Process Of Amyloid Formation Research Articles

A Generic Mechanism of Emergence of Amyloid Protofilaments from Disordered Oligomeric Aggregates

The presence of oligomeric aggregates, which is often observed during the process of amyloid formation, has recently attracted much attention because it has been associated with a range of neurodegenerative conditions including Alzheimer's and Parkinson's diseases. We provide a description of a sequence-indepedent mechanism by which polypeptide chains aggregate by forming metastable oligomeric intermediate states prior to converting into fibrillar structures. Our results illustrate that the formation of ordered arrays of hydrogen bonds drives the formation of β-sheets within the disordered oligomeric aggregates that form early under the effect of hydrophobic forces. Individual β-sheets initially form with random orientations and subsequently tend to align into protofilaments as their lengths increase. Our results suggest that amyloid aggregation represents an example of the Ostwald step rule of first-order phase transitions by showing that ordered cross-β structures emerge preferentially from disordered compact dynamical intermediate assemblies.

A Molecular Dynamics Study of the Interaction of d-Peptide Amyloid Inhibitors with Their Target Sequence Reveals a Potential Inhibitory Pharmacophore Conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.

Amyloid-Type Fiber Formation in Control of Enzyme Action: Interfacial Activation of Phospholipase A2

The lag-burst behavior in the action of phospholipase A2 (PLA2) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature Tm of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA2, evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA2, involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form “mature” fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

Altered Dimer Interface Decreases Stability in an Amyloidogenic Protein

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kappaI O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kappaI O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kappaI O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

Thioflavin T fluorescence anisotropy: An alternative technique for the study of amyloid aggregation

The process of amyloid polymerisation raises keen interest in particular because of the biomedical impact of this process. A variety of analytical methods have been developed to monitor amyloid formation. Thioflavin T (ThT) is the most commonly used dye for detection of amyloid aggregation. Nevertheless, ThT fluorescence enhancement is strongly dependent of fibril morphology. In this study using the HET-s prion fibril model, we show that amyloid formation can be monitored by measuring ThT fluorescence anisotropy. Kinetic parameters obtained by this method are identical to those determined by CD spectrometry. We propose that ThT anisotropy represent an interesting, simple and alternative technique to analyze the amyloid formation process.

Initial process of amyloid formation of apomyoglobin and effect of glycosphingolipid GM1

Using small-angle and wide-angle X-ray scattering techniques it has been possible to clarify the helix-to-sheet (cross-β) transition and the stacking process of the cross-β sheet of apomyoglobin as a model for amyloid. The present results indicate that the cross-β formation and the pleated sheet stacking start concurrently and that the stacking continues progressively after the saturation of the cross-β formation. The effect of glycosphingolipids on the above processes has also been studied. At high molar ratio of glycosphingolipid to apomyoglobin the growth of the amyloid is suppressed.

The value of the levels of acute phase reactants for the prediction of familial Mediterranean fever associated amyloidosis: a case control study

In order to determine the role of levels of acute phase proteins (APPs) for the development of amyloidosis in familial Mediterranean fever (FMF) patients, the levels of serum amyloid A (SAA), C reactive protein (CRP), fibrinogen and erythrocyte sedimentation rate were measured in paired sera of 36 FMF patients during and in between acute attacks, 39 of their healthy parents (obligate heterozgotes), and 15 patients with FMF associated amyloidosis. To compare the levels of APPs, 39 patients with chronic infections or inflammatory diseases who may develop secondary amyloidosis, 20 patients with acute infections who are known to have elevated acute phase response but will never develop amyloidosis and 19 healthy controls were included. The median levels of all APPs are increased in the patients with FMF during attacks and a significant decrease was observed after the attack was over. The level of SAA was above reference range in all FMF patients during the attack free period and the level of at least one other APP was also above normal in 64% of the patients. Both CRP and SAA levels were found to be higher in obligate heterozygotes compared to controls. The levels of SAA in patients with FMF during the attack-free period, obligate heterozygotes and patients with FMF-amyloidosis were found to be similar. The levels in each group were found to be higher than SAA levels found in healthy controls yet lower than the levels measured in the patients with acute infections and patients with chronic inflammation or chronic infections. In conclusion, our results show that SAA level reflects subclinical inflammation with high sensitivity but its value for the prediction of amyloid formation process seems to be low.

