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Herein we report for the first time fibril formation by pepsin at neutral pH and room temperature. Fibril formation was characterized by a number of techniques including fluorescence and circular dichroism spectroscopy, dynamic light scattering, and transmission electron microscopy.

We computationally and experimentally showed that tau protein fibrils can be formed at high temperature. When cooled, the fibrils dissociate back to monomers. Heparin promotes tau fibril formation and prevents its reversion. Our results revealed the physicochemical mechanism of reversible formation of tau fibrils.

Many proteins are known to be capable of forming amyloid fibrils, long thin fibrils that, if they possess sufficiently large aspect ratios and volume fractions, have been shown to form liquid crystals in several protein systems. However, denatured proteins exhibit polymorphism as they aggregate, and external factors affect which type of structure forms. Here we demonstrate the ability to form liquid crystalline phases in three different protein systems, without the need for the prior formation of fibrils, by the use of mechanical shear during the aggregation step to modify the aggregation process. It is shown that the rate of stirring affects the aspect ratio of the amyloid fibrils formed in vitro, and hence the ability to form liquid crystalline phases, as predicted by theory. A shift is observed with increasing stirring rate from the large spherulitic structures (structures formed via suprafibrillar aggregation under highly acidic conditions) to the alignment of amyloid fibrils into a nematic liquid crystal phase in insulin, β-lactoglobulin (BLG) and hen egg white lysozyme (HEWL). Because the stirring modifies the fibril formation, above a threshold stirring rate, the aspect ratio of insulin and BLG fibrils drops below the point at which liquid crystal-like phases can form. However, in the case of HEWL, the decrease in the fibril aspect ratio at higher stirring rates is still sufficiently small to enable liquid crystalline phases to form. The overall phase behaviour can be rationalised within the Onsager model for liquid crystallinity.

Since the formation of amyloid structures from proteins was recognized in numerous diseases, many efforts have been devoted to the task of finding effective anti-amyloidogenic compounds. In a number of these investigations, the existence of "generic" compounds is implicitly acknowledged. Curcumin seems to be one of these compounds, possessing key structural components effective toward fibrillation prevention, and its anti-amyloidogenic property has been reported for a number of model and disease-related proteins such as lysozyme and alpha-synuclein. In this study, insulin amyloid formation has been shown to be effectively influenced by micromolar concentrations of curcumin. Under amyloidogenic conditions (pH 2.5 and 37 °C), the compound was observed to inhibit fibril formation of insulin in a dose-dependent manner. Moreover, addition of curcumin to the protein incubated under such conditions at different time points resulted in reduced amounts of final fibrils. Disaggregation of pre-formed fibrils was also observed upon addition of curcumin, as well as reduction in final fibril amounts after seeding. Overall, this compound appears to be able to interact with native, intermediate and fibrillar forms. Docking experiments suggest a potential interacting site with the B-chain of insulin, as well as the possibility for beta-sheet breaker activity.

The molecular characteristics of purified pepsin solubilized collagen from rat liver was studied in control and dimethylnitrosamine administered animals. The alpha- and beta-chains of purified pepsin solubilized liver collagen were separated by subjecting the denatured collagen to SDS-polyacrylamide gel electrophoresis. The alpha 1(III) chains were resolved from the alpha 1(I) chains by interrupted electrophoresis with delayed reduction of the disulfide bonds of type III collagen. The aldehyde content of the purified pepsin solubilized collagen was estimated in control and experimental samples in order to assess the extent of collagen cross-links. Fibril formation curves were studied with purified pepsin solubilized collagen to see the rate of formation of cross-links within the fibrillar mesh. The results of the unreduced electrophoretic studies revealed a significant increase in the beta-subunit of type I collagen with a remarkable decrease of alpha/beta ratio in DMN treated animals. Reduction with beta-mercaptoethanol indicated the presence of type III collagen in the electrophoretic field with a proportionate increase on the 21st day. A significant increase in the aldehyde content and an increased rate of fibril formation were noticed in DMN induced fibrotic liver collagen. The data of the present investigation revealed that the DMN induced fibrotic liver collagen is more cross-linked than normal liver collagen and the deposition of type III collagen in more prominent than type I collagen in early fibrosis.

Alzheimer’s amyloid beta (Aβ) has long been known to be a highly insoluble and “sticky” protein. More recently it has become evident that several apolipoproteins (apo) can interact with Aβ. Apo J is a major carrier protein of normal soluble amyloid β (sAβ), which may well be the precursor of Aβ. On the other hand, apoE4 is linked to late-onset Alzheimer’s disease and can modulate Aβ fibril formation in vitro. Furthermore, the carboxyl terminus of apoE is a constituent of Alzheimer’s plaque amyloid. It is our working hypothesis that apoE, and possibly other chaperone proteins, may play a role in the conformational transition from soluble Aβ to amyloid fibrils.

