indeed, IAPP was found to bind to the 20–29 region, however a substantially
stronger interaction to a region within residues 11–20 was found. Moreover, peptide
fragments within this region were shown to readily form amyloid-like
structures, some of which are as short as pentapeptides, corresponding to
hIAPP(14–18) and hIAPP(15–19). Thus, we suggested that this new identified
region plays a central role in the recognition as well as the self-assembly of
IAPP amyloid formation process.During the
last few years, accumulating data has supported our previous assumption, both
directly and indirectly. However, the hIAPP(20–29) has still remained the major
studied amyloidogenic region. Here, we pinpoint these supporting evidences from
the recent literature in order to emphasize the relevance of the new
recognition and self-assembling region. Furthermore, we provide new proofs for
our hypothesis by investigating and comparing the amyloidogenic propensity of
the two regions derived from the amyloidogenic cat IAPP (cIAPP).


## 2. Materials and Methods

### 2.1. Peptide Solutions

Peptides were purchased from Peptron, Inc. (Taejeon, Korea).
Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 100 mM. To avoid any preaggregation, fresh stock solutions were prepared for each
experiment. Peptide stock solutions were diluted into 10 mM Tris buffer, pH 7.2 to
a final concentration of 2 or 5 mM and 2% or 5% DMSO, respectively.


### 2.2. Congo Red Staining and Birefringence

A 10μL suspension
of 5 mM peptide solution aged for 1 day was
allowed to dry overnight on a glass microscope slide. Staining was performed by
the addition of a 10 μL suspension of saturated Congo Red (CR) and NaCl in 80%
ethanol (v/v) solution. Birefringence was determined with an SZX-12 stereoscope (Olympus,
Hamburg, Germany) equipped with cross polarizers.


### 2.3. Transmission Electron Microscopy

A 10μL sample of 2 or 5 mM peptide solution
aged for 1 to 3 days was placed on a 400-mesh copper grid covered by carbon-stabilized
formvar film (SPI supplies, West Chester PA). After 1 minute, excess fluid was
removed, and the grid was then negatively stained with 2% uranyl acetate in
water for another 2 minutes. Samples were viewed in a JEOL 1200EX electron
microscope operating at 80 kV.


### 2.4. Fourier-Transform Infrared Spectroscopy

A 30μL sample of 2 mM peptide solution aged for 2 days was suspended on a polytetrafluoroethylene
(PTFE) card and dried by vacuum. Peptide deposit was resuspended with D2O
and subsequently dried. The resuspension procedure was repeated twice to ensure
maximal hydrogen to deuterium exchange. Infrared spectra were recorded using a
Nicolet Nexus 470 FT-IR spectrometer with a DTGS detector.


## 2.1. Peptide Solutions

Peptides were purchased from Peptron, Inc. (Taejeon, Korea).
Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 100 mM. To avoid any preaggregation, fresh stock solutions were prepared for each
experiment. Peptide stock solutions were diluted into 10 mM Tris buffer, pH 7.2 to
a final concentration of 2 or 5 mM and 2% or 5% DMSO, respectively.


## 2.2. Congo Red Staining and Birefringence

A 10μL suspension
of 5 mM peptide solution aged for 1 day was
allowed to dry overnight on a glass microscope slide. Staining was performed by
the addition of a 10 μL suspension of saturated Congo Red (CR) and NaCl in 80%
ethanol (v/v) solution. Birefringence was determined with an SZX-12 stereoscope (Olympus,
Hamburg, Germany) equipped with cross polarizers.


## 2.3. Transmission Electron Microscopy

A 10μL sample of 2 or 5 mM peptide solution
aged for 1 to 3 days was placed on a 400-mesh copper grid covered by carbon-stabilized
formvar film (SPI supplies, West Chester PA). After 1 minute, excess fluid was
removed, and the grid was then negatively stained with 2% uranyl acetate in
water for another 2 minutes. Samples were viewed in a JEOL 1200EX electron
microscope operating at 80 kV.


## 2.4. Fourier-Transform Infrared Spectroscopy

A 30μL sample of 2 mM peptide solution aged for 2 days was suspended on a polytetrafluoroethylene
(PTFE) card and dried by vacuum. Peptide deposit was resuspended with D2O
and subsequently dried. The resuspension procedure was repeated twice to ensure
maximal hydrogen to deuterium exchange. Infrared spectra were recorded using a
Nicolet Nexus 470 FT-IR spectrometer with a DTGS detector.


## 3. Results and Discussion

### 3.1. Identification of Other Amyloidogenic Regions within IAPP

The hIAPP(20–29) region was considered to be the central amyloidogenic domain within hIAPP. In
1999 and 2001, two other amyloidogenic regions, hIAPP(30–37) and hIAPP(8–20),
were identified to be able to form amyloid-like fibrils in aqueous medium as
well [19, 21]. Sequence examination of
these regions reveals a single amino acid variation between the rodent and
human sequences at position 18, displaying Arg or His, respectively. At the
C-terminus part, rIAPP(30–37) and hIAPP(30–37) are completely homologous. Thus,
it was suggested that in the rodent molecule, two potentially amyloidogenic
domains exist but are separated by the proline rich domain, residues 20–29,
which prevent β-sheet formation [4].Shortly after the publication of our results regarding the identification of very short
amyloid-forming peptides within the IAPP(14–19) region [20], another study has shown the
presence of short amyloidogenic peptides within the 8–20 region [22]. By scanning a series of
overlapping peptides from the 8–20 region, two peptide fragments, IAPP(12–17)
and IAPP(15–20), were found to form amyloid-like structures. This study gave a strong
independent support to our findings. All together these findings provided clear
evidence of the presence of more than one amyloidogenic domain within IAPP.


### 3.2. Contribution of Individual Residues to Amyloid Formation by Full-Length IAPP

The contribution of specific residues from different regions to hIAPP
fibrillization was also investigated in the context of full-length IAPP by
performing single and multiple amino acid substitutions and assessing their
influence on amyloid fibril formation. The ability of rIAPP variants of
single-residue substitutions with amino acids from the corresponding positions
of hIAPP was studied [23]. A single substitution of Arg to His at
position 18 (R18H), in the full-length rIAPP, was found sufficient to render its
competence for fibril formation at a small yield. Similar results were observed
with the single substitutions L23F and V26I. In addition, the combination of
two or three of these substitutions generally increased the ability to produce
fibrils. These results show that the presence of the three proline residues in
the rIAPP(20–29) domain is insufficient to abolish the ability to form fibrils.
Moreover, the ability of the R18H variant of rIAPP to self-associate into
amyloid fibrils suggests that other domains of IAPP except the 20–29 are
involved in the self-recognition process.The role of the histidine
at position 18 in amyloid formation was further examined by assessing hIAPP fibrillization at
various pH ranges [24]. The ionization state of
His-18 was found to substantially affect the rate of assembly as well as the morphology
of the amyloid fibrils formed by hIAPP. The aggregation process was faster at
high pH (8.8), when the histidine is deprotonated, than at low pH (4.0). This fact
may be physiologically relevant as mature hIAPP is stored in the β-cell
granules at a pH of 5.5 and released into the extracellular matrix where the pH
is of 7.4. Thus, the low pH in the pancreatic granules may protect hIAPP from
aggregation.Abedini and Raleigh, who performed the above-described study, further questioned the exclusive
importance of the 20–29 region by designing a variant of the amyloidogenic
hIAPP(8–37), containing three proline residues outside this region, at
positions 17, 19, and 30 [25]. The 3×P variant had dramatically greater solubility
and reduced tendency to form β-sheet structures compared to the wild-type
polypeptide, as assayed by a variety of amyloid-detecting techniques. The authors concluded that models of IAPP
fibrillization must take into account contributions of other regions within
IAPP.


### 3.3. Inhibition of hIAPP Amyloid Formation

Based on our identification of the hIAPP(13–18) recognition site, we designed peptide
inhibitors against IAPP fibrillization, which were targeted to this region. Using
the previously exemplified method of incorporatingβ-breaker elements into amyloidogenic
core peptides [26], we introduced a new
inhibition strategy using the α-aminoisobutyric acid (Aib), a β-breaker element with extreme structural
constrains [27]. Peptide fragments corresponding
to the recognition domain were modified with Aib. The modified peptides
completely lost their amyloidogenic potential. Furthermore, the Aib-modified
peptide showed a powerful inhibitory effect on the formation of amyloid fibrils
by the full-length hIAPP.Evidently, the 20–29 region was also used as a template for inhibitor design and served as a
target for inhibition. Kapurniotu etal. designed a nonamyloidogenic and
bioactive mimic of hIAPP, which contains a double N-methylation of full-length
hIAPP at positions G24 and I26, termed as IAPP-GI [28]. The presence of two N-methyl
rests on the same side of a β-strand interferes with the interstrand amide
hydrogen-bonding necessary for β-sheet formation [29]. The IAPP-GI analogue was
shown to be a nanomolar affinity inhibitor of hIAPP fibrillization and
cytotoxicity. However, although IAPP-GI did not form amyloid fibrils, it was
found to have a “pronounced self-association propensity” and to form
spheroids of up to 100 nm in diameter. Moreover, the Far-UV circular dichroism
(CD) analysis of IAPP-GI indicated the presence of β-sheet and/or β-turn
conformations. These insinuate the presence of another recognition and
self-assembly domain besides the 20–29 region that enabled the formation of the
observed structures. A reminiscent
scenario exists in the study of peptide nanotubes and nanostructures, which was
initiated from the search for a minimal amyloidogenic core domain of
Alzheimer's β-amyloid polypeptide. While the Phe-Phe dipeptide was shown to
form well-discrete peptide nanotubes [30], two other peptides, Cys-Phe-Phe and
diphenylglycine, which are very similar to the diphenylalanine, formed closed-cage
nanospheres [31]. We
speculate that the remarkable inhibition ability of the IAPP-GI analogue is actually
due to its high affinity recognition to the 3–18 site, as it is kept unmodified
and thus provides maximal compatibility.The search for
inhibitors against amyloid formation led to the identification of insulin as an
exceptional inhibitor of IAPP fibrillization [32–34]. This finding may be of high physiological
importance as IAPP and insulin are costored and cosecreted from the pancreatic
β-cell granules. Consequently, insulin is considered to form a complex with
IAPP which stabilizes it and prevents it from aggregation within the granules [35, 36]. In order to reveal the
molecular mechanism underlying the interaction between IAPP and insulin, we
performed a molecular mapping of the interaction interface. Using a
reductionist approach and peptide arrays, we located the cross-recognition sites
within both polypeptides [37]. Interestingly, insulin was
found to bind to the 13–18 region within IAPP. In addition, the identified recognition
site within insulin, which resides within the insulin B chain, was previously shown to have
sequence similarity to the hIAPP(13–18) region [35]. These findings reveal a
typical amyloid inhibition mechanism for insulin and reinforce the central role
of the 13–18 region: the binding of insulin to IAPP self-recognition site is
mediated through sequence similarity, it interferes with IAPP self-association
and prevents it from amyloid formation.


