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.     

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.     

The molecular basis of distinct aggregation pathways of islet amyloid polypeptide

Abnormal aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils is a hallmark of type 2 diabetes. In this study, we investigated the initial oligomerization and subsequent addition of monomers to growing aggregates of human IAPP at the residue-specific level using NMR, atomic force microscopy, mass spectroscopy, and computational simulations. We found that in solution IAPPs rapidly associate into transient low-order oligomers such as dimers and trimers via interactions between histidine 18 and tyrosine 37. This initial event is proceeded by slow aggregation into higher-order spherical oligomers and elongated fibrils. In these two morphologically distinct types of aggregates IAPPs adopt structures with markedly different residual flexibility. Here we show that the anti-amyloidogenic compound resveratrol inhibits oligomerization and amyloid formation via binding to histidine 18, supporting the finding that this residue is crucial for on-pathway oligomer formation.     

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.     

Serum albumin impedes the amyloid aggregation and hemolysis of human islet amyloid polypeptide and alpha synuclein

Protein aggregation is a ubiquitous phenomenon underpinning the origins of a range of human diseases. The amyloid aggregation of human islet amyloid polypeptide (IAPP) and alpha synuclein (αS), specifically, is a hallmark of type 2 diabetes (T2D) and Parkinson's disease impacting millions of people worldwide. Although IAPP and αS are strongly associated with pancreatic β-cell islets and presynaptic terminals, they have also been found in blood circulation and the gut. While extensive biophysical and biochemical studies have been focused on IAPP and αS interacting with cell membranes or model lipid vesicles, the roles of plasma proteins on the amyloidosis and membrane association of these two major types of amyloid proteins have rarely been examined. Using a thioflavin T kinetic assay, transmission electron microscopy and a hemolysis assay here we show that human serum albumin, the most abundant protein in the plasma, impeded the fibrillization and mitigated membrane damage of both IAPP and αS. This study offers a new insight on the native inhibition of amyloidosis.     

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.     

Heterogeneous triangular structures of human islet amyloid polypeptide (amylin) with internal hydrophobic cavity and external wrapping morphology reveal the polymorphic nature of amyloid fibrils

The misfolding and self-assembly of human islet amyloid polypeptide (hIAPP or amylin) into amyloid fibrils is pathologically linked to type II diabetes. The polymorphic nature of both hIAPP oligomers and fibrils has been implicated for the molecular origin of hIAPP toxicity to islet β-cells, but little is known about the polymorphic structure and dynamics of these hIAPP oligomers/fibrils at the atomic level. Here, we model the polymorphism of full length hIAPP(1-37) oligomers based on experimental data from solid-state NMR, mass per length, and electron microscopy using all-atom molecular dynamics simulation with explicit solvent. As an alternative to steric zipper structures mostly presented in the 2-fold symmetrical fibrils, the most striking structural feature of our proposed hIAPP oligomers is the presence of 3-fold symmetry along the fibril growth axis, in which three β-sheet-layers wind around a hydrophobic core with different periodicities. These 3-fold triangular hIAPP structures dramatically differ in the details of the β-layer assembly and core-forming sequence at the cross section, but all display a high structural stability with favorable layer-to-layer interactions. The 3-fold hIAPP structures can also serve as templates to present triple-stranded helical fibrils via peptide elongation, with different widths from 8.7 to 9.9 nm, twists from 2.8° to 11.8°, and pitches from 14.5 to 61.1 nm, in reasonable agreement with available biophysical data. Because similar 3-fold Aβ oligomers are also observed by both NMR experiments and our previous simulations, the 3-fold structure could be a general conformation to a broad range of amyloid oligomers and fibrils. Most importantly, unlike the conventional stacking sandwich model, the proposed wrapping-cord structures can readily accommodate more than three β-layers via a two dimension conformation search by rotating and translating the β-layers to adopt different favorable packings, which can greatly enrich the polymorphism of amyloid oligomers and fibrils.     

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation

Background

Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death.

Methodology/Principal Findings

We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elav^C155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures.

Conclusions/Significance

These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology.     

The effects of organic solvents on the membrane-induced fibrillation of human islet amyloid polypeptide and on the inhibition of the fibrillation

The organic solvent dimethylsulphoxide (DMSO) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) have been widely used as a pre-treating agent of amyloid peptides and as a vehicle for water-insoluble inhibitors. These solvents are left in many cases as a trace quantity in bulk and membrane environments with treated amyloid peptides or inhibitors. In the present work, we studied the effects of the two organic solvents on the aggregation behaviors of human islet amyloid polypeptide (hIAPP) and the performances of an all-D-amino-acid inhibitor D-NFGAIL in preventing hIAPP fibrillation both in bulk solution and at phospholipid membrane. We showed that the presence of 1% v/v DMSO or HFIP decreases the rate of fibril formation of hIAPP at the lipid membrane rather than accelerates the fibril formation as what happened in bulk solution. We also showed that the presence of 1% v/v DMSO or HFIP impairs the activity of the inhibitor at the lipid membrane surface dramatically, while it affects the efficiency of the inhibitor in bulk solution slightly. We found that the inhibitor inserts into the lipid membrane more deeply or with more proportion in the presence of the organic solvents than it does in the absence of the organic solvents, which may hinder the binding of the inhibitor to hIAPP at the lipid membrane. Our results suggest that the organic solvents should be used with caution in studying membrane-induced fibrillogenesis of amyloid peptides and in testing amyloid inhibitors under membrane environments to avoid incorrect evaluation to the fibrillation process of amyloid peptides and the activity of inhibitors.     

