Review: modulating factors in amyloid-beta fibril formation

Amyloid formation is a key pathological feature of Alzheimer's disease and is considered to be a major contributing factor to neurodegeneration and clinical dementia. Amyloid is found as both diffuse and senile plaques in the parenchyma of the brain and is composed primarily of the 40- to 42-residue amyloid-beta (Abeta) peptides. The characteristic amyloid fiber exhibits a high beta-sheet content and may be generated in vitro by the nucleation-dependent self-association of the Abeta peptide and an associated conformational transition from random to beta-conformation. Growth of the fibrils occurs by assembly of the Abeta seeds into intermediate protofibrils, which in turn self-associate to form mature fibers. This multistep process may be influenced at various stages by factors that either promote or inhibit Abeta fiber formation and aggregation. Identification of these factors and understanding the driving forces behind these interactions as well as the structural motifs necessary for these interactions will help to elucidate potential sites that may be targeted to prevent amyloid formation and its associated toxicity. This review will discuss some of the modulating factors that have been identified to date and their role in fibrillogenesis.     

Small-molecule aggregates inhibit amyloid polymerization

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.     

Chemical colloids versus biological colloids: a comparative study for the elucidation of the mechanism of protein fiber formation

Fiber formation from murine serum amyloid A1 (SAA) was compared to the linear aggregation and fiber formation of colloidal gold particles. Here we report the similarities of these processes. Upon incubation with acetic acid, SAA misfolds and adopts a new conformation, which we termed saa. saa apparently is less soluble than SAA in aqueous solution; it aggregates and forms nucleation units and then fibers. The fibers appear as a string of the nucleation units. Additionally, an external electric field promotes saa fiber formation. These properties of saa are reminiscent of colloidal gold formation from gold ions and one-dimensional aggregation of the gold colloids. Colloidal gold particles were also found to be capable of aggregating one-dimensionally under an electric field or in the presence of polylysine. These gold fibers resembled in structure that of saa fibers. In summary, protein aggregation and formation of fibers appear to follow the generalized principles derived in colloidal science for the aggregation of atoms and molecules, including polymers such as polypeptides. The analysis of colloidal gold formation and of one-dimensional aggregation provides a simple model system for the elucidation of some aspects of protein fiber formation.     

In vitro polymerization of a functional Escherichia coli amyloid protein

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.     

Small molecule screening in context: Lipid-catalyzed amyloid formation

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the β-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemer’s and Parkinson’s) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid-catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.     

Fiber-Dependent and -Independent Toxicity of Islet Amyloid Polypeptide

The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. Although its amyloid fiber aggregation kinetics and cytotoxicity to β-cells are well documented, few reports have directly assessed the role of fibers in cell-based toxicity experiments. Here, we report that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells. For example, fiber formation is more strongly suppressed in cell culture medium than in aqueous buffer. The serum component of the medium is responsible for this inhibition. Although amyloid formation was previously shown to be catalyzed by both synthetic and chloroform-extracted phospholipid surfaces, it is instead inhibited by membrane surfaces prepared directly from the plasma membranes of an immortal β-cell line. This disparity is reconciled by direct assessment of fibers in cell-culture-based toxicity experiments. We discovered that fibers are nontoxic if they are washed free of adsorbed nonfibrillar components. Moreover, toxicity is not only rescued when monomers are added back to fibers but is greater than what is observed from the precursor alone. Our results are interpreted in light of the capacity of the fiber surface to template amyloid nucleation.     

From chance to frequent encounters: origins of beta2-microglobulin fibrillogenesis

It is generally accepted that amyloid formation requires partial, but not complete unfolding of a polypeptide chain. Amyloid formation by beta-2 microglobulin (beta2m), however, readily occurs under strongly native conditions provided that there is exposure to specific transition metal cations. In this review, we discuss transition metal catalyzed conformational changes in several amyloidogenic systems including prion protein, Alzheimer's and Parkinson's diseases. For some systems, including beta2m from dialysis related amyloidosis (DRA), catalysis overcomes an entropic barrier to protein aggregation. Recent data suggest that beta2m samples conformations that are under thermodynamic control, resulting in local or partial unfolding under native conditions. Furthermore, exposure to transition metal cations stabilizes these partially unfolded states and promotes the formation of small oligomers, whose structures are simultaneously near-native and amyloid-like. By serving as a tether, Cu(2+) enables the encounter of amyloidogenic conformations to occur on time scales which are significantly more rapid than would occur between freely diffusing monomeric protein. Once amyloid formation occurs, the requirement for Cu(2+) is lost. We assert that beta2m amyloid fiber formation at neutral pH may be facilitated by rearrangements catalyzed by the transient and pair wise tethering of beta2m at the blood/dialysate interface present during therapeutic hemodialysis.     

Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.     

Semi-automated methods for simulation and measurement of amyloid fiber distributions obtained from transmission electron microscopy experiments

Transmission electron microscopy (TEM) is the standard procedure for qualitatively confirming the presence of amyloid fibers in a protein aggregation reaction product. However, extracting quantitative information about the amyloid size distribution from the electron micrographs is a nontrivial problem. Here we describe methods for (i) the simulation of pseudo-TEM images of amyloid fiber distributions having known characteristic properties and (ii) the semi-automated processing of experimental TEM images of amyloid fibers to produce two-dimensional histogram plots reflecting either the distribution of amyloid length and width or, alternatively, the distribution of width and fiber rigidity/persistence. The processing method is fully automatic when the density of fibers on the grid is sufficiently low (such that the adsorbed fibers do not touch) and is semi-automatic (requiring some user decision making) when the fibers are overlapping. Termed "ADM" (for Amyloid Distribution Measurement), the program suite is written in MATLAB code and is available on request from the author.     

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.     

The Role of Functional Amyloids in Bacterial Virulence

Amyloid fibrils are best known as a product of human and animal protein misfolding disorders, where amyloid formation is associated with cytotoxicity and disease. It is now evident that for some proteins, the amyloid state constitutes the native structure and serves a functional role. These functional amyloids are proving widespread in bacteria and fungi, fulfilling diverse functions as structural components in biofilms or spore coats, as toxins and surface-active fibers, as epigenetic material, peptide reservoirs or adhesins mediating binding to and internalization into host cells. In this review, we will focus on the role of functional amyloids in bacterial pathogenesis. The role of functional amyloids as virulence factor is diverse but mostly indirect. Nevertheless, functional amyloid pathways deserve consideration for the acute and long-term effects of the infectious disease process and may form valid antimicrobial targets.     

Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct

Alzheimer disease is characterized by the abnormal aggregation of amyloid beta peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid beta oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid beta aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid beta monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.     

From chance to frequent encounters: Origins of β2-microglobulin fibrillogenesis

It is generally accepted that amyloid formation requires partial, but not complete unfolding of a polypeptide chain. Amyloid formation by β-2 microglobulin (β2m), however, readily occurs under strongly native conditions provided that there is exposure to specific transition metal cations. In this review, we discuss transition metal catalyzed conformational changes in several amyloidogenic systems including prion protein, Alzheimer's and Parkinson's diseases. For some systems, including β2m from dialysis related amyloidosis (DRA), catalysis overcomes an entropic barrier to protein aggregation. Recent data suggest that β2m samples conformations that are under thermodynamic control, resulting in local or partial unfolding under native conditions. Furthermore, exposure to transition metal cations stabilizes these partially unfolded states and promotes the formation of small oligomers, whose structures are simultaneously near-native and amyloid-like. By serving as a tether, Cu²⁺ enables the encounter of amyloidogenic conformations to occur on time scales which are significantly more rapid than would occur between freely diffusing monomeric protein. Once amyloid formation occurs, the requirement for Cu²⁺ is lost. We assert that β2m amyloid fiber formation at neutral pH may be facilitated by rearrangements catalyzed by the transient and pair wise tethering of β2m at the blood/dialysate interface present during therapeutic hemodialysis.     

Insulin transport within skeletal muscle transverse tubule networks

It has recently been observed in situ in mice that insulin takes approximately 10 min to be transported 20 microm into the t-tubule networks of skeletal muscle fibers. The mechanisms for this slow transport are unknown. It has been suggested that the biochemical composition of the t-tubular space that may include large molecules acting as gels and increased viscosity in the narrow tubules may explain this slow diffusion. In this article, we construct a mathematical model of insulin transport within the t-tubule network to determine potential mechanisms responsible for this slow insulin transport process. Our model includes insulin diffusion, insulin binding to insulin receptors, t-tubule network tortuosity, interstitial fluid viscosity, hydrodynamic wall effects, and insulin receptor internalization and recycling. The model predicted that depending on fiber type there is a 2-15 min delay in the arrival time of insulin between the sarcolemma and inner t-tubules (located 20 microm from the sarcolemma) after insulin injection. This is consistent with the experimental data. Increased viscosity in the narrow t-tubules and large molecules acting as gels are not the primary mechanisms responsible for the slow insulin diffusion. The primary mechanisms responsible for the slow insulin transport are insulin binding to insulin receptors and network tortuosity.     

