Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition

β-Amyloid Is Different in Normal Aging and in Alzheimer Disease

The mechanism of neurodegeneration caused by beta-amyloid in Alzheimer disease is controversial. Neuronal toxicity is exerted mostly by various species of soluble beta-amyloid oligomers that differ in their N- and C-terminal domains. However, abundant accumulation of beta-amyloid also occurs in the brains of cognitively normal elderly people, in the absence of obvious neuronal dysfunction. We postulated that neuronal toxicity depends on the molecular composition, rather than the amount, of the soluble beta-amyloid oligomers. Here we show that soluble beta-amyloid aggregates that accumulate in Alzheimer disease are different from those of normal aging in regard to the composition as well as the aggregation and toxicity properties

Detection of Populations of Amyloid-Like Protofibrils with Different Physical Properties

We used tapping mode atomic force microscopy to study the morphology of the amyloid protofibrils formed at fixed conditions (low pH with high ionic strength) by self-assembly of the N-terminal domain of the hydrogenase maturation factor HypF. Although all protofibrils in the sample share a beaded structure and similar values of height and width, an accurate analysis of contour length and end-to-end distance and the comparison of experimental data with theoretical predictions based on the worm-like chain model show that two different populations of protofibrils are present. These populations are characterized by different physical properties, such as persistence length, bending rigidity and Young's modulus. Fluorescence quenching measurements on earlier globular intermediates provide an independent evidence of the existence of different populations. The finding that differences in mechanical properties exist even within the same sample of protofibrils indicates the presence of different subpopulations of prefibrillar aggregates with potentially diverse tendencies to react with undesired molecular targets. This study describes a strategy to discriminate between such different subpopulations that are otherwise difficult to identify with conventional analyses

Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate: FASTER UNFOLDING AND CHANGE OF PATHWAY*

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and β-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis

Amyloid Formation of a Protein in the Absence of Initial Unfolding and Destabilization of the Native State

In 5% (v/v) trifluoroethanol, pH 5.5, 25°C one of the acylphosphatases from Drosophila melanogaster (AcPDro2) forms fibrillar aggregates that bind thioflavin T and Congo red and have an extensive β-sheet structure, as revealed by circular dichroism. Atomic force microscopy indicates that the fibrils and their constituent protofilaments have diameters compatible with those of natural amyloid fibrils. Spectroscopic and biochemical investigation, carried out using near- and far-UV circular dichroism, intrinsic and 1-anilino-8-naphthalenesulfonic acid-derived fluorescence, dynamic light scattering, and enzymatic activity assays, shows that AcPDro2 has, before aggregation, a secondary structure content packing around aromatic and hydrophobic residues, hydrodynamic diameter, and catalytic activity indistinguishable from those of the native protein. The native protein was found to have the same conformational stability under native and aggregating conditions, as determined from urea-induced unfolding. The kinetic analysis supports models in which AcPDro2 aggregates initially without need to unfold and subsequently undergoes a conformational change into amyloid-like structures. Although fully or partially unfolded states have a higher propensity to aggregate, the residual aggregation potential that proteins maintain upon complete folding can be physiologically relevant and be directly involved in the pathogenesis of some protein deposition diseases

Agitation and High Ionic Strength Induce Amyloidogenesis of a Folded PDZ Domain in Native Conditions

Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37°C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular β-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (ΔGU-FH2O). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation

Natively Folded HypF-N and Its Early Amyloid Aggregates Interact with Phospholipid Monolayers and Destabilize Supported Phospholipid Bilayers

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2–3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability