A blend of two resveratrol derivatives abolishes hIAPP amyloid growth and membrane damage

Type II diabetes mellitus (T2DM) is characterized by the presence of amyloid deposits of the human islet amyloid polypeptide (hIAPP) in pancreatic β-cells. A wealth of data supports the hypothesis that hIAPP's toxicity is related to an abnormal interaction of amyloids with islet cell membranes. Thus, many studies aimed at finding effective therapies for T2DM focus on the design of molecules that are able to inhibit hIAPP's amyloid growth and the related membrane damage as well. Based on this view and inspired by its known anti-amyloid properties, we have functionalized resveratrol with a phosphoryl moiety (4′-O-PR) that improves its solubility and pharmacological properties. A second resveratrol derivative has also been obtained by conjugating resveratrol with a dimyristoylphosphatidyl moiety (4′-DMPR). The use of both compounds resulted in abolishing both amyloid growth and amyloid mediated POPC/POPS membrane damage in tube tests. We propose that a mixture of a water-soluble anti-aggregating compound and its lipid-anchored derivative may be employed as a general strategy to prevent and/or to halt amyloid–related membrane damage.     

Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

BackgroundPolybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radiolabeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.MethodsPeptide-ligand interactions were studied using CD spectroscopy and solution-phase binding assays with radiolabeled p5 analogues. The interaction of a subset of peptides was further studied by using molecular dynamics simulations.ResultsDisruption of the peptide helical structure reduced peptide binding to heparin and human amyloid extracts. The all-D enantiomer and the β-sheet-structured peptide bound all substrates as well as, or better than, p5. The interaction of helical and β-sheet structured peptides with Aβ fibrils was modeled and shown to involve both ionic and non-ionic interactions.ConclusionsThe α-helical secondary structure of peptide p5 is important for heparin and amyloid binding; however, helicity is not an absolute requirement as evidenced by the superior reactivity of a β-sheet peptide. The differential binding of the peptides with heparin and amyloid fibrils suggests that these molecular interactions are different. The all-D enantiomer of p5 and the β-sheet peptide are candidates for amyloid targeting reagents in vivo.General SignificanceEfficient binding of polybasic peptides with amyloid is dependent on the linearity of charge spacing in the context of an α-helical secondary structure. Peptides with an α-helix or β-sheet propensity and with similar alignment of basic residues is optimal.     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.     

Cytotoxic Aggregation and Amyloid Formation by the Myostatin Precursor Protein

Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer''s and Parkinson''s diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer''s amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by β-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.     

The Sulfated Triphenyl Methane Derivative Acid Fuchsin Is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing β-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on β-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the β-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.     

A novel in vitro filter trap assay identifies tannic acid as an amyloid aggregation inducer for HET-s

In this work we present an easy and low cost in vitro filter trap assay to quickly identify direct actors on amyloid prion aggregation. We chose the recombinant purified prion protein HET-s from Podospora anserina as a reference. HET-s was labelled with a fluorophore prior to aggregation assays in a 96 well micro-array system. Aggregation assays were carried out in presence of a number of chemical compounds, followed by a filter trap assay through a cellulose acetate membrane and the straight detection of retained fluorescent amyloid fibres. We tested 22 chemical compounds from which 11 have already been described to affect various prions and other amyloid proteins. Four compounds showed direct effects on the aggregation of HET-s. ZnCl seemed to prevent the formation of amyloid fibres. Puzzlingly, three members of the group of tannins (tannic acid, epigallocatechin and epigallocatechin-gallate) had accelerant properties on amyloid aggregation. Resistance of the prion forming domain (PFD) in Proteinase K proteolysis assays underlined that tannic acid favours amyloid fibre formation of HET-s.     

Aggregation Modulators Interfere with Membrane Interactions of?β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β₂-microglobulin (β₂m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β₂m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.     

