Photoresponsive nanogels formed by the self-assembly of spiropyrane-bearing pullulan that act as artificial molecular chaperones

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation.     

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.     

Type IV collagen prevents amyloid beta-protein fibril formation

The potential of targeting through molecular therapeutics the underlying amyloid beta-protein (A beta) fibrillogenesis causing the initiation and progression of Alzheimer's disease (AD) offers an opportunity to improve the disease. Type IV collagen (collagen IV) is localized in senile plaques in patients with AD. By using thioflavin T fluorescence spectroscopy and electron microscopy, we found that collagen IV inhibited A beta1-40 (A beta40) fibril formation. The critical concentration of collagen IV for this inhibition was 5 microg/mL. Circular dichroism data indicate that collagen IV prevents formation of a beta-structured aggregate of A beta40. These studies demonstrated that collagen IV is apparently a potent inhibitor of A beta fibril formation.     

Protein engineering as a strategy to avoid formation of amyloid fibrils

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.     

Inhibition of amyloid fibril formation and disassembly of pre-formed fibrils by natural polyphenol rottlerin

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ_1–40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC₅₀ and DC₅₀ values were calculated in the range of 1.3–36.4 μM and 15.6–25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ_1–40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.     

Studying the effects of chaperones on amyloid fibril formation

The results of cell and animal model studies demonstrate that molecular chaperones play an important role in controlling the processes of protein misfolding and amyloid formation in vivo. In addition, chaperones are involved in the appearance, propagation and clearance of prion phenotypes in yeast. The effect of chaperones on amyloid formation has been studied in great detail in recent years in order to elucidate the underlying mechanisms. An important approach is the direct study of effects of chaperones on amyloid fibril formation in vitro. This review introduces the methods and techniques that are commonly used to control and monitor the time course of fibril formation, and to detect interactions between chaperones and fibril-forming proteins. The techniques we address include thioflavin T binding fluorescence and filter retardation assays, size-exclusion chromatography, dynamic light scattering, and biosensor assays. Our aim in this review is to provide guidance on how to embark on study of the effect of chaperones on amyloid fibril formation, and how to avoid common problems that may be encountered, using examples and experience from the authors' lab and from the wider literature.     

Quasielastic light scattering study of amyloid beta-protein fibril formation

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Inhibition of amyloid formation

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance.     

Effects of confinement on insulin amyloid fibrils formation

Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. Moreover, the analysis of the initial stages of the fibrillation process in aqueous solutions revealed a large spatial heterogeneity, which is completely absent when the fibrillation is carried out in the hydrogel. We attribute this heterogeneity to the diffusion in solution of large amyloidal aggregates, which must be formed very fast compared to the average times for the whole sample. These findings are interpreted in the framework of recently suggested heterogeneous nucleation mechanisms. Moreover, they may be useful for the development of new insulin pharmaceutical formulations, more stable against adverse conditions.     

Inhibition of familial cerebral amyloid angiopathy mutant amyloid beta-protein fibril assembly by myelin basic protein

Deposition of fibrillar amyloid beta-protein (Abeta) in the brain is a prominent pathological feature of Alzheimer disease and related disorders, including familial forms of cerebral amyloid angiopathy (CAA). Mutant forms of Abeta, including Dutch- and Iowa-type Abeta, which are responsible for familial CAA, deposit primarily as fibrillar amyloid along the cerebral vasculature and are either absent or present only as diffuse non-fibrillar plaques in the brain parenchyma. Despite the lack of parenchymal fibril formation in vivo, these CAA mutant Abeta peptides exhibit a markedly increased rate and extent of fibril formation in vitro compared with wild-type Abeta. Based on these conflicting observations, we sought to determine whether brain parenchymal factors that selectively interact with and modulate CAA mutant Abeta fibril assembly exist. Using a combination of immunoaffinity chromatography and mass spectrometry, we identified myelin basic protein (MBP) as a prominent brain parenchymal factor that preferentially binds to CAA mutant Abeta compared with wild-type Abeta. Surface plasmon resonance measurements confirmed that MBP bound more tightly to Dutch/Iowa CAA double mutant Abeta than to wild-type Abeta. Using a combination of biochemical and ultrastructural techniques, we found that MBP inhibited the fibril assembly of CAA mutant Abeta. Together, these findings suggest a possible role for MBP in regulating parenchymal fibrillar Abeta deposition in familial CAA.     

Inhibition of receptor-mediated endocytosis demonstrates generation of amyloid beta-protein at the cell surface

Sequential cleavages of the amyloid beta-protein precursor (APP) by the beta- and gamma-secretases generate the amyloid beta-protein (A beta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in A beta generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The alpha-secretase cleavage products of APP were increased by dyn K44A, in that alpha-APPs in medium and the C83 C-terminal stub in the membrane both rose. The beta-secretase cleavage of APP, C99, also increased modestly. The use of specific gamma-secretase inhibitors to study the accumulation of alpha- and beta-cleavage products independent of their processing by gamma-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both alpha- and beta-secretases. Unexpectedly, endogenous A beta secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential alpha- and gamma-cleavage) also rose. We conclude that endogenous A beta can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the gamma-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Cholesterol-dependent formation of GM1 ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1/Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. To elucidate the molecular mechanism underlying GM1/Abeta formation, the effects of lipid composition on the binding of Abeta to GM1-containing lipid bilayers were examined in detail using fluorescent dye-labeled human Abeta-(1-40). Increases in not only GM1 but also cholesterol contents in the lipid bilayers facilitated the binding of Abeta to the membranes by altering the binding capacity but not the binding affinity. An increase in membrane-bound Abeta concentration triggered its conformational transition from helix-rich to beta-sheet-rich structures. Excimer formation of fluorescent dye-labeled GM1 suggested that Abeta recognizes a GM1 "cluster" in membranes, the formation of which is facilitated by cholesterol. The results of the present study strongly suggested that increases in intramembrane cholesterol content, which are likely to occur during aging, appear to be a risk factor for amyloid fibril formation.     

Conformational dynamics of amyloid beta-protein assembly probed using intrinsic fluorescence

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.     

Atrial natriuretic peptide deposited as atrial amyloid fibrils

Deposition of amyloid in the atria is exceedingly common in the aging heart. We have extracted amyloid fibrils from atria and purified a major protein which had N-terminal amino acid sequence identical to that of atrial natriuretic peptide (ANP). Antisera to ANP and to the amyloid fibril protein both labelled atrial muscle cells and atrial amyloid in an identical way.     

Evidence for stepwise formation of amyloid fibrils by the mouse prion protein

The full-length mouse prion protein, moPrP, is shown to form worm-like amyloid fibrils at pH 2 in the presence of 0.15 M NaCl, in a slow process that is accelerated at higher temperatures. Upon reduction in pH to 2, native moPrP transforms into a mixture of soluble β-rich oligomers and α-rich monomers, which exist in a slow, concentration-dependent equilibrium with each other. It is shown that only the β-rich oligomers and not the α-rich monomers, can form worm-like amyloid fibrils. The mechanism of formation of the worm-like amyloid fibrils from the β-rich oligomers has been studied with four different physical probes over a range of temperatures and over a range of protein concentrations. The observed rate of fibrillation is the same, whether measured by changes in ellipticity at 216 nm, in thioflavin fluorescence upon binding, or in the mean hydrodynamic radius. The observed rate is significantly slower when monitored by total scattering intensity, suggesting that lateral association of the worm-like fibrils occurs after they form. The activation energy for worm-like fibril formation was determined to be 129 kJ/mol. The observed rate of fibrillation increases with an increase in protein concentration, but saturates at protein concentrations above 50 μM. The dependence of the observed rate of fibrillation on protein concentration suggests that aggregate growth is rate-limiting at low protein concentration and that conformational change, which is independent of protein concentration, becomes rate-limiting at higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. Longer but fewer worm-like fibrils are seen to form at low protein concentration, and shorter but more worm-like fibrils are seen to form at higher protein concentrations. This observation suggests that the β-rich oligomers grow progressively in size to form critical higher order-oligomers from which the worm-like amyloid fibrils then form.     

Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Plasticity of amyloid fibrils

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20th century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloids from the point of view of the polymer chemist may shed new light on a number of issues, such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.     

Metals accelerate the formation and direct the structure of amyloid fibrils of NAC

Non-beta amyloid component of Alzheimer's disease amyloid or NAC is a highly amyloidogenic peptide consisting of 35 amino acids which was first identified associated with senile plaques in the Alzheimer's disease brain. It is a fragment of the presynaptic protein alpha-synuclein and, as such, it is implicated in the aetiologies of both Alzheimer's (AD) and Parkinson's (PD) disease. Metals are involved in the aggregation of amyloidogenic peptides such as beta amyloid (Abeta), British amyloid peptide (ABri) and alpha-synuclein though nothing is yet known about how they might influence the aggregation of NAC. We show herein that NAC will form beta-pleated conformers at a peptide concentration of only 2.0 microM and that metals, and Zn(II) and Cu(II) in particular, accelerate the formation of these fibrils. Cu(II) and Zn(II) did not influence the diameter or general structure of the fibrils which were formed though many more shorter fibrils were observed in their presence and these shorter fibrils were highly thioflavin T positive and they were efficient catalysts of the redox cycling of added Fe(II). By way of contrast, beta-pleated conformers of NAC which were formed in the presence of Al(III) showed much lower levels of thioflavin T fluorescence and were poorer catalysts of the redox cycling of added Fe(II) and these properties were commensurate with an increased abundance of a novel amyloid morphology which consisted of twisted fibrils with a periodicity of about 100 nm. These spirals of twisted fibrils were especially abundant in the presence of added Al(III) and it is speculated that NAC binding of Al(III) may be important in their formation and subsequent stability.