Antibodies specific for the pancreatic islet amyloid polypeptide associated with type 2 diabetes mellitus

Antibodies raised to a lysine solubilized peptide composed of residues 20-29 of the pancreatic islet amyloid polypeptide react selectively and specifically with this polypeptide and with islet amyloid deposits in Type 2 diabetes mellitus. These antibodies may prove useful in studies employing radioimmunoassay of body fluids and islet cell cultures in order to define if a pathogenic relationship exists between the islet amyloid polypeptide and Type 2 diabetes mellitus.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Aggregation of an amyloidogenic fragment of human islet amyloid polypeptide

Native human islet amyloid polypeptide (hIAPP) has been identified as the major component of amyloid plaques found in the pancreatic islets of Langerhans of persons affected by type 2 diabetes mellitus. Early studies of hIAPP determined that a segment of the molecule, amino acids 20-29, is responsible for its aggregation into amyloid fibrils. The present study demonstrates that the aggregation of hIAPP 20-29-Trp is a nucleation-dependent process, displaying a distinct lag time before the onset of rapid aggregation. Moreover, the lag time can be eliminated by seeding the sample of unaggregated peptide with preformed fibrils. In contrast to the expectation from the conventional model of nucleation-dependent aggregation, however, the lag time of hIAPP aggregation does not depend on peptide concentration. To explain this observation, a modified version of the standard model of nucleation-dependent aggregation is presented in which the monomeric peptide concentration is buffered by an off-aggregation-pathway formation of peptide micelles.     

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.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Identifying structural features of fibrillar islet amyloid polypeptide using site-directed spin labeling

Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet beta-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid beta peptide fibrils are discussed.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.     

The sequence dependence of fiber organization. A comparative molecular dynamics study of the islet amyloid polypeptide segments 22-27 and 22-29

Amyloid fiber formation and the possible polymorphism of molecular arrangements depend on the polypeptide length and composition. Here, we seek the chemical clues underlying these processes. Our starting point is based on the experimental observation that some short peptide segments are able to develop fibers that are very similar to those of their original parent proteins. We focus our study on the NFGAILSS peptide, derived from the human islet amyloid polypeptide (residues 22-29). This peptide turned out to be a perfect example, illustrating the fact that the amyloid microscopic organization is highly complex, rather than simply involving hydrogen bond formation. Furthermore, obtaining a reliable molecular model has allowed us to analyze the differences between the amyloid structure we have obtained for this peptide and that obtained for the previously studied, two residues shorter, segment (residues 22-27, NFGAIL). This comparative study yields some clues about chemical events that govern the aggregation of proteins into oriented fibers, such as molecular packing between sheets and the degree of interaction specificity. We characterize the important role played by the hydrophobic and aromatic residues in the inter-sheet association and present new approaches toward the understanding of the nature of events that are likely to take place during fibril formation. These include analysis of interaction patterns derived from specific sheet-associated packing.     

Phospholipid catalysis of diabetic amyloid assembly

Islet amyloid polypeptide (IAPP) is a 37-residue hormone that forms cytotoxic amyloid fibers in the endocrine pancreas of patients with type II diabetes (NIDDM). A potential origin for cytotoxicity is disruption of lipid membranes by IAPP as has been observed in vitro. The cause of amyloid formation during NIDDM is not known, nor is the mechanism by which membrane disruption occurs in vitro. Here, we use kinetic studies in conjunction with assessments of lipid binding and electron microscopy to investigate the interactions of IAPP with phospholipid bilayers and the morphological effects of membranes on IAPP fibers. Fibrillogenesis of IAPP is catalyzed by synthetic and human tissue-derived phospholipids, leading to >tenfold increases in the rate of fibrillogenesis. The molecular basis of this phenomenon includes a strong dependence on the concentration and charge density of the membrane. IAPP binds to lipid membranes of mixed anionic (DOPG) and zwitterionic (DOPC) content. The transition for binding occurs over a physiologically relevant range of anionic content. Membrane binding by IAPP occurs on timescales that are short compared to fibrillogenesis and results in assembly into preamyloid states via ordered interactions at the N but not C terminus of the protein. These assemblies lead both to gross morphological changes in liposomes and to alterations in the appearance of early fibers when compared to liposome-free fibril formation. Intact bilayer surfaces are regenerated upon dissociation of fibers from the membrane surface. These findings offer a structural mechanism of membrane destabilization and suggest that changes in lipid metabolism could induce IAPP fiber formation in NIDDM.     

Contribution of the intrinsic disulfide to the assembly mechanism of islet amyloid

Amyloidogenesis from soluble protein requires conformational and oligomeric assembly steps. In systems where the precursor protein is natively unfolded, such as islet amyloid polypeptide (IAPP), forces and structural changes relevant to protein unfolding are not thought to participate in the assembly mechanism. Thus, fiber core structure elements should provide the dominant contributions to assembly kinetics. Here we show, however, that residues outside the amyloid core can influence the mechanism of IAPP fiber assembly. IAPP possesses an intramolecular disulfide bond between residues 2 and 7. This short-range disulfide prohibits the N-terminal region from adopting the beta-strand structure of an amyloid. We examined the role of this disulfide in fiber formation by generating a truncated construct (IAPP(8-37)) and a stable reduced form of the full-length protein (IAPP(CAM)). The fiber structures and assembly kinetics of these variants were assessed via optical and mass spectroscopy. Our data confirm that the disulfide does not contribute to the amyloid fiber core structure. Remarkably, however, it plays a central role in the assembly mechanism. First, loss of the disulfide substantially reduces fiber formation by secondary nucleation, i.e., the ability of pre-existing fibers to participate in the formation of new fibers. Second, the bypass of nucleation by seed addition is a two-step process, termed activation. Loss of the disulfide eliminates this two-step nature of seeded kinetics.     

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.     

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.     

Identification of minimal peptide sequences in the (8-20) domain of human islet amyloid polypeptide involved in fibrillogenesis

We have examined a series of overlapping peptide fragments from the 8-20 region of human islet amyloid polypeptide (IAPP) with the objective of defining the smallest fibril-forming domain. Peptide fragments corresponding to LANFLV (residues 12-17) and FLVHSS (residues 15-20) were strong enhancers of beta-sheet transition and fibril formation. Negative stain electron microscopy illustrated the ability of these peptide fragments to form fibrils independently when incubated alone in solution. Circular dichroism analysis revealed that when full-length human IAPP was incubated in the presence of these two fragments, fibrillogenesis was accelerated. While the two fragments, LANFLV and FLVHSS, were able to enhance the recruitment of additional IAPP molecules during fibril formation, the "seeding" activity of these peptides had no effect on altering IAPP-induced cytotoxcity as determined by cell culture studies. Therefore, this study has identified two internal IAPP peptide fragments within the 8-20 domain that may have a role in enhancing the folding and aggregation of human IAPP. These fragments are the smallest sequences identified, within the 8-20 region of hIAPP, that can independently form fibrils, and that can interact with IAPP to assemble into fibrils with characteristics similar as those formed by human IAPP alone.     

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.     

Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.     

Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

Direct detection of transient alpha-helical states in islet amyloid polypeptide

The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone cosecreted with insulin by the beta-cells of the pancreas. In humans with type 2 diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with beta-cell death. Here, we examine the rat IAPP sequence variant under physiological solution conditions. Rat IAPP is mechanistically informative for fibrillogenesis, as it samples intermediate-like states but does not progress to form amyloid. A central challenge was the development of a bacterial expression system to generate isotopically labeled IAPP without terminal tags, but which does include a eukaryotic post-translational modification. While optical spectroscopy shows IAPP to be natively unfolded, NMR chemical shifts of backbone and beta-carbon resonances reveal the sampling of alpha-helical states across a continuous stretch comprising approximately 40% of the protein. In addition, the manifestation of nonrandom coil chemical shifts is confirmed by the relative insensitivity of the amide proton chemical shifts to alterations in temperature. Intriguingly, the residues displaying helical propensity are conserved with the human sequence, suggesting a functional role for this conformational bias. The inability of rat IAPP to self assemble can be ascribed, in part, to several slowly exchanging conformations evident as multiple chemical shift assignments in the immediate vicinity of three proline residues residing outside of this helical region.