Conformational Tuning of Amylin by Charged Styrene-Maleic-Acid Copolymers

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in β-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin’s C terminus (residues 31–37) that belongs to one of the two β-sheet regions (residues 14–19 and 31–36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.     

Conformation-dependent scFv antibodies specifically recognize the oligomers assembled from various amyloids and show colocalization of amyloid fibrils with oligomers in patients with amyloidoses

Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a na?ve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aβ1-40, prion peptide 106-126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aβ1-40 and prion peptide 106-126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses.     

Full length amylin oligomer aggregation: insights from molecular dynamics simulations and implications for design of aggregation inhibitors

Amyloid oligomers are considered to play essential roles in the pathogenesis of amyloid-related degenerative diseases including type 2 diabetes. Using an explicit solvent all atomic MD simulation, we explored the stability, conformational dynamics and association force of different single-layer models of the full-length wild-type and glycine mutants of amylin (pentamer) obtained from a recent high resolution fibril model. The RMSF profile shows enhanced flexibility in the disorder (Lys1-Cys7) and turn region (Ser19-Gly23), along with smallest fluctuation at the residues (Asn14-Phe15-Leu16-Val17-His18) of β₁ region and (Ala25-Ile26-Leu27-Ser28-Ser29) of the β2 region. We obtained a significant difference in backbone RMSD between the wild-type and the mutants, indicating that mutations affected the stability of the peptide. The RMSD and RMSF profiles indicate the edge and loop residues are the primary contributors to the overall conformational changes. The degree of structural similarity between the oligomers in the simulation and the fibril conformation is proposed as the possible explanation for experimentally observed shortening of the nucleation lag phase of amylin with oligomer seeding. On the basis of structure-stability findings, the β₁ and β₂ portions are optimal target for further anti-amyloid drug design. The MM-PBSA binding energy calculation reveals the binding of amylin: amylin strands in single layer is dominated by contributions from van der Waals interactions. The non-polar solvation term is also found to be favorable. While the electrostatic interactions and polar solvation energy was found to be favorable for the interaction for the larger aggregate and unfavorable for the smaller aggregates. A per-residue decomposition of the binding free energy has been performed to identify the residues contributing most to the self-association free energy. Residues found in the β-sheet regions were found to be key residue making the largest favorable contributions to the single-layer association. The result from our simulation could be used in rational design of new amylinomimetic agent, amylin aggregation inhibitors and amylin-specific biomarkers.     

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.     

Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol

Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.     

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

Amorphous protein aggregates are oligomers that lack specific, high-order structures. Soluble amorphous aggregates are smaller than ~1 µm. Despite their lack of high-order structure, amorphous protein aggregates exhibit specific biophysical properties such as reversibility of formation, density, conformation, and biochemical stability. Our mutational analysis using a Solubility Controlling Peptide (SCP) tag strongly suggests that amorphous aggregation of small globular proteins can significantly increase in vivo immune response and that the magnitude of enhanced immune responses depends on the aggregates’ biophysical and biochemical properties. We propose that SCP tags might help develop subunit (protein) adjuvant-free (immunostimulant-free) vaccines by controlling the aggregation propensity of target proteins.     

Conformational Stability of Fibrillar Amyloid-Beta Oligomers via Protofilament Pair Formation – A Systematic Computational Study

Amyloid- (A) oligomers play a crucial role in Alzheimer’s disease due to their neurotoxic aggregation properties. Fibrillar A oligomerization can lead to protofilaments and protofilament pairs via oligomer elongation and oligomer association, respectively. Small fibrillar oligomers adopt the protofilament topology, whereas fibrils contain at least protofilament pairs. To date, the underlying growth mechanism from oligomers to the mature fibril still remains to be elucidated. Here, we performed all-atom molecular dynamics simulations in explicit solvent on single layer-like protofilaments and fibril-like protofilament pairs of different size ranging from the tetramer to the 48-mer. We found that the initial U-shaped topology per monomer is maintained over time in all oligomers. The observed deviations of protofilaments from the starting structure increase significantly with size due to the twisting of the in-register parallel -sheets. This twist causes long protofilaments to be unstable and leads to a breakage. Protofilament pairs, which are stabilized by a hydrophobic interface, exhibit more fibril-like properties such as the overall structure and the twist angle. Thus, they can act as stable conformational templates for further fibril growth. Key properties like the twist angle, shape complementarity, and energetics show a size-dependent behavior so that small oligomers favor the protofilament topology, whereas large oligomers favor the protofilament pair topology. The region for this conformational transition is at the size of approximately twelve A monomers. From that, we propose the following growth mechanism from A oligomers to fibrils: (1) elongation of short protofilaments; (2) breakage of large protofilaments; (3) formation of short protofilament pairs; and (4) elongation of protofilament pairs.     

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.     