Ecology and Biogenesis of Functional Amyloids in Pseudomonas

Functional amyloids can be found in the extracellular matrix produced by many bacteria during biofilm growth. They mediate the initial attachment of bacteria to surfaces and provide stability and functionality to mature biofilms. Efficient amyloid biogenesis requires a highly coordinated system of amyloid subunits, molecular chaperones and transport systems. The functional amyloid of Pseudomonas (Fap) represents such a system. Here, we review the phylogenetic diversification of the Fap system, its potential ecological role and the dedicated machinery required for Fap biogenesis, with a particular focus on the amyloid exporter FapF, the structure of which has been recently resolved. We also present a sequence covariance-based in silico model of the FapC fiber-forming subunit. Finally, we highlight key questions that remain unanswered and we believe deserve further attention by the scientific community.     

Aggregation Modulators Interfere with Membrane Interactions of β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β₂-microglobulin (β₂m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β₂m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.     

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.     

Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and Aβ(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40°. Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Aβ(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Aβ(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth     

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.     

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin �� Heavy Chains

We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of Aβ, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig γ heavy chains. A γ₁ heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloid-laden tissue sections. F1 did not bind to native Aβ monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig γ heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble Aβ oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig γ heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules.     

Oleuropein aglycone: A polyphenol with different targets against amyloid toxicity

Background

Many data highlight the benefits of the Mediterranean diet and its main lipid component, extra-virgin olive oil (EVOO). EVOO contains many phenolic compounds that have been found effective against several aging- and lifestyle-related diseases, including neurodegeneration. Oleuropein, a phenolic secoiroid glycoside, is the main polyphenol in the olive oil. It has been reported that the aglycone form of Oleuropein (OleA) interferes in vitro and in vivo with amyloid aggregation of a number of proteins/peptides involved in amyloid, particularly neurodegenerative, diseases avoiding the growth of toxic oligomers and displaying protection against cognitive deterioration.

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

Discovery of Dihydrochalcone as Potential Lead for Alzheimer’s Disease: In Silico and In Vitro Study

By the virtual screening method we have screened out Dihydrochalcone as a top-lead for the Alzheimer’s disease using the database of about 32364 natural compounds. The binding affinity of this ligand to amyloid beta (A) fibril has been thoroughly studied by computer simulation and experiment. Using the Thioflavin T (ThT) assay we have obtained the inhibition constant IC50 M. This result is in good agreement with the estimation of the binding free energy obtained by the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulation with the force field CHARMM 27 and water model TIP3P. Cell viability assays indicated that Dihydrochalcone could effectively reduce the cytotoxicity induced by A. Thus, both in silico and in vitro studies show that Dihydrochalcone is a potential drug for the Alzheimers disease.     

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid β-protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Aβ small oligomers and fibrillar aggregates to simulate the action of diffusible Aβ oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Aβ. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Aβ peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

The Interaction of αB-Crystallin with Mature α-Synuclein Amyloid Fibrils Inhibits Their Elongation

αB-Crystallin is a small heat-shock protein (sHsp) that is colocalized with α-synuclein (αSyn) in Lewy bodies—the pathological hallmarks of Parkinson's disease—and is an inhibitor of αSyn amyloid fibril formation in an ATP-independent manner in vitro. We have investigated the mechanism underlying the inhibitory action of sHsps, and here we establish, by means of a variety of biophysical techniques including immunogold labeling and nuclear magnetic resonance spectroscopy, that αB-crystallin interacts with αSyn, binding along the length of mature amyloid fibrils. By measurement of seeded fibril elongation kinetics, both in solution and on a surface using a quartz crystal microbalance, this binding is shown to strongly inhibit further growth of the fibrils. The binding is also demonstrated to shift the monomer-fibril equilibrium in favor of dissociation. We believe that this mechanism, by which a sHsp interacts with mature amyloid fibrils, could represent an additional and potentially generic means by which at least some chaperones protect against amyloid aggregation and limit the onset of misfolding diseases.     

Retardation of Aβ Fibril Formation by Phospholipid Vesicles Depends on Membrane Phase Behavior

An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Aβ). Recombinant Aβ(M1-40) and Aβ(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Aβ fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane.