The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain

Several proteins that interact with cell surface glycolipids share a common fold with a solvent-exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH-pi stacking interaction). Stacking interactions between aromatic residues (pi-pi stacking) also play a pivotal role in the assembly process, including many cases of amyloid fibril formation. We found a structural similarity between a typical glycolipid-binding domain (the V3 loop of HIV-1 gp120) and the minimal amyloid-forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air-water interface, the NFGAILSS peptide specifically interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addition, we observed that the full-length human islet amyloid polypeptide (1-37) did interact with a monolayer of lactosylceramide, and that the glycolipid film significantly affected the aggregation process of the peptide. As glycolipid-V3 interactions are efficiently inhibited by suramin, a polyaromatic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amyloid fibril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken together, our results indicate that the minimal amyloid-forming fragment of human islet amyloid polypeptide is a glycolipid-binding domain, and provide further experimental support for the role of aromatic pi-pi and CH-pi stacking interactions in the molecular control of the amyloidogenesis process.     

Micelle formation by a fragment of human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid plaques found in the pancreatic islets of persons with type 2 diabetes mellitus. HIAPP belongs to the group of amyloidogenic proteins, characterized by their aggregation and deposition as fibrillar amyloid in various body tissues. The aggregation of amyloidogenic proteins is thought to occur via a common pathway, but currently no unifying kinetic model exists. In previous work, we presented a model of amyloid fibril formation formulated from our observations of the aggregation of an amyloidogenic fragment of hIAPP, amino acids 20-29. Our model is based on nucleation-dependent aggregation, modified by the formation of off-pathway hIAPP micelles. In the present study we confirm the presence of peptide micelles, and experimentally determine the critical micelle concentration in solutions of hIAPP fragments using three different techniques: conductivity, pH, and fluorescence. All three techniques yield a critical micelle concentration of 3-3.5 micro M peptide. Furthermore, based on changes in the fluorescence intensity of a labeled peptide fragment as well as a decrease in solution pH as a result of deprotonation of the amino terminus, we conclude that the amino terminus of the fragment undergoes a significant change of environment upon micellization.     

Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) β-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.     

The role of His-18 in amyloid formation by human islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is the major protein component of the amyloid deposits found in type-II diabetes. IAPP is stored in a relatively low pH environment in the pancreatic secretory granules prior to its release to the extracellular environment. Human IAPP contains a single histidine at position 18. Aggregation of IAPP is considerably faster at a lower pH (4.0 +/- 0.3) than at high pH (8.8 +/- 0.3), as judged by turbidity and thioflavine-T fluorescence studies. The rate of aggregation at low pH increases drastically in the presence of salt. CD experiments show that the conversion of largely unstructured monomers to beta-sheet-rich structures is faster at high pH. TEM studies show that fibrils are formed at both pH values but are more prevalent at pH 8.8 (+/-0.3). Both the free N terminus of IAPP and His-18 will titrate over the pH range studied. An N-terminal acetylated fragment consisting of residues 8-37 of human IAPP was also studied to isolate contributions from the protonation of His-18. Previous studies have shown that this fragment forms protofibrils that are very similar to those formed by intact IAPP. The effects of varying the protonation state of His-18 in the 8-37 analogue indicate that the rate of aggregation and fibril formation is noticeably faster when His-18 is deprotonated, similar to the wild type. However, the pH-dependent effects are larger for full-length IAPP than for the disulfide-truncated, acetylated analogue. TEM studies indicate differences in the morphology of the deposits formed at high and low pH. These results are discussed in light of recent structural models of IAPP fibrils.     

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12–18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.     

Peptide sequence and amyloid formation; molecular simulations and experimental study of a human islet amyloid polypeptide fragment and its analogs

We present a combined experimental and theoretical investigation of the tendencies to form amyloid fibrils by a hexapeptide derivative of the human islet amyloid polypeptide, the NFGAIL (22-27) fragment and its mutants. We performed a complete alanine scan of this fragment and studied the capability of the wild-type and its mutant analogs to form ordered fibrils by ultrastructural and biophysical analyses. In parallel, we conducted a meticulous characterization of each sequence-complex at an atomistic level by performing nine independent molecular dynamics simulations for a total of 36 ns. These allowed us to rationalize the experimental observations and to establish the role of every residue in the fibrillogenesis. The main factor that determines the formation of regular fibrils is a coherent organization of the intersheet space. In particular, phenylalanine side chains cement the macromolecular assemblies due to their aromatic chemical character and restricted conformational flexibility when interacting with aliphatic residues.     

Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.     

Genetic background determines the extent of islet amyloid formation in human islet amyloid polypeptide transgenic mice

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.     