Shedding a negative image

Although the idea of a protein as an infectious agent emerged almost 40 years ago, the hypothesis only became mainstream after Stanley Prusiner's seminal paper describing how proteins could cause disease (Prusiner, 1982). Prusiner, now a professor of neurology at the University of California, San Francisco, USA, won the 1997 Nobel Prize in Physiology or Medicine for his discovery of these proteinaceous infectious particles, known as prions. After some initial resistance, the scientific community has now largely accepted that prions, by misfolding and aggregating into larger clumps, cause neurodegenerative diseases such as scrapie in sheep, bovine spongiform encephalitis (BSE) in cattle and Creutzfeldt–Jakob Disease (CJD) in humans. However, it is only in the past few years that scientists have begun to understand the molecular mechanisms behind infectious proteins. Recent work on how prions propagate their structural conformation by recruiting other proteins helps to explain the pathology behind the neurodegenerative diseases that they cause, and also expands the concept to other proteins and diseases. As scientists gain a better understanding of prions, these proteins are shedding some of their predominantly negative image as pathological aberrations of nature. In fact, prions might have important biological roles in both prokaryotic and higher organisms. An exciting development came earlier this year with the discovery that the ability of prion particles to spread infection depends on their brittleness (Tanaka et al , 2006). Jonathan Weissman, from the Howard Hughes Medical Institute at the University of California, San Francisco, USA, and colleagues showed that the more brittle—and therefore less stable—prion particles break into smaller fragments more frequently. This creates new seeding particles that, in turn, grow into larger aggregates by recruiting new molecules of the same protein. Weissman and his colleagues conducted their studies on yeast prions; although their ability to cause infection is doubted by many …

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.

Characterization of the Formation of Amyloid Protofibrils from Barstar by Mapping Residue-specific Fluorescence Dynamics

The small protein barstar aggregates at low pH to form soluble oligomers, which can be transformed into fibrillar aggregates at an elevated temperature. To characterize structurally, with residue-specific resolution, the process of amyloid formation of barstar, as well as to monitor the increase in size that accompanies the aggregation process, time-resolved fluorescence anisotropy decay measurements have been introduced as a valuable probe. Seven different single-cysteine-containing mutant forms of barstar were made, to each of which a fluorophore was attached at the thiol group. The rotational dynamics of these seven fluorophores, as well as of the sole intrinsic tryptophan residue in the protein, were determined in the amyloid protofibrils formed, as well as in the soluble oligomers from which the protofibrils arise upon heating. Mapping of the fast rotational dynamics onto the sequence of the protein yields dynamic amplitude maps that allowed identification of the segments of the chain that possess local structure in the soluble oligomer and amyloid protofibrils. The patterns of these maps of the soluble oligomer and protofibrils are seen to be similar; and protofibrils display more local structure than do the soluble oligomers, at all residue positions studied. The observation that transformation from soluble oligomers to protofibrils does not perturb local structure significantly at eight different residue positions, suggests that the soluble oligomers transform directly into protofibrils, without undergoing drastic structural rearrangements.

Small Transthyretin (TTR) Ligands as Possible Therapeutic Agents in TTR Amyloidoses

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.

Rapid formation of amyloid from α‐monomeric recombinant human PrP in vitro

The infectious agent of prion diseases is identified with PrP(Sc), a beta-rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrP(C). To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low-level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP(90-231) in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrP(C)-like state in vitro, that the lag time of the reaction can be further shortened by the presence of a "seed" of pre-formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.

Oxidative Metabolites Accelerate Alzheimer's Amyloidogenesis by a Two-Step Mechanism, Eliminating the Requirement for Nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.

Self-organization of Short Peptide Fragments: From Amyloid Fibrils to Nanoscale Supramolecular Assemblies

Numerous supramolecular protein assemblies had been demonstrated to have either physiological or pathological activities. The most significant case of disease-associated self-organized structures is that of amyloid fibrils. The formation of these fibrils is the hallmark of major human disorders, including Alzheimer's disease and type II diabetes. In this review we illustrate the molecular properties of the amyloid fibrils as supramolecular assemblies in the nanometric scale. We present recent advances in the elucidation of the molecular recognition and self-assembly processes that lead to the formation of these toxic structures, and we describe how the mechanistic study of amyloid formation process has led to unexpected discoveries of peptide-based nanostructures.

Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic β-cells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.

Cognitive impairment in PDAPP mice depends on ApoE and ACT-catalyzed amyloid formation

Biochemical and genetic studies indicate that the inflammatory proteins, apolipoprotein E (ApoE) and α 1-antichymotrypsin (ACT) are important in the pathogenesis of Alzheimer’s disease (AD). Using several lines of multiply transgenic/knockout mice we show here that murine ApoE and human ACT separately and synergistically facilitate both diffuse Aβ immunoreactive and fibrillar amyloid deposition and thus also promote cognitive impairment in aged PDAPP(V717F) mice. The degree of cognitive impairment is highly correlated with the ApoE- and ACT-dependent hippocampal amyloid burden, with PDAPP mice lacking ApoE and ACT having little amyloid and little learning disability. A analysis of young mice before the onset of amyloid formation shows that steady-state levels of monomeric Aβ peptide are unchanged by ApoE or ACT. These data suggest that the process or product of amyloid formation is more critical than monomeric Aβ for the neurological decline in AD, and that the risk factors ApoE and ACT participate primarily in disease processes downstream of APP processing.

Exploring amyloid formation by a de novo design.

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.

Formation of amyloid in human pancreatic islets transplanted to the liver and spleen of nude mice

In previous studies we have shown that apparently normal human islets, transplanted under the renal capsule of nude mice, frequently and rapidly develop amyloid deposits derived from the beta-cell hormone islet amyloid polypeptide (IAPP). In the present study, we show for the first time that human islets, transplanted into the liver or spleen of nude mice, also develop islet amyloid rapidly. Ultrastructural studies of such islets showed that the first aggregation of IAPP takes place within the beta-cells and that extracellular deposits show up later in the amyloid formation process. We also found that the amount of amyloid formed in human islet grafts placed under the kidney capsule increased with extended (26 weeks) observation time. Moreover, prolonged in vitro culture (14 days) prior to the implantation under the renal capsule seemed to enhance the formation of amyloid in the grafted islets. Since aggregated IAPP has been shown to be toxic to beta-cells, the finding of amyloid deposits in transplanted islets offers a possible explanation to the frequent loss of function of islets transplanted into diabetic patients.

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide.

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

Can serum amyloid A or macrophage colony stimulating factor serve as marker of amyloid formation process?

Amyloid formation depends on amyloid precursor production and is influenced by the activity of the underlying disorder and mediated by some proinflammatory cytokines. In this pilot study we tried to find some specific markers that could establish the activity of the disease. We investigated 45 samples of sera and 38 samples of urine from patients (pts) with secondary amyloidosis (AA), primary amyloidosis (AL), systemic autoimmune diseases with renal impairment (Vasc) and healthy controls (Co). Pts with AA had increased plasma levels of TNF alpha (9.97 +/- 4.22 vs. 2.63 +/- 1.34 pg/mL, p < 0.001) and SAA (43.14 +/- 16.0 vs. 3.42 +/- 0.7 ng/mL, p < 0.05) in comparison with Co. Plasma levels of M-CSF in the AA group were significantly increased in comparison with Co (1077.34 +/- 238.6 vs. 137.71 +/- 19.6, pg/mL, p < 0.001) and also in comparison with Vasc (482.24 +/- 86.7 pg/mL, p < 0.05). Urinary excretions of TNF alpha (8.92 +/- 8.1 vs. 0.17 +/- 0.11 microgram/mol creatinine, p < 0.01), sIL-6R (1.39 +/- 1.14 vs. 0.07 +/- 0.05 g/mol creatinine, p < 0.01) and M-CSF (650.2 +/- 153.7 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in AA were significantly increased in comparison with Co. Pts with AL had increased plasma levels of M-CSF (819.83 +/- 264.2 vs. 137.71 +/- 19.6 pg/mL, p < 0.05) and urinary excretion of M-CSF (865.0 +/- 188.4 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in comparison with Co. SAA has a low specificity for amyloidosis but is a sensitive acute phase reactant. TNF alpha, a proinflammatory cytokine, may reflect the activity of the underlying diseases in secondary amyloidosis. M-CSF was increased both in plasma and urine in amyloidosis groups and seems to be the most promising (possibly specific) marker of amyloidosis.