胰岛淀粉样多肽的研究进展

由蛋白错误折叠后聚集所产生的淀粉样蛋白沉积是导致老年痴呆症、疯牛病、2型糖尿病等多种疾病的重要因素。由胰岛淀粉样多肽(islet amyloid polypeptide,IAPP)所形成的淀粉样蛋白沉积,具有破坏胰岛β细胞膜结构、诱导β细胞凋亡和损伤β细胞功能的作用,被认为是2型糖尿病的重要致病原因之一。对IAPP的聚集性、聚集体的结构,以及其对β细胞的毒性作用研究,不但有助于明确2型糖尿病的发病机制,而且最新研究也表明抑制IAPP的聚集可有效减少β细胞的凋亡,提高胰岛移植的成功率。因此,IAPP已成为2型糖尿病治疗中一个具有良好前景的靶点。该文对IAPP研究的最新进展进行了简要介绍。     

Membrane interaction of islet amyloid polypeptide

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.     

Membrane interaction of islet amyloid polypeptide

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.     

Synergistic action of polyanionic and non-polar cofactors in fibrillation of human islet amyloid polypeptide

Recent studies have led us to suppose that synergistic action of multiple solute cofactors could play substantial roles in amyloid-type fibrillogenesis of pathogenic polypeptides. To support this view, we performed aggregation experiments of human islet amyloid polypeptide (IAPP) in media containing both polyanions and non-polar solvents. The results demonstrated that the fibrillation at sub-micromolar IAPP occurred only when polyanionic and non-polar solutes coexist. A simple sum of two independent cofactor's effects could not account for the synergistic action. We propose that this synergy of polyanionic and nonpolar milieus could substantially modify the amyloidgenesis in the human body.     

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 A apart. Electron diffraction also revealed the characteristic 4.7 A meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 A that has been previously visualised from a single Abeta fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded beta-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils.     

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide

Insoluble amyloid formation by islet amyloid polypeptide (IAPP) in the islets of Langerhans of the pancreas is a major pathophysiological feature of noninsulin dependent diabetes mellitus (NIDDM) or type II diabetes. Because in vivo formed amyloid colocalizes with areas of cell degeneration and IAPP amyloid aggregates are cytotoxic per se, the process of IAPP amyloid formation has been strongly associated with the progressive pancreatic cell degeneration and thus much of the pathology of type II diabetes. IAPP is a pancreatic polypeptide of 37 residues that, in its soluble form, is believed to play a role as a regulator of glucose homeostasis. The molecular cause and mechanism of the conversion of soluble IAPP into insoluble amyloid aggregates in vivo and its role in disease progress still remain to be clarified. Nevertheless, in the past few years significant progress has been made in understanding the amyloidogenesis pathway of IAPP in vitro and gaining insight into the structural and conformational "requirements" of IAPP amyloidogenesis and related cytotoxic effects. Importantly, several of the studies have revealed significant similarities of the above features of IAPP to other amyloidogenic polypeptides such as the beta-amyloid polypeptide Abeta. This suggests that, at the molecular level, amyloidogenesis, and possibly related cell degeneration and disease pathogenesis by completely different polypeptide sequences, may obey to common structural and conformational "rules" and follow similar molecular pathways. This review describes studies on the structural and conformational features of IAPP amyloid formation and cytotoxicity, and the application of the obtained knowledge for the understanding of the molecular mechanism of the IAPP amyloidogenesis pathway and the related cytotoxicity.     

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.     

Mechanism of islet amyloid polypeptide fibrillation at lipid interfaces studied by infrared reflection absorption spectroscopy

Islet amyloid polypeptide (IAPP) is a pancreatic hormone and one of a number of proteins that are involved in the formation of amyloid deposits in the islets of Langerhans of type II diabetes mellitus patients. Though IAPP-membrane interactions are known to play a major role in the fibrillation process, the mechanism and the peptide's conformational changes involved are still largely unknown. To obtain new insights into the conformational dynamics of IAPP upon its aggregation at membrane interfaces and to relate these structures to its fibril formation, we studied the association of IAPP at various interfaces including neutral as well as charged phospholipids using infrared reflection absorption spectroscopy. The results obtained reveal that the interaction of human IAPP with the lipid interface is driven by the N-terminal part of the peptide and is largely driven by electrostatic interactions, as the protein is able to associate strongly with negatively charged lipids only. A two-step process is observed upon peptide binding, involving a conformational transition from a largely alpha-helical to a beta-sheet conformation, finally forming ordered fibrillar structures. As revealed by simulations of the infrared reflection absorption spectra and complementary atomic force microscopy studies, the fibrillar structures formed consist of parallel intermolecular beta-sheets lying parallel to the lipid interface but still contain a significant number of turn structures. We may assume that these dynamical conformational changes observed for negatively charged lipid interfaces play an important role as the first steps of IAPP-induced membrane damage in type II diabetes.     

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.     

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.