Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces

Many amyloid proteins misfold into β-sheet aggregates upon interacting with biomembranes at the onset of diseases, such as Parkinson's disease and type II diabetes. The molecular mechanisms triggering aggregation depend on the orientation of β-sheets at the cell membranes. However, understanding how β-sheets adsorb onto lipid/aqueous interfaces is challenging. Here, we combine chiral sum frequency generation (SFG) spectroscopy and ab initio quantum chemistry calculations based on a divide-and-conquer strategy to characterize the orientation of human islet amyloid polypeptides (hIAPPs) at lipid/aqueous interfaces. We show that the aggregates bind with β-strands oriented at 48° relative to the interface. This orientation reflects the amphiphilic properties of hIAPP β-sheet aggregates and suggests the potential disruptive effect on membrane integrity.     

Lipid membranes modulate the structure of islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.     

Fibril structure of human islet amyloid polypeptide

Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.     

Interaction between amyloid beta-protein aggregates and membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.     

Ganglioside-induced amyloid formation by human islet amyloid polypeptide in lipid rafts

Human islet amyloid polypeptide (hIAPP) is the primary component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus. However, it is unknown how amyloid fibrils are formed in vivo. In this study, we demonstrate that gangliosides play an essential role in the formation of amyloid deposits by hIAPP on plasma membranes. Amyloid fibrils accumulated in ganglioside- and cholesterol-rich microscopic domains (‘lipid rafts’). The depletion of gangliosides or cholesterol significantly reduced the amount of amyloid deposited. These results clearly showed that the formation of amyloid fibrils was mediated by gangliosides in lipid rafts.     

Cholesterol modulates the interaction of the islet amyloid polypeptide with membranes

The deposition of insoluble amyloid fibrils resulting from the aggregation of the human islet amyloid polypeptide (hIAPP) within the islet of Langerhans is a pathological feature of type 2 diabetes mellitus (T2DM). Increasing evidence indicates that biological membranes play a key role in amyloid aggregation, modulating among others the kinetics of amyloid formation, and being the target of toxic species generated during amyloid formation. In T2DM patients, elevated levels of cholesterol, an important determinant of the physical state of biological membranes, are observed in β-cells and are thought to directly impair β-cell function and insulin secretion. However, it is not known whether cholesterol enhances membrane-interaction or membrane-insertion of hIAPP. In this study, we investigated the effect of cholesterol incorporated in zwitterionic and anionic membranes. Our circular dichroism and liquid state NMR data reveal that 10–30% of cholesterol slightly affects the aggregational and conformational behaviour of hIAPP. Additional fluorescence results indicate that 10 and 20% of cholesterol slightly slow down the kinetics of oligomer and fibril formation while anionic lipids accelerate this kinetics. This behavior might be caused by differences in membrane insertion and therefore in membrane binding of hIAPP. The membrane binding affinity was evaluated using ¹H NMR experiments and our results show that the affinity of hIAPP for membranes containing cholesterol is significantly smaller than that for membranes containing anionic lipids. Furthermore, we found that hIAPP-induced membrane damage is synchronized to fibril formation in the absence and in the presence of cholesterol.     

Binding of islet amyloid polypeptide to supported lipid bilayers and amyloid aggregation at the membranes

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of β-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.     

Effects of interfaces on aggregates of peptides derived from pancreatic islet amyloid polypeptide

Recent experimental studies indicate that oligomeric complexes of misfolded proteins and peptides are the primary agents of cytotoxicity in amyloid-related diseases. Given the prevalence of mixed-polarity interfaces in physiological environments, an understanding of the mechanisms of interactions between amorphous (pre-fibrillar) aggregates and surfaces is important for completing our knowledge of the behaviour of peptide aggregation phenomena. We have employed fully solvated molecular dynamics simulations to study the morphology, interactions and peptide conformations of disordered aggregates of the amyloidogenic NFGAIL (derived from human islet amyloid polypeptide) and non-amyloidogenic AGAIL peptides upon adsorption to vapour–water, decane–water, bilayer and solid–water interfaces. All of the interfaces studied promote elongation and surface-spreading of both peptide aggregates, with the liquid–liquid interface being particularly efficient at altering the gross morphology of disordered aggregates. NFGAIL aggregates are more effective at disrupting lipid bilayers compared to AGAIL. Additionally, the interfaces studied cause greater changes in peptide conformations within the NFGAIL aggregates compared to AGAIL. We propose that simulations may elucidate the capability of interfaces to effect changes in the behaviour of disordered peptide aggregates, which may also serve to provide measures of the intrinsic fibrillogenicity of a given peptide sequence.     

Molecular mechanics and quantum chemical calculations unveil the combating effect of baicalein on human islet amyloid polypeptide aggregates

ABSTRACT

Formation of insoluble toxic aggregates by hIAPP polypeptide is found to be a core component for disease pathogenicity of patients suffering from type II diabetes. Naturally occurring polyphenols that possess anti-aggregation property are being majorly studied in the current scenario for treating various conformational diseases. Herein, we study the disaggregation mechanism of naturally occurring polyphenol baicalein, obtained from the roots of S. baicalensis and Indian trumpet flower on hIAPP dimer using quantum chemical calculation and discrete molecular dynamics. Our study reported that a drastic loss in the secondary structural propensity of hIAPP was seen upon binding of baicalein. Notably, the hydrophobic core and the phenolic groups present in the ends of baicalein molecule play a key role in inhibiting the aggregates formed upon binding to the amyloidogenic core region of hIAPP. Thus, our study provides a comprehensive understanding over the disaggregation effect of baicalein on hIAPP dimer from a computational point of view and thereby bridging the gap for future therapeutic strategy in designing the anti-aggregation compounds that inhibit hIAPP amyloids.     

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.     

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.     

Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.     

Cleavage agents for soluble oligomers of human islet amyloid polypeptide

Soluble oligomers of human islet amyloid polypeptide (h-IAPP) are implicated in the initiation of beta-cell apoptosis leading to type 2 diabetes mellitus (T2DM). Cleavage of the h-IAPP included in an oligomer may provide a novel method for reducing the level of h-IAPP oligomers, offering a new therapeutic option for T2DM. From the combinatorial library of triazine derivatives prepared by exploiting the Co(III) complex of cyclen as the cleavage center for peptide bonds, eight compounds were selected as cleavage agents for oligomers of h-IAPP. After reaction with cleavage agents for 36 h at 37 degrees C and pH 7.50, up to 20 mol% of h-IAPP (initial concentration: 4.0 microM) was cleaved, although the target oligomers existed as transient species. Considerable activity was manifested at agent concentrations as low as 100 nM.     

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.     

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.     

Nucleobindin 1 caps human islet amyloid polypeptide protofibrils to prevent amyloid fibril formation

Many human diseases are associated with amyloid fibril deposition, including type 2 diabetes mellitus where human islet amyloid polypeptide (hIAPP) forms fibrils in the pancreas. We report here that engineered, soluble forms of the human Ca(2+)-binding protein nucleobindin 1 (NUCB1) prevent hIAPP fibril formation and disaggregate preexisting hIAPP fibrils. Scanning transmission electron microscopy (STEM) and atomic force microscopy indicate that NUCB1 binds to and stabilizes heterogeneous prefibrillar hIAPP species. The NUCB1-stabilized prefibrillar species were isolated by size-exclusion chromatography and analyzed by STEM, dynamic light scattering, and multi-angle light scattering. The stabilized prefibrillar species show a size range of 2-6 million Da and have other similarities to hIAPP protofibrils, but they do not progress to become mature fibrils. The effects of NUCB1 are absent in the presence of Ca(2+). We postulate that the engineered forms of NUCB1 prevent hIAPP fibril formation by a mechanism where protofibril-like species are "capped" to prevent further fibril assembly and maturation. This mode of action appears to be different from other protein-based inhibitors, suggesting that NUCB1 may offer a new approach to inhibiting amyloid formation and disaggregating amyloid fibrils.     

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.     

Cloning and expression of human islet amyloid polypeptide in cultured cells

Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells.     

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.     

Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide

In type 2 diabetes mellitus (T2DM), misfolded human islet amyloid polypeptide (hIAPP) forms amyloid deposits in pancreatic islets. These amyloid deposits contribute to the dysfunction of β-cells and the loss of β-cell mass in T2DM patients. Inhibition of hIAPP fibrillization has been regarded as a potential therapeutic approach for T2DM. Silibinin, a major active flavonoid extracted from herb milk thistle (Silybum marianum), has been used for centuries to treat diabetes in Asia and Europe with unclear mechanisms. In this study, we tested whether silibinin has any effect on the amyloidogenicity of hIAPP. Our results provide first evidence that silibinin inhibits hIAPP fibrillization via suppressing the toxic oligomerization of hIAPP and enhances the viability of pancreatic β-cells, therefore silibinin may serve as a potential therapeutic agent for T2DM.