Signaling in morphogenesis: transport cues in morphogenesis

Extracellular transport processes play critical roles in morphogenesis. While diffusive transport effects on morphogenesis are well illustrated in examples like blood capillary architecture and in cell morphogenetic responses to the local extracellular protein environment, the effects of fluid convection, although important in many developing and regenerating tissues, are not well understood. Convective forces are present whenever a hydrated tissue undergoes dynamic mechanical strain, and so convection could not only dominate the transport of large molecules like proteins, but might also serve as a mechanism for mechanosensing. The complex interdependence of mechanical forces, protein transport and extracellular morphogen gradients needs to be elucidated in a comprehensive way in order for the importance of transport on morphogenesis to be fully appreciated.     

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20 μM). Importantly, when the 5 μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.     

Inhibition of amyloidosis using low-molecular-weight heparins

BACKGROUND: Amyloid diseases are characterized by the tissue deposition of extracellular proteinaceous material, which results in organ dysfunction and death. Colocalization of heparan sulfate (HS) proteoglycans to amyloid deposits suggests that they may be an early event in amyloid formation and play an important role in fibril formation. Structural analysis has demonstrated that HS interacts with amyloidogenic proteins resulting in structural changes that allow for an increase in beta-sheet content, possibly enhancing fibrillogenesis. Recent studies have shown that small-molecule anionic sulfonates or sulfates can arrest inflammation-associated (AA) amyloid induction. MATERIALS AND METHODS: In the present study, we examine the effect of low-molecular-weight heparins (LMWHs) on the development of amyloid in the mouse model of AA amyloid. In addition, in vitro fibril formation assays were performed to determine the effect of LMWHs on fibrillogenesis. RESULTS: Injection of mice with clinically relevant doses of LMWHs (enoxaparin and dalteparin) demonstrated a reduction in AA amyloid deposition. These compounds were capable of arresting the progression of AA amyloid and eventually resulting in regression of the amyloid deposits. In vitro analysis indicated that LMWHs prevented AA and Abeta peptide fibril formation by impeding the structural changes necessary for fibril formation. CONCLUSIONS: Our findings suggest that the LMWHs may provide beneficial effects against the development of amyloidoses, including Alzheimer's disease.     

Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters

To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.     

The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation?

Amyloidosis is a class of diseases caused by protein aggregation and deposition in various tissues and organs. In this paper, a yeast amyloid-forming protein Sup35 was used as a model for understanding amyloid fiber formation. The dynamics of amyloid formation by Sup35 were studied with scanning force microscopy. We found that: 1) the assembly of Sup35 fibers begins with individual NM peptides that aggregate to form large beads or nucleation units which, in turn, form dimers, trimers, tetramers and longer linear assemblies appearing as a string of beads; 2) the morphology of the linear assemblies differ; and 3) fiber assembly suggests an analogy to the aggregation of colloidal particles. A dipole assembly model is proposed based on this analogy that will allow further experimental testing.     

In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease beta-protein

Two synthetic peptides corresponding to the reported 28-residue sequence of Alzheimer's Disease beta-protein (SP28) and to residues 12-28 (SP17) were used to form fibrils in vitro. Synthetic fibrils bound Congo Red and closely resembled amyloid fibrils isolated from leptomeninges and senile plaques of Alzheimer's brain by electron microscopy. A polyclonal antiserum to SP28 specifically decorated both synthetic and native amyloid by colloidal gold immunoelectron microscopy. Amyloid fibrils isolated from tissue were insoluble on SDS-Polyacrylamide gels, and tended to aggregate while synthetic amyloid fibrils were completely solubilized, releasing only monomers of SP28 and SP17. Anti-SP28 immunostained cerebrovascular and plaque core amyloid, but not neurofibrillary tangles, in tissue section. Western blot analysis showed that anti-SP28 reacted with a 4 kDa band released from amyloid core-enriched preparations and leptomeninges. By contrast, a 16 kDa band corresponding to the tetramer of beta-protein was not recognized. These data suggest that as little as a 17 residue sequence of beta-protein may be required to form fibrils and that the complete sequence of the 4 kDa beta-protein may be important in determining insolubility and the formation of intermediate size polymers.