Using site-specific antibodies, we assessed the effect of aggregation of various length forms of A beta on the immunoreactive profile of the peptides. All of the antibodies tested reacted with monomeric/dimeric forms of A beta 1-42 and its further aggregates. However, antibodies directed against the 1-24 region of A beta reacted weakly or not at all with A beta 1-39/40 monomers or dimers, but immunoreactivity was enhanced substantially following peptide incubation and aggregation. These results suggest that the conformation of the N-terminal region of monomeric and dimeric A beta 1-39/40 is different from that of aggregated forms, whereas the longer A beta 1-42 does not significantly change its N-terminal conformation during beta-sheet fibril formation. These immunochemical results are consistent with previous structural data, and help to explain the differential effects of A beta 1-39/40 and 1-42 on fibril formation in brain.

A 39-43-amino acid residue-long fragment (beta-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimer's disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer beta-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of A beta 1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.

In a healing wound fibronectin appears in various stages. It is present in the fibrin clot which closes and protects the wound and it is synthesized in the granulation tissue. Consequently, it plays a role in several processes which is possibly due to its multifunctional nature.

A new method for determining the size of critical nucleus of fibril formation of polypeptide chains is proposed. Based on the hypothesis that the fibril grows by addition of a nascent peptide to the preformed template, the nucleus size N(c) is defined as the number of forming template peptides above which the time to add a new monomer becomes independent of the template size. Using lattice models one can show that our method and the standard method which is based on calculation of the free energy, provide the same result for N(c).

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.

The WHO has listed Alzheimer's disease among the major neurological disorders with an estimated 35 million people affected worldwide. Amyloid-beta is mostly believed to be the causative factor in Alzheimer's disease and the severity of the disease correlates with the tendency of amyloid-beta to form aggregation patterns-plaques. Lacking effective medication, the identification of any underlying mechanistic principles regarding plaque formation appears to be crucial. Here we carry out computer simulations to study the effect of C-60 on structure and stability of an idealised pentameric construct of amyloid-beta units (a model fibril). A binding site on top of the structurally ordered stack of beta-sheets is identified that triggers structural alterations at the turn region of the hook-like beta-sheet assembly. Significant structural alterations are: (i) the destruction of regular helical twist, (ii) the loss of a stabilizing salt bridge and (iii) the loss of a stabilizing hydrophobic interaction close to the turn. Consequently, the main effect of C-60 is the induction of sizable destabilization in native fibril structure. These structural insights may serve as a molecular guide for further rational drug design of effective inhibitors targeting fibril formation in Alzheimer's disease.

Alzheimer's disease (AD) is the most common chronic, progressive neurodegenerative disease, which affects an increasing number of elderly patients. So far, there is no cure for the disease. Early diagnosis to predict dementia outcome is of great importance. Biomarkers, as physiological indicators of AD, hold great promise for clinical diagnosis and drug discovery. Sensitive and selective methods that are amenable to the detection of biomarkers are much preferred. Surface plasmon resonance (SPR) is a sensitive optical technique capable of measuring analyte concentrations as well as kinetics of biomolecular interactions. It exhibits a variety of fascinating properties, such as high sensitivity, label-free and real-time analysis, and low sample consumption. In this review, we address the recent development on sensitive detection of a series of biomarkers for AD using SPR. Furthermore, amyloid-β (Aβ) fibril formation and interaction monitored by SPR have been documented. The challenges and perspectives inherent in the detection of the biomarkers are also highlighted.

Table 1 Structures of resveratrol (1) and resveratrol analogs tested for their capacity to prevent TTR-induced cytotoxicity in AC16 cells. Between parentheses is shown the compound number and between brackets are shown the values of acid-mediated WT TTR fibril formation in an in vitro assay measured by turbidity. 100% fibril formation corresponds to values of turbidity of WT TTR alone; 0% indicates no turbidity was detected thus, total inhibition of TTR amyloid fibril and aggregate formation. The details of the assay and most of the fibril formation data have been previously published and are shown here for reference.

Possible abnormalities of the collagenous matrix in osteoporosis might provide important informations on the pathogenesis of this common disorder. Furthermore, observations from osteogenesis imperfecta suggest that alterations in collagen modification may cause problems in fibril formation and hence mineralization. In particular, several studies on this inherited disease revealed alterations in the collagenous composition (Brenner et al. 1989a) and both changes in the primary structure as well as overmodification of the collagen molecule (Prockop et al. 1989; Kirsch et al. 1981). A transient overmodification of lysine residues has also been found in hyperplastic callus tissue (Brenner et al. 1989b) and is seen during fetal development (Kirsch et al. 1981; Brenner et al. 1989b). Therefore, we wondered whether similar abnormalities can be found in the collagenous bone matrix of adult patients with osteopenia. For this purpose, we performed a combined biochemical and morphometric study.

The effect of beta-sheet propensity on the structural features of peptide aggregates was investigated using an off-lattice coarse-grained peptide model. A phase diagram as a function of temperature and beta-sheet propensity reveals a diverse family of supramolecular assemblies. Highly rigid peptides (peptides with high beta-sheet propensity) are seen to assemble predominantly into fibrillar structures. Increasing the flexibility of the peptide (reducing beta-sheet propensity) leads to a variety of structures, including fibrils, beta-barrel structures, and amorphous aggregates. Nonfibrillar entities have been suggested as primary causative agents in amyloid diseases and our simulations indicate that mutations that decrease beta-sheet propensity will decrease fibril formation and favor the formation of such toxic oligomers. Parallels between beta-sheet aggregates and nematic liquid crystals are discussed.

β2-Microglobulin (β2m) is a 99-residue protein with a classical immunoglobulin fold that constitutes the light chain of the MHC-I complex. In long-term hemodialysis patients, the concentration of β2m in the blood serum is highly elevated and the protein forms amyloid fibrils that typically deposit in osteoarticular tissues. Although there have been numerous studies that have addressed the process of fibril formation and the structure of the fibrils formed by a small fragment of the protein, atomic level structural information on the full-length fibrils is still lacking. Here, we present solid-state NMR data on the long straight fibrils formed by the full-length protein at pH 2.5, including resonance assignments of a significant number of the residues as well as experiments aimed at characterizing the fibril dynamics. The relationship between these findings and the aggregation behavior of β2m will be discussed.

Spontaneous self-assembly and amyloid formation are a general property of many polypeptides and the information controlling these processes is encoded in the sequence. This determines the form and structural features of the interacting partners that regulate the properties of the final aggregates. Understanding the correlations between sequence, structure and dynamics in peptides and proteins at an atomistic level of resolution still represents one of the grand challenges of modern biological chemistry. In this context, computer simulations represent a valuable approach to understand recognition and spontaneous self-organization, processes that cannot be directly observed experimentally. Herein, we will discuss cases illustrating the extent to which simulations can be used to understand the self-organization properties of natural and designed amyloidogenic peptide sequences. The simulations provide evidence for the influence of specific interactions with well defined stereochemical constraints on fibril formation. The results from our and other groups suggest that simulations can be applied to detect the critical physico-chemical determinants of a certain process and can be helpful in the design of new chemical systems and experiments.

Heparin is involved in the pathogenesis of prion diseases, affecting the process of fibril formation. It has been shown that whether it accelerates or inhibits fibrilogenesis depends on its concentration: prion peptide PrP 185-208 aggregates in the presence of 0.04 mg ml−1 heparin, but concentrations ten times lower or higher cause no aggregation. Polyamidoamine, polypropyleneimine and phosphorus dendrimers that previously exhibited anti-prion activity have been shown to interact with heparin. The interactions between cationic dendrimers and anionic heparin are mainly electrostatic. The present study shows that these interactions are indirectly responsible for the inhibition or enhancement of fibril formation by dendrimers.

Studies have indicated that the 20-29 segment of islet amyloid polypeptide (IAPP) determines the fibril formation. Variations between species in this region explain why only some species develop islet amyloid. This assumption is strongly supported by our study where we have used synthetic peptides in an fibril formation test system. The sequence AILS (IAPP25–28) is the most important amyloido-genic part of the IAPP molecule.

In the past few years molecular biology has contributed to the ongoing research on the transthyretin (TTRj related amyloidoses. Several non exclusive intervening factors in amyloidogenesis are investigated by these techniques; they include the amyloidogenic potential of TTR, due to either modifications on the conformation of the protein and/or proteolysis at specific and constant sites, and finally interaction with tissue or circulating factors. Newer tools like PCR technology, genetic engineering in bacteria and transgenic animals are now used to approach the pathogenic mechanisms underlying these diseases.

The involvement of transthyretin (TTR) in Familial Amyloidotic Polyneuropathy (FAP) has been widely documented. Extensive biochemical studies on the TTR from Portuguese FAP-diagnosed patients and the TTR from control patients have revealed them to be indistinguishable with respect to many physical parameters (Mascarenhas, M.J. (1983)). However, further sequence analysis studies exposed a Val 30 —> Met substitution. Since then, 7 further FAP-related TTR mutations have been discovered all of which result in the same fatal amyloid deposition. In an effort to understand how a single substitution can result in extensive fibril formation we have carried out a computer graphics analysis of each mutation by subsituting it into the high resolution structure of native TTR and studying the molecule for any subsequent changes imposed on it. This resulted in the discovery of a region in the TTR molecule in which all the mutations lie that overlaps the interacting surface in the crystallographic unit cell. This suggested a similar arrangement of molecules within the fibrils and crystals. To obtain experimental evidence for the orientation of molecules within the fibrils we have implemented x-ray fibre diffraction techniques using Met 30 fibrils extracted from human kidney tissue. Preliminary results support the proposed orientation of molecules within the fibrils indicating that the thyroxine-binding channel lies parallel to the fibre axis.

Publisher Summary This chapter discusses noninvasive investigational techniques for studying clinical bone disease. The interpretation of plasma concentrations of calcium and phosphate has been improved by new concepts of the renal and skeletal handling of these circulating ions. As the surface osteocytes are responsive to parathyroid hormone (PTH) and calcitonin (CT), the bone does not act as a simple sink to soak up excess calcium in the blood or restore that is missing should the level fall. Urinary cyclic AMP, with phosphate, is measured routinely in the Ellsworth–Howard test of renal responsiveness to intravenously administered pure PTH. Collagens are derived from procollagens, precursor molecules to which are attached N-terminal and C-terminal extension peptides that are cleaved by specific endopeptidases prior to fibril formation.