Inhibitors can arrest the membrane activity of human islet amyloid polypeptide independently of amyloid formation.

Human islet amyloid polypeptide (hIAPP), co-secreted with insulin from pancreatic beta cells, misfolds to form amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Like many amyloidogenic proteins, hIAPP is membrane-active: this may be significant in the pathogenesis of NIDDM. Non-fibrillar hIAPP induces electrical and physical breakdown in planar lipid bilayers, and IAPP inserts spontaneously into lipid monolayers, markedly increasing their surface area and producing Brewster angle microscopy reflectance changes. Congo red inhibits these activities, and they are completely arrested by rifampicin, despite continued amyloid formation. Our results support the idea that non-fibrillar IAPP is membrane-active, and may have implications for therapy and for structural studies of membrane-active amyloid.     

Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vitro

Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer's disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into beta-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 microM peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 degrees C. The state at 45 degrees C was also found to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of beta-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases.     

Optimized experimental pre-treatment strategy for temporary inhibition of islet amyloid polypeptide aggregation

Islet amyloid polypeptide (IAPP) is a neuroendocrine hormone from pancreatic β-cells. Misfolded, aggregated IAPP is believed to be toxic to islet cells and amyloid deposits in the pancreas are pathological hallmarks of type 2 diabetes. Rapid fibrillization of this peptide makes it difficult to study in its soluble form, impeding a better understanding of its role. In this study, a variety of popular pretreatment methods were tested for their ability to delay aggregation of IAPP, including solutions of hexafluoroisopropanol, sodium hydroxide, hydrochloric acid, phosphate buffered saline, ammonium hydroxide, as well as tris buffer at different pH and containing either calcium (II), zinc (II), or iron (II). Aggregation was assessed using the thioflavin T fluorescence assay as well as by transmission electron microscopy. Tris buffer at pH 8.1 containing Zn(II) was found to have the best balance of temporary inhibition of aggregation and biological relevance.     

Differential effects of serine side chain interactions in amyloid formation by islet amyloid polypeptide

Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular β‐strands. Ser 19 and Ser 20 are in the partially ordered β‐turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2‐aminobutyric acid (2‐Abu) to examine the site‐specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2‐Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2‐Abu mutation modestly slows the rate of amyloid formation. 2‐Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross‐column interactions made by residue 29 are not essential for amyloid formation and also show that cross‐strand networks of hydrogen‐bonded Ser side chains, so called Ser‐ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2‐Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

On the plasticity of amyloid formation: The impact of destabilizing small to large substitutions on islet amyloid polypeptide amyloid formation

Amyloids are partially ordered, proteinaceous, β‐sheet rich deposits that have been implicated in a wide range of diseases. An even larger set of proteins that do not normally form amyloid in vivo can be induced to do so in vitro. A growing number of structures of amyloid fibrils have been reported and a common feature is the presence of a tightly packed core region in which adjacent monomers pack together in extremely tight interfaces, often referred to as steric zippers. A second common feature of many amyloid fibrils is their polymorphous nature. We examine the consequences of disrupting the tight packing in amyloid fibrils on the kinetics of their formation using the 37 residue polypeptide hormone islet amyloid polypeptide (IAPP, amylin) as a model system. IAPP forms islet amyloid in vivo and is aggressively amyloidogenic in vitro. Six Cryo‐EM structures of IAPP amyloid fibrils are available and in all Gly24 is in the core of the structured region and makes tight contacts with other residues. Calculations using the ff14SBonlysc forcefield in Amber20 show that substitutions with larger amino acids significantly disrupt close packing and are predicted to destabilize the various fibril structures. However, Gly to 2‐amino butyric acid (2‐carbon side chain) and Gly to Leu substitutions actually enhance the rate of amyloid formation. A Pro substitution slows, but does not prevent amyloid formation.     

How the imidazole ring modulates amyloid formation of islet amyloid polypeptide: A chemical modification study

BackgroundThe misfolding of human islet amyloid polypeptide (hIAPP) is an important pathological factor on the onset of type 2 diabetes. A number of studies have been focused on His¹⁸, the only histidine of hIAPP, whose imidazole ring and the protonation state might impact hIAPP amyloid formation, but the exact mechanism remains unclear.MethodsWe used diethylpyrocarbonate (DEPC) to specifically modify His¹⁸ and obtained mono-ethyloxyformylated hIAPP (DMI). Thioflavin T based fluorescence, transmission electronic microscopy, circular dichroism spectroscopy, fluorescence dye leakage, Fourier transform infrared spectroscopy and replica-exchange molecular dynamics (REMD) simulation were applied to study the impact of DEPC-modification on hIAPP amyloid formation.ResultsAfter an ethyl-acetate group was introduced to the His¹⁸ of hIAPP by diethylpyrocarbonate (DEPC) modification, the pH dependent hIAPP fibrillation went to the opposite order and the number of intra-molecular hydrogen bonds decreased, while the possibility of His¹⁸ participating in the formation of α-helical structures increased. Furthermore, the membrane–peptide interaction and ion–peptide interaction were both impaired.ConclusionsThe intramolecular hydrogen bond formation by His¹⁸ and the possibility of His¹⁸ participating in the formation of α-helical structures greatly modulated the manner of hIAPP amyloid formation. The imidazole ring directly participates in the hIAPP–membrane/ion interaction.General significanceDEPC modification is an alternative approach to investigate the role of the imidazole ring during amyloid formation.     

Amyloid formation by pro-islet amyloid polypeptide processing intermediates: examination of the role of protein heparan sulfate interactions and implications for islet amyloid formation in type 2 diabetes

Amyloid formation has been implicated in a wide range of human diseases including Alzheimer's disease, Parkinson's disease, and type 2 diabetes. In type 2 diabetes, islet amyloid polypeptide (IAPP, also known as amylin) forms cytotoxic amyloid deposits in the pancreas, and these are believed to contribute to the pathology of the disease. The mechanism of islet amyloid formation is not understood; however, recent proposals have invoked a role for incompletely processed proIAPP. In this model, incompletely processed proIAPP containing the N-terminal pro region is excreted and binds to heparan sulfate proteoglycans (HSPGs) of the basement membrane thereby establishing a high local concentration which can act as a seed for amyloid formation. Here we report biophysical proof-of-principle experiments designed to test the viability of this model. The model predicts that interactions with HSPGs should accelerate amyloid formation by the proIAPP processing intermediate, and this is indeed what is observed. Interaction with heparan sulfate leads to the rapid formation of an intermediate state with partial helical content which then converts, on a slower time scale, to amyloid fibrils. TEM shows that fibrils formed by the proIAPP processing intermediate in the presence and in the absence of heparan sulfate have the classic features of amyloid. Fibrils formed by the proIAPP processing intermediate are competent to seed amyloid formation by mature IAPP. The seeding experiments support a second major premise of the model, namely, that fibrils formed by the processing intermediate are capable of seeding amyloid formation by the mature peptide.     

Analysis of the inhibition and remodeling of islet amyloid polypeptide amyloid fibers by flavanols

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in transplanted islets. The flavanol (-)-epigallocatechin-3-gallate [EGCG; (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] is an effective inhibitor of amyloid formation by IAPP; however, the interactions required for the inhibition of IAPP amyloid formation and for the remodeling of amyloid fibers are not known. A range of features have been proposed to be critical for EGCG protein interactions, including interactions with aromatic residues, interactions with amino groups, or sulfhydryls. Using a set of IAPP analogues, we show that none of these are required. Studies in which EGCG is added to the lag phase of amyloid formation shows that it interacts with intermediates as well as with monomers and amyloid. The features of EGCG required for effective inhibition were examined. The stereoisomer of EGCG, (-)-gallocatechin gallate (GCG), is an effective inhibitor, although less so than EGCG. Removing the gallate ester moiety leads to EGC which is a less effective inhibitor. Removing only the 3-hydroxyl group of the trihydroxyphenyl ring leads to a compound that has more pronounced effects on the lag phase than EGC but is less effective at reducing the amount of amyloid. Elimination of both the 3-hydroxy group and the gallate ester results in loss of activity. EGCG remodels IAPP amyloid fibers but does not fully resolubilize them to unstructured monomers, and the remodeling is not the reverse of amyloid assembly. The ability of the compounds to remodel IAPP amyloid closely follows their relative ability to inhibit amyloid formation.     

Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species

Amyloid deposits in the islets of Langerhans occur in association with type 2 diabetes mellitus (DM) in humans and cats and consist of a 37-amino-acid polypeptide known as islet amyloid polypeptide (IAPP). In order to find an explanation for the situation that islet amyloid (IA) does not develop in common rodent species, we have deduced the amino acid sequence of the IAPP molecule in mouse, rat and hamster. We find that a specific region of the molecule diverges to a high degree. Synthetic peptides corresponding to this region of human and hamster IAPP were compared for their ability to form amyloid fibrils in vitro. Whereas the human peptide readily formed fibrils with amyloid character, the hamster peptide completely lacked this property. We suggest this to be a likely explanation for the differences in IA formation between humans and rodents and discuss our findings in relation to the type 2 DM syndrome.     

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and β-cell death. Here, we used variants of the hIAPP_1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, ²H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone.     

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.