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Beta-synuclein exhibits high sequence homology and structural similarity with alpha-synuclein, a protein implicated in the pathogenesis of Parkinson's disease. We investigated the chaperone function of beta-synuclein and its anti-fibrillar activity in comparison with alpha-synuclein. beta-Synuclein suppressed the heat-induced aggregation of aldolase, alcohol dehydrogenase, and citrate synthase, and its anti-aggregative activity was remarkably higher than that of alpha-synuclein. Heat-induced inactivation of citrate synthase was significantly protected by beta-synuclein. Moreover, beta-synuclein inhibited the amyloid formation of both Abeta(1-40) and alpha-synuclein. It is, therefore, suggested that beta-synuclein can prevent abnormal protein aggregations more effectively than alpha-synuclein by acting as a molecular chaperone.

Phenylazo benzenesulfonamides were designed and synthesized as beta-amyloid (Abeta40) fibril assembly inhibitors, and evaluated for inhibition of Abeta40 aggregation and neurotoxicity using rat cortical neurons. Compound 2 (LB-152) was the most potent compound in this study, and the para-NMe(2) group on the end of the phenylazo moiety may play an important role in preventing Abeta40 fibril formation. LB-152 provides a new lead for further development of potential beta-amyloid aggregation inhibitors to treat AD.

In amyloid related diseases, proteins form fibrillar aggregates with highly ordered beta-sheet structure regardless of their native conformations. Formation of such amyloid fibrils can be reproducible in vitro using isolated proteins/peptides, suggesting that amyloid fibril formation takes place as a result of protein conformational change. In vitro studies revealed that perturbation of the native structure is important for the fibril formation, and it is suggested that the mechanisms of amyloid fibril formation share the mechanisms of protein folding. In particular, amyloid fibril formation is similar to one of the common features of proteins, i.e. amorphous aggregation upon partial unfolding, which is likely driven by hydrophobic interactions through exposed protein interior. However, these molecular associations are distinct phenomena, and identifying factors that lead to amyloid fibril formation would precede our understanding of the mechanisms of amyloid fibrillization. The necessity of understanding the nature of protein denatured states is also suggested.

The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature (Td) of the SC solution was found to be 18.6 degrees C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4 degrees C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM (f-50 gel). In particular, the melting temperature of the f-50 gel was 47 degrees C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f-50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation.

Peptide fragments taken from residues 18‒54 of short consensus repeat 3 (SCR3) from the human complement receptor CR1 have been found in aqueous solution to slowly aggregate and form fibrils. NMR studies of the monomeric form of these peptides show that they are essentially unfolded in aqueous solution and that they all have an increased helicity in TFE solutions. The behaviour of residues 28‒31 from SCR3 is particularly interesting. These residues have a highβ-sheet propensity in the native protein and a seven peptide containing their sequence is found to form fibrils despite its short length. However, NMR studies show that these residues adopt a well-definedα-helix in 80% TFE and under these conditions fibril formation has not been observed. These data demonstrate the strong dependence of conformational propensities on environment.

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

The 39-42 amino acid long, amphipathic amyloid-beta peptide (Abeta) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Abeta presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Abeta(1-40) membrane assemblies for two different scenarios with potential implication in AD: Abeta peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Abeta(1-40) by stabilizing its hydrophobic transmembrane C-terminal part (residue 29-40) in an alpha-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Abeta(1-40) is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Abeta(1-40) peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide-membrane assemblies are involved in Abeta's pathophysiology with the finely balanced type of Abeta-lipid interactions against release of Abeta from neuronal membranes being overcompensated by an Abeta-membrane assembly which causes toxic beta-structured aggregates in AD. Therefore, pathological interactions of Abeta peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Abeta-membrane assemblies inherited from Abeta's origin.

Proteins adopt an amazing array of sequence-dependent structures that enable them to perform the many chemical functions critical to life. Over the past decade, however, it has become clear that many different protein sequences can also form misfolded, insoluble aggregates known as amyloid fibrils, with common structural elements. Amyloid fibrils appear to be involved in a number of diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, and type II diabetes (1). Even ordinary globular proteins (e.g., myoglobin) can form amyloid fibrils under certain conditions (2), suggesting that fibril formation is a previously unappreciated general property of many proteins. Thus, it is of fundamental interest to understand how so many different protein sequences can adopt this alternative structure and it is of medical interest to understand and control disease-related amyloid formation. Knowledge of the three-dimensional structure of amyloid fibrils is critical for understanding the mechanism of fibrillogenesis and for design of possible inhibitors. Unfortunately, amyloid fibrils are noncrystalline and insoluble, and therefore are not amenable to x-ray crystallography and solution NMR, the classic tools of structural biology. In a recent issue of PNAS, Petkova et al. (3) report a structural model for Alzheimer's β-amyloid fibrils deduced primarily from solid-state NMR experiments. This work provides a significant step forward in understanding β-amyloid formation and showcases the power of solid-state NMR for obtaining structural information on important but challenging biomolecules.

Proteins adopt an amazing array of sequence-dependent structures that enable them to perform the many chemical functions critical to life. Over the past decade, however, it has become clear that many different protein sequences can also form misfolded, insoluble aggregates known as amyloid fibrils, with common structural elements. Amyloid fibrils appear to be involved in a number of diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, prion diseases, and type II diabetes (1). Even ordinary globular proteins (e.g., myoglobin) can form amyloid fibrils under certain conditions (2), suggesting that fibril formation is a previously unappreciated general property of many proteins. Thus, it is of fundamental interest to understand how so many different protein sequences can adopt this alternative structure and it is of medical interest to understand and control disease-related amyloid formation. Knowledge of the three-dimensional structure of amyloid fibrils is critical for understanding the mechanism of fibrillogenesis and for design of possible inhibitors. Unfortunately, amyloid fibrils are noncrystalline and insoluble, and therefore are not amenable to x-ray crystallography and solution NMR, the classic tools of structural biology. In a recent issue of PNAS, Petkova et al. (3) report a structural model for Alzheimer's β-amyloid fibrils deduced primarily from solid-state NMR experiments. This work provides a significant step forward in understanding β-amyloid formation and showcases the power of solid-state NMR for obtaining structural information on important but challenging biomolecules.

Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. Fibrillation was accompanied by a dramatic increase in the beta-structure content and a characteristic increase in the thioflavin T fluorescence intensity. S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded alpha-lactalbumin precipitated out of solution as amorphous aggregates. Our data suggest that the transformation of a protein into an essentially unfolded (thus, highly flexible) conformation is required for successful fibril formation, whereas more rigid (but still flexible) species may form amorphous aggregates.

The accumulation of amyloid-beta and concomitant oxidative stress are major pathogenic events in Alzheimer’s disease. Indole-3-propionic acid (IPA, OXIGON™) is a potent anti-oxidant devoid of pro-oxidant activity. IPA has been demonstrated to be an inhibitor of beta-amyloid fibril formation and to be a potent neuroprotectant against a variety of oxidotoxins. This review will summarize the known properties of IPA and outline the rationale behind its selection as a potential disease-modifying therapy for Alzheimer’s disease.

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

The association of amyloid fibril formation with a number of important diseases, and the extensive study of this process in vitro, has resulted in a large literature containing a vast amount of information about the fibril formation process. This includes mutations and experimental conditions that promote or protect against fibril formation. A database (fibril_one) was designed to hold information relating to the formation of fibrils. It was populated by extensive searches of the literature and other databases. A powerful World Wide Web query interface to the database was developed, enabling a simple and effective method to view amyloidogenic mutations associated with specific proteins. The Web interface was used to identify trends in the data. This revealed that mutations promoting fibril formation through altered folding tend to be associated with destabilization of the native fold. In particular, tendencies of mutations to disrupt the native secondary structure and packing in the hydrophobic core were discovered to be significant. Query access to the database is available freely on the World Wide Web at http://www.bioinformatics.leeds.ac.uk/group/online/fibril_one.

This chapter reviews the main material properties required for a pressure-sensitive adhesive (PSA) to be tacky and discusses the way in which experimental parameters affect the practical evaluation of tackiness. The bonding and debonding of a PSA from a surface is a complicated process. A broad spectrum of relaxation times is necessary to ensure quick bonding (short relaxation times) and reasonable fibril stability (long relaxation times), as well as to impart a broad temperature window of use. In addition to these important material parameters of the PSA, the surface can also play a role in controlling tackiness in certain cases. There are two major causes of poor adhesion of a PSA to a surface: low G c and poor contact. If the interfacial G c (static, but also dynamic) is too low, and once cavities are nucleated at the interface, they can easily propagate, coalesce, and debonding occurs without any fibril formation. The other case occurs when the elastic modulus of the PSA is too high or when the surface is too rough for the adhesive to conform to it during the short contact time. The essential difference between a low energy surface and a high energy surface can be visualized through the comparative analysis of probe tack tests.

In a previous work by Alvarez-Martinez et al. (2011), the authors pointed out some fallacies in the mainstream interpretation of the prion amyloid formation. It appeared necessary to propose an original hypothesis able to reconcile the in vitro data with the predictions of a mathematical model describing the problem. Here, a model is developed accordingly with the hypothesis that an intermediate on-pathway leads to the conformation of the prion protein into an amyloid competent isoform thanks to a structure, called micelles, formed from hydrodynamic interaction. The authors also compare data to the prediction of their model and propose a new hypothesis for the formation of infectious prion amyloids., Author Summary Understanding the mechanism of prions is an important issue. Indeed, it involves a mechanism modifying the structure of the proteins that are of high interest in theoretical biology. Knowing the underlying mechanism that leads to prion disease could help further investigations in the world of amyloid disease and for example the so-called Alzheimer's disease. The theory of prion, also known as Protein-Only, has been widely studied. Nevertheless no mathematical models are able to reproduce the phenomena in silico. This suggests a lack of information in the theory. Here we propose a new model, built with a new approach theory that fits experimental data in a very satisfactory way. This model, together with experiments, maintains the idea that an intermediate conformation of the protein helps the disease to spread. Besides, this work is an excellent example of a strong interaction between mathematical modelling and biological approach. Indeed, because of a strong discrepancy between theoretical results of the early original model and biological data on pathological prion formation, the team of biologists decided to investigate more closely their experiments. They came out with a new discovery: the crucial role of micelles in the pathological conformation of the prion protein.

Diseases such as type 2 diabetes, Alzheimer's and Parkinson's share as common feature the accumulation of mis-folded disease-specific protein aggregates into fibrillar structures, or plaques. These fibrils may either be toxic by themselves, or act as reservoirs for smaller cytotoxic oligomers. This suggests to investigate molecules as potential therapeutics that either reduce fibril formation or increase fibril stability. One example is rat amylin, which can inhibit aggregation of human amylin, a hallmark of type 2 diabetes. In the present paper, we use molecular dynamics to compare the stability of various preformed aggregates, built out of either human amylin, rat amylin, or mixtures of both. We considered two types of fibril-like oligomers: a single-layer in-register conformation, and a double-layer conformation in which the first U-shaped layer consists of rat amylin and the second layer of human amylin. Our results explain the weak amyloid-inhibiting properties of rat amylin and suggest that membrane leakage due to pore formation is responsible for the toxicity of rat amylin observed in a recent experiment. Together, our results put in question the use of rat amylin or the similar FDA approved drug pramlintide as an inhibitor of human amylin aggregation. They also point to mixed human-rat amylin fibril-like oligomers as possible model-systems for studies of amyloid formation that involve cross-species transmission.

Lewy bodies are the pathological hallmark of Parkinson's disease (PD). While fibrillar α-synuclein (αS) is the main protein component of Lewy bodies, these structures also contain lipids. To elucidate the presence of lipids in Lewy bodies, we investigated the interaction of lipids with monomeric and fibrillar αS. In vitro, lipid membranes accelerated αS fibril formation under physiological conditions. Moreover lipids and small vesicles co-localized with supra-fibrillar structures and individual αS fibrils suggesting that aggregation initiates at the membrane. The presence of lipids in Lewy bodies may therefore be an indication that cell membranes are the major target in aggregation induced neuronal cell death

Human islet amyloid polypeptide (hIAPP) aggregates and forms amyloid fibrils in the pancreas of patients with type2 diabetes. Anionic membranes bind hIAPP and accelerate the fibril formation. During this process, hIAPP disrupts the membrane, a toxic process that has been associated with the N-terminal 19 residues and proposed to lead to the destruction of the insulin-producing β-cells in the pancreas. The mechanism by which the membrane is disrupted is therefore key to our understanding of toxicity of hIAPP, and to design of effective disease modifying treatments.Employing molecular dynamics (MD) simulations, we have investigated the molecular events key to hIAPP binding to anionic phospholipid bilayers. The diffusion of lipids in conventional membrane models is too slow to allow for hIAPP-membrane binding on the time-scale feasible with conventional MD simulations. In order to overcome the slow diffusion of lipids in conventional membranes which might not allow for adequate sampling of the binding of hIAPP binding to the membrane, we have employed a highly mobile membrane mimetic (HMMM) model.Multiple independent MD simulations, amounting to 1.2 μs, both for the N-terminal 19-residue peptide, and for the full (37-residue) hIAPP, resulted in spontaneous binding to mixed anionic/zwitterionic HMMM membranes. Initial contacts between the peptide and lipids, which form rapidly (within the first 10 ns of the simulations), are mediated by a group of positively charged residues, Lys1, Arg11, and His18, indicating that the N-terminal part of hIAPP is responsible for its interaction with anionic membranes. These simulations have provided an unprecedented level of statistic on lipid-protein interaction for hIAPP peptides, characterizing residues and lipid chemical groups key for bindig and interaction, as well as the structure and dynamics of the peptide in its membrane-bound form.

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimer's disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis. We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, flavonoids and biflavonoids were screened against fibril formation by a recombinant antibody variable domain (V1). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 microM in 79 microM of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 microM using ThT as a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the V(L) protein dimer. These studies suggested that these compounds could potentially stabilize the VL dimer and therefore reduce its tendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

The aggregation of proteins into fibrils plays a crucial role in neurological conditions such as Parkinson's disease. Further insight into fibril formation has now been gained that reveals the effect of hydrophobic surfaces, including air.

Peptide chemistry has experienced enormous progress over the last decades, and the synthesis-oriented peptide scientist is confronted today with research domains at the interface of chemistry, biology and medicine. In this review, we look at some of our contributions over four decades, developed at four places in Germany and Switzerland. Special attention is given to four concepts which can be considered as nucleation seeds for some of today's research topics in peptide chemistry: (1) The use of polyethylene glycols (PEG) as solubilizing carriers in liquid-phase peptide synthesis which has resulted in the well known 'PEGylation' of peptides and proteins, (2) the construction of artificial proteins using topological templates, termed template-assembled synthetic proteins (TASP) which has stimulated the use of templates in peptide and protein design and mimicry, (3) pseudo-prolines which serve today as routine technology for the prevention of peptide self-association and beta-sheet formation in peptide synthesis, thus making accessible solid-phase synthesis of peptides up to the size of small proteins, and finally (4) studies on the onset of secondary structure and conformational transitions in designed oligo-peptides which resulted in the creation of 'switch-peptides' as folding precursors, allowing delineation of the molecular mechanism of protein folding and misfolding as an early step in peptide self-assembly and fibril formation relevant in degenerative diseases.

Models of structural transition in prion protein (PrP) focus on the domain visualised by solution NMR. Accumulating evidence suggests that the adjacent and highly conserved nonpolar segment, as well as PrP-membrane interactions, should also be considered. Calculations predict that membrane-induced structural destabilisation is mediated by stabilisation of the unfolded form. Comparative analysis of PrP structures leads to a model for PrP dimerisation that incorporates the nonpolar segment. A prediction that PrP will interact with the PrP-like protein (Dpl) to form a heterodimer, but that Dpl will not form a homodimer, can be tested. Modelling is discussed in the context of ataxias associated with the expression of Dpl or truncated PrP in transgenic animals lacking wild-type PrP. A PrP(C) dimer model forms the basis for considering the geometry of PrP(Sc) fibril formation.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Both Alzheimer’s disease (AD) and Parkinson’s disease (PD) exist in rare familial forms caused by mutations in genes that encode the predominant fibrillar protein component of the characteristic lesion: amyloid plaques and Lewy bodies, respectively. This convergence of pathological and genetic information has given rise to the idea that the fibrils themselves are responsible for neuronal death. Biophysical studies, summarized herein, have demonstrated that the mutations accelerate the overall process of fibril formation, including the accumulation of an intermediate species, designated the protofibril. We propose that protofibrils, rather than fibrils, are the pathogenic entities in both AD and PD.