### 3.4. Interaction of IAPP with the Membrane

As in the case of other amyloid-related disease,
the mechanism by which IAPP causes cell destruction is assumed to involve interaction
with the cell membrane. Different studies have investigated the possible
interaction of IAPP with the membrane, mostly by the use of membrane mimetics
such as liposomes and phospholipid assemblies. In a recent study, hIAPP was
claimed to insert into phospholipid monolayer as a monomer [38]. Interestingly, it was suggested that the
N-terminus of hIAPP is largely responsible for the insertion. Experiments using
fragments of hIAPP showed that a peptide consisting of the 19 N-terminal
residues of hIAPP efficiently inserts into a phospholipids monolayer,  whereas
the 20–29 residue peptide inserts much less efficiently. These results offer a
possible role for the N-terminal region also in the sense of interaction with
the pancreatic β-cells which may be part of the mechanism underlying type II diabetes
pathogenesis.


### 3.5. Comparison between the Two Amyloidogenic Regions Derived from the Cat IAPP

Cats are among the few mammalian species that are known to suffer from type II diabetes and from
accumulation of IAPP islet amyloid deposits [39, 40]. The cat IAPP (cIAPP)
sequence differs from that of hIAPP in only four amino acids, at positions 17,
18, 23, and 29. The decapeptide corresponding to cIAPP(20–29) was previously
shown to be able to aggregate into amyloidal structures as well [16, 41, 42], thus supporting the apparent
fibrillization role of the 20–29 region. However, both the dog and cat IAPP are
identical over the 20–29 region [43] and dogs are not known to
develop type II diabetes.In order to re-evaluate the role of the two identified recognition sites of IAPP in the
fibrillization process, we tested the self-assembly propensity of peptide fragments
derived from the cat IAPP, cIAPP(22–29), and cIAPP(14–20). We also tested the
aggregation of the corresponding human sequence peptides for reference. The
peptide sequences are presented in Table1. The peptides were dissolved into
aqueous solution, under the exact same conditions, incubated for one to three
days and tested for amyloid formation by electron microscopy (EM) and Congo Red
(CR) birefringence. Their secondary conformation was also evaluated by
Fourier-transform infrared spectroscopy (FTIR).Table 1
The studied peptides. Peptide fragments corresponding to regions within the human
and cat IAPP were tested for the ability to form amyloid-like structures.
NameOriginSequenceResiduesC1catNLGAILSP22–29H1humanNFGAILSS22–29C2catNFLIRSS14–20H2humanNFLVHSS14–20Differences between the peptides appeared already
before applying the amyloid tests. Upon dilution of the peptides into aqueous
solution, the H1 peptide immediately precipitated into eye-visible aggregates. The other peptide solutions appeared
clear to the unarmed-eye. Examination of the peptide samples under the electron
microscope revealed the presence of well-ordered tubular structures for all the
peptides except the C1 peptide (Figure1). Apparently, the peptides were found to form nanotubes rather than
fibrils. The formation of tubular structures by H2 and H1 was previously shown [18, 20, 44]. Here we found that the C2 peptide can also form
highly defined tubular structures. Thin fibrils were also detected in proximity
with the nanotubes or in independent bundles (not shown). The C2 sample
contained mostly nanotubes, the H2 sample contained nanotubes and fibrils, and the
H1 sample contained also large aggregates. The C1 peptide however did not form
fibrils or tubes, only less ordered spherical structures and aggregates were
observed. The spherical structures may be reminiscing the ones observed for the
IAPP analogue IAPP-GI [28], which were discussed above, however they are
less ordered than the last ones.Morphology of the formed structures. Electron microscopic examination of negatively
stained samples of the studied peptides: (a) C2, (b) H2, (c) C1, and (d) H1. The
C1 peptide failed to form tubular structures as did the other peptides. (e) and
(f) are high magnification micrographs of the marked rectangle areas of C1 and H1, respectively.
(a)(b)Upon staining with the Congo Red dye, the C2 and
H2 samples exhibited clear gold-green birefringence (Figure2). In the H1
sample, the dye appears to be bound, however, no green color was detected.
Possibly, the large amount of aggregates, which were present in addition to the
fibrillar and tubular structures, were shielding over them. No birefringence or CR binding was shown in
the C1 sample, the slide looked quite clean. Secondary structure analysis of
the peptides by FTIR showed large β-sheet content for all the peptides (Figure 3).
The β-sheet conformation is characteristic of amyloid-like structures, but is not
exclusive for them. The C1 peptide, negatively detected for amyloid structures,
also displayed β-sheet conformation which may be attributed to the spheroid
structures observed by the EM or to its soluble form.Congo Red binding and birefringence. Microscopic examination under
cross-polerizers upon staining with Congo Red of samples of the studied
peptides: (a) C2, (b) H2, (c) C1, and (d) H1. The C2 and H2 peptides exhibited
green birefringence characteristic of amyloidal structures.
(a)(b)(c)(d)Secondary conformation of the formed structures. Fourier-transform infrared (FTIR)
spectroscopy analysis of the secondary conformation of the peptides: (a) C2, (b) H2, (c) C1,
and (d) H1. All the peptides exhibited spectra typical forβ-sheet secondary structure.
(a)(b)(c)(d)The comparison between the two cat derived
peptides reveals that the C2 peptide, containing residues 14–20, is much more
competent for self-assembly into amyloid-like structures than the C1 peptide,
composed of residues 22–29. Although a longer version of the C1 peptide,
composed of residues 20–29, was previously shown to form fibrillar structures
[41, 42], in the present experimental setup it was unable
to form such fibrils. We consider this diversity to result from the length of
the peptide and different experimental conditions, which are in the present
assay of more physiologically-relevant environment and of lower concentration.
Nevertheless, when compared to the C2 peptide, C1 is dramatically less potent
for fibril formation. While the C1 peptide may contain self-association
properties as observed by the presence of spherical structures and β-sheet
content, the C2 peptide exhibited self-assembly potency into well-ordered
macromolecular fibrils, which were not displayed by the C1 peptide. This
observation provides fresh supporting evidence for the central role of the
13–18 region within the IAPP molecule in amyloid formation: while in hIAPP both
regions are highly amyloidogenic, in the cat IAPP, there is a clear difference
between the two peptides.Different behavior was also observed for the two
human derived peptides H1 and H2, which may insinuate their diverse roles in
the amyloidogenic process. The H1 peptide seems to be highly aggregative since
visible precipitates were observed upon dilution into aqueous solution as well
as under the EM. The H2 peptide however seems to have higher ability to form
well-ordered structures which requires specific recognition. Its solution was
visibly clear and EM examination revealed only fibrilar and tubular structures
without aggregates. In addition, green birefringence upon CR binding, which is
characteristic of ordered structures, was observed in the H2 sample but not in
the H1 sample. Consistent with this observation is the different recognition
affinity of the two peptide fragments to the full-length IAPP as we have
previously showed by the peptide array assay [20].The C1 peptide is not as aggregative as the H1 peptide, although it contains some structural
features as observed by the FTIR and EM analyses. A comparison between these two sequences
in their longer versions of residues 20–29 was previously  performed by Ashburn and Lansbury [42]. In this study, both peptides were found to form
amyloid fibrils, however, with remarkably different kinetics. The cIAPP(20–29)
peptide formed fibrils approximately 13-fold more slowly than its human
version. This correlates with our results regarding the different potency for
fibrillization of the two sequences. It is likely that with a longer incubation
period, the cIAPP(22–29) would also fibrillate eventually.The cIAPP sequence differs from the hIAPP sequence
by a Leu instead of a Phe at position 23 and a Pro instead of a Ser at
position 29. Both modifications were shown to separately influence the kinetics
of the peptide fibrillization [42]. It seems obvious that a Ser to Pro substitution
will decrease the fibrillization potency, since Pro is a strong β-breaker
element. Regarding the Phe to Leu substitution, we speculate that the Phe
residue facilitates the fibrillization process through aromatic interactions. According
to our established hypothesis, aromatic residues and interactions largely enhance
the process of amyloid formation by providing directionality and stability to
the growing fibril [45–49].


### 3.6. Intra-β-Sheet Mechanism for IAPP Amyloid Formation

The accumulated data presented in this paper prove
that none of the two amyloidogenic regions within IAPP can be ignored. A
possible mechanism underlying the self-assembly of IAPP into amyloid fibrils
includes a role for both regions. It was already suggested in 2001 by Jaikaran
and Clark that the structural transition of IAPP into amyloid fibrils involves
in interactions betweenβ-stranded regions within the monomer [4]. According to their model, an intramolecular β-sheet
is formed by three β-strands composed of the segments 8–20, 24–29, and 32–37. Earlier
secondary structure prediction based on IAPP sequence provided support to their
model by predicting β-turns at positions 20 and 31, as well as β-strands for
residues 14–18 and 26–29 [50]. More recently, another model for the structure
of IAPP within the amyloid fibril was proposed. Accordingly, the same three
β-strands are present, however, arranged in a zigzag planar s-shaped β-sheet,
instead of the previously proposed e-shaped structure [51].The presence
of an intramolecularβ-sheet in the fibrillar structure of IAPP is supported by
the identification of long-range interactions between distinct residues within
the IAPP molecule. Using fluorescence resonance energy transfer (FRET), the tyrosine
at position 37 was shown to be close in space to the two phenylalanine residues
(F23 and F15) at the fibrillogenic state of hIAPP [52]. Another interesting point
arises from the genetic background of type II diabetes. Although the disease is
predominantly sporadic, a mutation in hIAPP sequence, the S20G mutation, which
is identified among the Japanese population, was found to be related to early
onset of the disease [53]. The S20G mutant was also
shown to exhibit increased fibrillization and cytotoxicity compared to
wild-type hIAPP [54]. Indeed, according to the
intra-β-sheet models, the mutation is placed in a β-turn region, which is
favorable for the glycine residue.


## 3.1. Identification of Other Amyloidogenic Regions within IAPP

The hIAPP(20–29) region was considered to be the central amyloidogenic domain within hIAPP. In
1999 and 2001, two other amyloidogenic regions, hIAPP(30–37) and hIAPP(8–20),
were identified to be able to form amyloid-like fibrils in aqueous medium as
well [19, 21]. Sequence examination of
these regions reveals a single amino acid variation between the rodent and
human sequences at position 18, displaying Arg or His, respectively. At the
C-terminus part, rIAPP(30–37) and hIAPP(30–37) are completely homologous. Thus,
it was suggested that in the rodent molecule, two potentially amyloidogenic
domains exist but are separated by the proline rich domain, residues 20–29,
which prevent β-sheet formation [4].Shortly after the publication of our results regarding the identification of very short
amyloid-forming peptides within the IAPP(14–19) region [20], another study has shown the
presence of short amyloidogenic peptides within the 8–20 region [22]. By scanning a series of
overlapping peptides from the 8–20 region, two peptide fragments, IAPP(12–17)
and IAPP(15–20), were found to form amyloid-like structures. This study gave a strong
independent support to our findings. All together these findings provided clear
evidence of the presence of more than one amyloidogenic domain within IAPP.


## 3.2. Contribution of Individual Residues to Amyloid Formation by Full-Length IAPP

The contribution of specific residues from different regions to hIAPP
fibrillization was also investigated in the context of full-length IAPP by
performing single and multiple amino acid substitutions and assessing their
influence on amyloid fibril formation. The ability of rIAPP variants of
single-residue substitutions with amino acids from the corresponding positions
of hIAPP was studied [23]. A single substitution of Arg to His at
position 18 (R18H), in the full-length rIAPP, was found sufficient to render its
competence for fibril formation at a small yield. Similar results were observed
with the single substitutions L23F and V26I. In addition, the combination of
two or three of these substitutions generally increased the ability to produce
fibrils. These results show that the presence of the three proline residues in
the rIAPP(20–29) domain is insufficient to abolish the ability to form fibrils.
Moreover, the ability of the R18H variant of rIAPP to self-associate into
amyloid fibrils suggests that other domains of IAPP except the 20–29 are
involved in the self-recognition process.The role of the histidine
at position 18 in amyloid formation was further examined by assessing hIAPP fibrillization at
various pH ranges [24]. The ionization state of
His-18 was found to substantially affect the rate of assembly as well as the morphology
of the amyloid fibrils formed by hIAPP. The aggregation process was faster at
high pH (8.8), when the histidine is deprotonated, than at low pH (4.0). This fact
may be physiologically relevant as mature hIAPP is stored in the β-cell
granules at a pH of 5.5 and released into the extracellular matrix where the pH
is of 7.4. Thus, the low pH in the pancreatic granules may protect hIAPP from
aggregation.Abedini and Raleigh, who performed the above-described study, further questioned the exclusive
importance of the 20–29 region by designing a variant of the amyloidogenic
hIAPP(8–37), containing three proline residues outside this region, at
positions 17, 19, and 30 [25]. The 3×P variant had dramatically greater solubility
and reduced tendency to form β-sheet structures compared to the wild-type
polypeptide, as assayed by a variety of amyloid-detecting techniques. The authors concluded that models of IAPP
fibrillization must take into account contributions of other regions within
IAPP.


## 3.3. Inhibition of hIAPP Amyloid Formation

Based on our identification of the hIAPP(13–18) recognition site, we designed peptide
inhibitors against IAPP fibrillization, which were targeted to this region. Using
the previously exemplified method of incorporatingβ-breaker elements into amyloidogenic
core peptides [26], we introduced a new
inhibition strategy using the α-aminoisobutyric acid (Aib), a β-breaker element with extreme structural
constrains [27]. Peptide fragments corresponding
to the recognition domain were modified with Aib. The modified peptides
completely lost their amyloidogenic potential. Furthermore, the Aib-modified
peptide showed a powerful inhibitory effect on the formation of amyloid fibrils
by the full-length hIAPP.Evidently, the 20–29 region was also used as a template for inhibitor design and served as a
target for inhibition. Kapurniotu etal. designed a nonamyloidogenic and
bioactive mimic of hIAPP, which contains a double N-methylation of full-length
hIAPP at positions G24 and I26, termed as IAPP-GI [28]. The presence of two N-methyl
rests on the same side of a β-strand interferes with the interstrand amide
hydrogen-bonding necessary for β-sheet formation [29]. The IAPP-GI analogue was
shown to be a nanomolar affinity inhibitor of hIAPP fibrillization and
cytotoxicity. However, although IAPP-GI did not form amyloid fibrils, it was
found to have a “pronounced self-association propensity” and to form
spheroids of up to 100 nm in diameter. Moreover, the Far-UV circular dichroism
(CD) analysis of IAPP-GI indicated the presence of β-sheet and/or β-turn
conformations. These insinuate the presence of another recognition and
self-assembly domain besides the 20–29 region that enabled the formation of the
observed structures. A reminiscent
scenario exists in the study of peptide nanotubes and nanostructures, which was
initiated from the search for a minimal amyloidogenic core domain of
Alzheimer's β-amyloid polypeptide. While the Phe-Phe dipeptide was shown to
form well-discrete peptide nanotubes [30], two other peptides, Cys-Phe-Phe and
diphenylglycine, which are very similar to the diphenylalanine, formed closed-cage
nanospheres [31]. We
speculate that the remarkable inhibition ability of the IAPP-GI analogue is actually
due to its high affinity recognition to the 3–18 site, as it is kept unmodified
and thus provides maximal compatibility.The search for
inhibitors against amyloid formation led to the identification of insulin as an
exceptional inhibitor of IAPP fibrillization [32–34]. This finding may be of high physiological
importance as IAPP and insulin are costored and cosecreted from the pancreatic
β-cell granules. Consequently, insulin is considered to form a complex with
IAPP which stabilizes it and prevents it from aggregation within the granules [35, 36]. In order to reveal the
molecular mechanism underlying the interaction between IAPP and insulin, we
performed a molecular mapping of the interaction interface. Using a
reductionist approach and peptide arrays, we located the cross-recognition sites
within both polypeptides [37]. Interestingly, insulin was
found to bind to the 13–18 region within IAPP. In addition, the identified recognition
site within insulin, which resides within the insulin B chain, was previously shown to have
sequence similarity to the hIAPP(13–18) region [35]. These findings reveal a
typical amyloid inhibition mechanism for insulin and reinforce the central role
of the 13–18 region: the binding of insulin to IAPP self-recognition site is
mediated through sequence similarity, it interferes with IAPP self-association
and prevents it from amyloid formation.


## 3.4. Interaction of IAPP with the Membrane

As in the case of other amyloid-related disease,
the mechanism by which IAPP causes cell destruction is assumed to involve interaction
with the cell membrane. Different studies have investigated the possible
interaction of IAPP with the membrane, mostly by the use of membrane mimetics
such as liposomes and phospholipid assemblies. In a recent study, hIAPP was
claimed to insert into phospholipid monolayer as a monomer [38]. Interestingly, it was suggested that the
N-terminus of hIAPP is largely responsible for the insertion. Experiments using
fragments of hIAPP showed that a peptide consisting of the 19 N-terminal
residues of hIAPP efficiently inserts into a phospholipids monolayer,  whereas
the 20–29 residue peptide inserts much less efficiently. These results offer a
possible role for the N-terminal region also in the sense of interaction with
the pancreatic β-cells which may be part of the mechanism underlying type II diabetes
pathogenesis.


