The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

Effects of macromolecular crowding on protein folding and aggregation

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.     

Surface-catalyzed amyloid fibril formation

Light chain (or AL) amyloidosis is characterized by the pathological deposition of insoluble fibrils of immunoglobulin light chain fragments in various tissues, walls of blood vessels, and basement membranes. In the present investigation, the in vitro assembly of a recombinant amyloidogenic light chain variable domain, SMA, on various surfaces was monitored using atomic force microscopy. SMA formed fibrils on native mica at pH 5.0, conditions under which predominantly amorphous aggregates form in solution. Fibril formation was accelerated significantly on surfaces compared with solution; for example, fibrils grew on surfaces at significantly faster rates and at much lower concentrations than in solution. No fibrils were observed on hydrophobic or positively charged surfaces or at pH >7.0. Two novel types of fibril growth were observed on the surface: bidirectional linear assembly of oligomeric units, and linear growth from preformed amorphous cores. In addition to catalyzing the rate of fibrillation, the mechanism of fibril formation on the surfaces was significantly different from in solution, but it may be more physiologically relevant because in vivo the deposits are associated with surfaces.     

Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.     

Effect of surfaces on amyloid fibril formation

Using atomic force microscopy (AFM) we investigated the interaction of amyloid beta (Aβ) (1–42) peptide with chemically modified surfaces in order to better understand the mechanism of amyloid toxicity, which involves interaction of amyloid with cell membrane surfaces. We compared the structure and density of Aβ fibrils on positively and negatively charged as well as hydrophobic chemically-modified surfaces at physiologically relevant conditions. We report that due to the complex distribution of charge and hydrophobicity amyloid oligomers bind to all types of surfaces investigated (CH₃, COOH, and NH₂) although the charge and hydrophobicity of surfaces affected the structure and size of amyloid deposits as well as surface coverage. Hydrophobic surfaces promote formation of spherical amorphous clusters, while charged surfaces promote protofibril formation. We used the nonlinear Poisson-Boltzmann equation (PBE) approach to analyze the electrostatic interactions of amyloid monomers and oligomers with modified surfaces to complement our AFM data.     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

High pressure induces scrapie-like prion protein misfolding and amyloid fibril formation

Our understanding of conformational conversion of proteins in diseases is essential for any diagnostic and therapeutic approach. Although not fully understood, misfolding of the prion protein (PrP) is implicated in the pathogenesis of prion diseases. Despite several efforts to produce the pathologically misfolded conformation in vitro from a recombinant PrP, no positive result has yet been obtained. Within the "protein-only hypothesis", the reason for this hindrance may be that the experimental conditions used did not allow selection of the pathway adopted in vivo resulting in conversion into the infectious form. Here, using a pressure perturbation approach, we show that recombinant PrP is converted to a novel misfolded conformer, which is prone to aggregate and ultimately form amyloid fibrils. A short incubation at high pressure (600 MPa) of the truncated form of hamster prion protein (SHaPrP(90-231)) resulted in the formation of pre-amyloid structures. The mostly globular aggregates were characterized by ThT and ANS binding, and by a beta-sheet-rich secondary structure. After overnight incubation at 600 MPa, amyloid fibrils were formed. In contrast to pre-amyloid structures, they showed birefringency of polarized light after Congo red staining and a strongly decreased ANS binding capacity, but enhanced ThT binding. Both aggregate types were resistant to digestion by PK, and can be considered as potential scrapie-like forms or precursors. These results may be useful for the search for compounds preventing pathogenic PrP misfolding and aggregation.     

Pathways and intermediates of amyloid fibril formation

The lack of understanding of amyloid fibril formation at the molecular level is a major obstacle in devising strategies to interfere with the pathologies linked to peptide or protein aggregation. In particular, little is known on the role of intermediates and fibril elongation pathways as well as their dependence on the intrinsic tendency of a polypeptide chain to self-assembly by β-sheet formation (β-aggregation propensity). Here, coarse-grained simulations of an amphipathic polypeptide show that a decrease in the β-aggregation propensity results in a larger heterogeneity of elongation pathways, despite the essentially identical structure of the final fibril. Protofibrillar intermediates that are thinner, shorter and less structured than the final fibril accumulate along some of these pathways. Moreover, the templated formation of an additional protofilament on the lateral surface of a protofibril is sometimes observed as a collective transition. Conversely, for a polypeptide model with a high β-aggregation propensity, elongation proceeds without protofibrillar intermediates. Therefore, changes in intrinsic β-aggregation propensity modulate the relative accessibility of parallel routes of aggregation.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Freezing of a fish antifreeze protein results in amyloid fibril formation

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.     

Shear flow induces amyloid fibril formation

Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by beta-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native beta-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.     

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.     

The role of protein stability, solubility, and net charge in amyloid fibril formation

Ribonuclease Sa and two charge-reversal variants can be converted into amyloid in vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Implications of macromolecular crowding for protein assembly

Recent studies have led to increased appreciation of the influence of excluded volume in solutions of high total macromolecular content ('macromolecular crowding') upon the various classes of reaction that lead to the assembly of proteins and protein complexes. In general, crowding is expected to stabilize native protein structure relative to less compact non-native structures and to favor the formation of functional complexes of native proteins. Under certain pathological conditions, 'overcrowding' may enhance the formation of nonfunctional aggregates of non-native protein (e.g. amyloid and inclusion bodies).     

Inhibition of protein aggregation and amyloid formation by small molecules

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