Designed hairpin peptides interfere with amyloidogenesis pathways: fibril formation and cytotoxicity inhibition, interception of the preamyloid state

Hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites modulate the aggregation of two unrelated amyloidogenic systems, human pancreatic amylin (hAM) and α-synuclein (α-syn), associated with type II diabetes and Parkinson's disease, respectively. In the case of hAM, we have previously reported that inhibition of amyloidogenesis is observed as an increase in the lag time to amyloid formation and a diminished thioflavin (ThT) fluorescence response. In this study, a reduced level of hAM fibril formation is confirmed by transmission electron microscopy imaging. Several of the hairpins tested were significantly more effective inhibitors than rat amylin. Moreover, a marked inhibitory effect on hAM-associated cytotoxicity by the more potent hairpin peptide is demonstrated. In the case of α-syn, the dominant effect of active hairpins was, besides a weakened ThT fluorescence response, the earlier appearance of insoluble aggregates that do not display amyloid characteristics with the few fibrils observed having abnormal morphology. We attribute the alteration of the α-synuclein aggregation pathway observed to the capture of a preamyloid state and diversion to nonamyloidogenic aggregates. These β-hairpins represent a new class of amyloid inhibitors that bear no sequence similarity to the amyloid-producing polypeptides that are inhibited. A mechanistic rationale for these effects is proposed.     

Oleuropein aglycon prevents cytotoxic amyloid aggregation of human amylin

Pancreatic amyloid deposits of amylin are a hallmark of Type II diabetes and considerable evidence indicates that amylin oligomers are cytotoxic to β-cells. Many efforts are presently spent to find out naturally occurring molecules, or to design synthetic ones, able to hinder amylin aggregation or to protect cells against aggregate cytotoxicity. In this context, a protective effect of some polyphenols against amyloid cytotoxicity was reported. Actually dietary polyphenols are endowed with multiple health benefits, and extra virgin olive oil is attracting increasing interest as a source of these substances. Here, we investigated the effects on amylin aggregation and cytotoxicity of the secoiridoid oleuropein aglycon, the main phenolic component of extra virgin olive oil. We found that oleuropein, when present during the aggregation of amylin, consistently prevented its cytotoxicity to RIN-5F pancreatic β-cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide test and caspase-3 activity assay. A lack of interaction with the cell membrane of amylin aggregates grown in the presence of oleuropein was shown by fluorescence microscopy and synthetic lipid vesicle permeabilization. Moreover, our ThT assay, circular dichroism analysis and electron microscopy images suggested that oleuropein interferes with amylin aggregation, resulting in a different path skipping the formation of toxic pre-fibrillar aggregates. These results provide a molecular basis for some of the benefits potentially coming from extra virgin olive oil consumption and pave the way to further studies on the possible pharmacological use of oleuropein to prevent or to slow down the progression of type II diabetes.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Stable and metastable states of human amylin in solution

Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an α-helical segment comprising residues 9-17 and a short antiparallel β-sheet comprising residues 24-28 and 31-35; one with an extended antiparallel β-hairpin with the turn region comprising residues 20-23; and one with no particular structure. Using detailed calculations, we determine the relative stability of these various conformations, finding that the β-hairpin conformation is the most stable, followed by the α-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and linewidths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its interaction with cell membranes are discussed.     

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

Interpreting the aggregation kinetics of amyloid peptides

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.     

Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy

Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.     

Cholesterol Regulates Assembly of Human Islet Amyloid Polypeptide on Model Membranes

Amylin, a 37-aa pancreatic hormone, is the major constituent of islet amyloid, a hallmark of type II diabetes mellitus. Recent studies have revealed a pivotal role of anionic phospholipids in membrane-catalyzed amylin fibrillogenesis and aggregation. However, cholesterol, an integral component of eukaryotic cell membranes, also could have a role. In this study, we have examined the effect of cholesterol on amylin polymerization both on planar membranes and in solution. Using time-lapse atomic force microscopy, we have studied the dynamics and macromolecular organization of amylin on anionic and neutral planar membranes that lack or include cholesterol. On cholesterol-depleted planar membranes, amylin formed highly symmetrical tetrameric and pentameric pore-like supramolecular structures composed of 25- to 35-nm intermediate-sized globular structures or oligomers. Conversely, on membranes incorporating cholesterol, amylin formed highly compact ∼ 200- to 500-nm protein clusters that constituted seeds or nuclei for continuing amylin binding and aggregation. However, cholesterol inhibited amylin nucleation with a 7-fold decrease in the number of amylin particles. Consequently, cholesterol-containing membranes accumulated significantly less amyloid with some membrane areas completely free of amyloid particles. The inhibitory effect of cholesterol on amylin aggregation in solution was also demonstrated as a 16-fold decrease in the aggregation rate. Consistent with this, circular dichroism spectroscopy revealed a stable, soluble random-coil conformation for amylin in the presence of cholesterol that could explain the inhibitory effect of cholesterol on amylin polymerization in solution and on membranes. The modulatory effect of cholesterol was largely independent of membrane charge or phospholipids, suggesting a novel cholesterol-regulated amylin polymerization process.     

Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology.

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet beta-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1-37, to beta-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20-29, the peptides human or rat IAPP 30-37 and 8-20, assume beta-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular beta-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a 'nidus' adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect beta-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions

Human amylin (hA1–37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17–29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17–29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17–29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17–29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17–29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17–29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (?44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17–29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17–29 toxicities are especially attributable to oligomeric aggregates. hA17–29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.