## 3.5. Comparison between the Two Amyloidogenic Regions Derived from the Cat IAPP

Cats are among the few mammalian species that are known to suffer from type II diabetes and from
accumulation of IAPP islet amyloid deposits [39, 40]. The cat IAPP (cIAPP)
sequence differs from that of hIAPP in only four amino acids, at positions 17,
18, 23, and 29. The decapeptide corresponding to cIAPP(20–29) was previously
shown to be able to aggregate into amyloidal structures as well [16, 41, 42], thus supporting the apparent
fibrillization role of the 20–29 region. However, both the dog and cat IAPP are
identical over the 20–29 region [43] and dogs are not known to
develop type II diabetes.In order to re-evaluate the role of the two identified recognition sites of IAPP in the
fibrillization process, we tested the self-assembly propensity of peptide fragments
derived from the cat IAPP, cIAPP(22–29), and cIAPP(14–20). We also tested the
aggregation of the corresponding human sequence peptides for reference. The
peptide sequences are presented in Table1. The peptides were dissolved into
aqueous solution, under the exact same conditions, incubated for one to three
days and tested for amyloid formation by electron microscopy (EM) and Congo Red
(CR) birefringence. Their secondary conformation was also evaluated by
Fourier-transform infrared spectroscopy (FTIR).Table 1
The studied peptides. Peptide fragments corresponding to regions within the human
and cat IAPP were tested for the ability to form amyloid-like structures.
NameOriginSequenceResiduesC1catNLGAILSP22–29H1humanNFGAILSS22–29C2catNFLIRSS14–20H2humanNFLVHSS14–20Differences between the peptides appeared already
before applying the amyloid tests. Upon dilution of the peptides into aqueous
solution, the H1 peptide immediately precipitated into eye-visible aggregates. The other peptide solutions appeared
clear to the unarmed-eye. Examination of the peptide samples under the electron
microscope revealed the presence of well-ordered tubular structures for all the
peptides except the C1 peptide (Figure1). Apparently, the peptides were found to form nanotubes rather than
fibrils. The formation of tubular structures by H2 and H1 was previously shown [18, 20, 44]. Here we found that the C2 peptide can also form
highly defined tubular structures. Thin fibrils were also detected in proximity
with the nanotubes or in independent bundles (not shown). The C2 sample
contained mostly nanotubes, the H2 sample contained nanotubes and fibrils, and the
H1 sample contained also large aggregates. The C1 peptide however did not form
fibrils or tubes, only less ordered spherical structures and aggregates were
observed. The spherical structures may be reminiscing the ones observed for the
IAPP analogue IAPP-GI [28], which were discussed above, however they are
less ordered than the last ones.Morphology of the formed structures. Electron microscopic examination of negatively
stained samples of the studied peptides: (a) C2, (b) H2, (c) C1, and (d) H1. The
C1 peptide failed to form tubular structures as did the other peptides. (e) and
(f) are high magnification micrographs of the marked rectangle areas of C1 and H1, respectively.
(a)(b)Upon staining with the Congo Red dye, the C2 and
H2 samples exhibited clear gold-green birefringence (Figure2). In the H1
sample, the dye appears to be bound, however, no green color was detected.
Possibly, the large amount of aggregates, which were present in addition to the
fibrillar and tubular structures, were shielding over them. No birefringence or CR binding was shown in
the C1 sample, the slide looked quite clean. Secondary structure analysis of
the peptides by FTIR showed large β-sheet content for all the peptides (Figure 3).
The β-sheet conformation is characteristic of amyloid-like structures, but is not
exclusive for them. The C1 peptide, negatively detected for amyloid structures,
also displayed β-sheet conformation which may be attributed to the spheroid
structures observed by the EM or to its soluble form.Congo Red binding and birefringence. Microscopic examination under
cross-polerizers upon staining with Congo Red of samples of the studied
peptides: (a) C2, (b) H2, (c) C1, and (d) H1. The C2 and H2 peptides exhibited
green birefringence characteristic of amyloidal structures.
(a)(b)(c)(d)Secondary conformation of the formed structures. Fourier-transform infrared (FTIR)
spectroscopy analysis of the secondary conformation of the peptides: (a) C2, (b) H2, (c) C1,
and (d) H1. All the peptides exhibited spectra typical forβ-sheet secondary structure.
(a)(b)(c)(d)The comparison between the two cat derived
peptides reveals that the C2 peptide, containing residues 14–20, is much more
competent for self-assembly into amyloid-like structures than the C1 peptide,
composed of residues 22–29. Although a longer version of the C1 peptide,
composed of residues 20–29, was previously shown to form fibrillar structures
[41, 42], in the present experimental setup it was unable
to form such fibrils. We consider this diversity to result from the length of
the peptide and different experimental conditions, which are in the present
assay of more physiologically-relevant environment and of lower concentration.
Nevertheless, when compared to the C2 peptide, C1 is dramatically less potent
for fibril formation. While the C1 peptide may contain self-association
properties as observed by the presence of spherical structures and β-sheet
content, the C2 peptide exhibited self-assembly potency into well-ordered
macromolecular fibrils, which were not displayed by the C1 peptide. This
observation provides fresh supporting evidence for the central role of the
13–18 region within the IAPP molecule in amyloid formation: while in hIAPP both
regions are highly amyloidogenic, in the cat IAPP, there is a clear difference
between the two peptides.Different behavior was also observed for the two
human derived peptides H1 and H2, which may insinuate their diverse roles in
the amyloidogenic process. The H1 peptide seems to be highly aggregative since
visible precipitates were observed upon dilution into aqueous solution as well
as under the EM. The H2 peptide however seems to have higher ability to form
well-ordered structures which requires specific recognition. Its solution was
visibly clear and EM examination revealed only fibrilar and tubular structures
without aggregates. In addition, green birefringence upon CR binding, which is
characteristic of ordered structures, was observed in the H2 sample but not in
the H1 sample. Consistent with this observation is the different recognition
affinity of the two peptide fragments to the full-length IAPP as we have
previously showed by the peptide array assay [20].The C1 peptide is not as aggregative as the H1 peptide, although it contains some structural
features as observed by the FTIR and EM analyses. A comparison between these two sequences
in their longer versions of residues 20–29 was previously  performed by Ashburn and Lansbury [42]. In this study, both peptides were found to form
amyloid fibrils, however, with remarkably different kinetics. The cIAPP(20–29)
peptide formed fibrils approximately 13-fold more slowly than its human
version. This correlates with our results regarding the different potency for
fibrillization of the two sequences. It is likely that with a longer incubation
period, the cIAPP(22–29) would also fibrillate eventually.The cIAPP sequence differs from the hIAPP sequence
by a Leu instead of a Phe at position 23 and a Pro instead of a Ser at
position 29. Both modifications were shown to separately influence the kinetics
of the peptide fibrillization [42]. It seems obvious that a Ser to Pro substitution
will decrease the fibrillization potency, since Pro is a strong β-breaker
element. Regarding the Phe to Leu substitution, we speculate that the Phe
residue facilitates the fibrillization process through aromatic interactions. According
to our established hypothesis, aromatic residues and interactions largely enhance
the process of amyloid formation by providing directionality and stability to
the growing fibril [45–49].


## 3.6. Intra-β-Sheet Mechanism for IAPP Amyloid Formation

The accumulated data presented in this paper prove
that none of the two amyloidogenic regions within IAPP can be ignored. A
possible mechanism underlying the self-assembly of IAPP into amyloid fibrils
includes a role for both regions. It was already suggested in 2001 by Jaikaran
and Clark that the structural transition of IAPP into amyloid fibrils involves
in interactions betweenβ-stranded regions within the monomer [4]. According to their model, an intramolecular β-sheet
is formed by three β-strands composed of the segments 8–20, 24–29, and 32–37. Earlier
secondary structure prediction based on IAPP sequence provided support to their
model by predicting β-turns at positions 20 and 31, as well as β-strands for
residues 14–18 and 26–29 [50]. More recently, another model for the structure
of IAPP within the amyloid fibril was proposed. Accordingly, the same three
β-strands are present, however, arranged in a zigzag planar s-shaped β-sheet,
instead of the previously proposed e-shaped structure [51].The presence
of an intramolecularβ-sheet in the fibrillar structure of IAPP is supported by
the identification of long-range interactions between distinct residues within
the IAPP molecule. Using fluorescence resonance energy transfer (FRET), the tyrosine
at position 37 was shown to be close in space to the two phenylalanine residues
(F23 and F15) at the fibrillogenic state of hIAPP [52]. Another interesting point
arises from the genetic background of type II diabetes. Although the disease is
predominantly sporadic, a mutation in hIAPP sequence, the S20G mutation, which
is identified among the Japanese population, was found to be related to early
onset of the disease [53]. The S20G mutant was also
shown to exhibit increased fibrillization and cytotoxicity compared to
wild-type hIAPP [54]. Indeed, according to the
intra-β-sheet models, the mutation is placed in a β-turn region, which is
favorable for the glycine residue.


## 4. Conclusions

The formation of amyloid deposits by IAPP appears to play a central role in the pathogenesis
of type II diabetes. Knowledge regarding the exact mechanism of this process may
be highly relevant for the development of therapeutic agents for the treatment
of the disease. Although it is clear that many environmental factors are
involved in this process, understanding the amyloid formation route at the
molecular level may be highly valued. In this paper, we have summarized results
from several research groups which support the notion that the 20–29 region
within IAPP cannot be considered as the sole amyloidogenic core of the
polypeptide. By reviewing previously published data, by us and others, as well
as providing experimental data regarding the amyloidogenicity of the 13–18
region derived from the cat IAPP, we emphasized the essential role of the 13–18
region in the fibrillization process of IAPP. We conclude that an intramolecularβ-sheet structure,
formed by at least the two discussed regions, may provide a very good mechanism
to the discussed observations.



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*Source: 256954-2008-06-10.xml*