Growing awareness that the underlying cause of many diseases involves the formation of amyloid plaques, from Alzheimer’s through to the spongiform encephalopathies, has prompted the search for new methods to study the structural conversion of normally soluble protein to amyloid fibril. Recent evidence has suggested that the formation of amyloid fibrils may involve structural rearrangement of native monomeric protein through partially folded intermediates [1]. Mass spectrometry (MS) is playing an increasing role in characterizing the nature of proteins that are present in the fibril [2] and also in looking at intermediates that form during fibril formation [3, 4].

Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer's disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into beta-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 microM peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 degrees C. The state at 45 degrees C was also found to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of beta-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases.

Publisher Summary The deposition of amyloid fibrils is responsible for the pathology of a number of diseases. Their resistance to biological clearance is a hallmark of amyloidosis; however, systemic amyloidoses such as AL (immunoglobulin light chain) and AA (serum amyloid A protein) have been shown to reduce or resolve on removal of the source of precursor protein or on treatment with a pharmacologic agent that blocks fibril formation. Thus, interfering with amyloid fibril formation by a small organic molecule pharmacologic therapy could be a treatment option. A variety of such ligands have been described in the chapter, although they lack suitable profiles as therapeutics. For Alzheimer's disease (AD), whose amyloidosis affects the largest number of people, peptides and monoclonal antibodies have also been proposed as potential therapeutic agents, although it is unclear how blood–brain barrier penetration and cellular penetration will be achieved with these large, polar molecules.

In the present study, we investigated whether or not laminin inhibits A β 42 fibril formation in the same manner as A β 40. Both a thioflavine-T fluorometric assay and electron microscopy by negative staining demonstrated laminin to have a concentration-dependent inhibitory effect on A β 42 fibril formation. The amyloid fibril formation was inhibited approximately by 70% due to the presence of 1.0 mg/ml laminin co-incubated with 1.0 mg/ml A β 42 peptide (molar ratio; A β 42 peptide:laminin=200:1). These results thus suggested that laminin or its derivatives may be effective as therapeutic agents to either prevent or slow down the progression of amyloidogenesis in Alzheimer's disease.

Senile plaques are the main neuropathological feature of AD, and are formed by extracellular amyloid β-peptide (Aβ) fibrils and dystrophic neuntes. Several cellular proteins have been found within Aβ deposits, including apolipoprotein E, α1,-antichymotrypsin and acetylcholinesterase (AChE). AChE has been found to co-localize with Aβ deposits such as those present in pre-amyloid diffuse deposits, mature senile plaques and cerebral blood vessels. Primate studies have shown that AChE may be deposited in plaques during the early stages of amyloid formation. Therefore, the diffuse deposition of AChE together with Aβ, may represent an early step in the development of amyloid fibrils.

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.

Alzheimer’s disease (AD) is the most common form of dementia in adults. It determines a progressive loss of intellectual function and appears both sporadically and in an autosomal dominant (familial) form. Presently there is no treatment for AD.

Previously, we have developed a retro-inverso peptide inhibitor (RI-OR2, rGffvlkGr) that blocks the in vitro formation and toxicity of the Aβ oligomers which are thought to be a cause of neurodegeneration and memory loss in Alzheimer's disease. We have now attached a retro-inverted version of the HIV protein transduction domain 'TAT' to RI-OR2 to target this new inhibitor (RI-OR2-TAT, Ac-rGffvlkGrrrrqrrkkrGy-NH2) into the brain. Following its peripheral injection, a fluorescein-labelled version of RI-OR2-TAT was found to cross the blood brain barrier and bind to the amyloid plaques and activated microglial cells present in the cerebral cortex of 17-months-old APPswe/PS1ΔE9 transgenic mice. Daily intraperitoneal injection of RI-OR2-TAT (at 100 nmol/kg) for 21 days into 10-months-old APPswe/PS1ΔE9 mice resulted in a 25% reduction (p

4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of the interactions between known small molecule inhibitors and TTR by molecular docking. A series of 2,4,6-triaryl-4H-1,4-oxazines was synthesized by the cyclization of N,N-bis(phenacyl)anilines with POCl3 in pyridine. Inhibition of TTR amyloid fibril was evaluated by a fibril formation assay. The results indicate that 4H-1,4-oxazines significantly inhibit TTR amyloid fibril at a concentration of 7.2 μM.