The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning-based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of ß-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid ß and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid ß with hydrophobic surfaces in ß-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen-deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary ß-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

Prion Protein Octarepeat Domain Forms Transient ß-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the ß-turn structure, while the binding of additional metal ions induces the formation of ß-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of ß-hairpin structures. As the formation of ß-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.

A Hairpin Motif in the Amyloid-ß Peptide Is Important for Formation of Disease-Related Oligomers.

The amyloid-ß (Aß) peptide is associated with the development of Alzheimer's disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aß oligomers is dependent on the presence of a specific ß-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the ß-strand-forming segments of Aß decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aß aggregation and suggest that inhibition of Aß folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers.

Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro.

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.

Amyloid-ß oligomers are captured by the DNAJB6 chaperone: Direct detection of interactions that can prevent primary nucleation.

A human molecular chaperone protein, DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), efficiently inhibits amyloid aggregation. This inhibition depends on a unique motif with conserved serine and threonine (S/T) residues that have a high capacity for hydrogen bonding. Global analysis of kinetics data has previously shown that DNAJB6 especially inhibits the primary nucleation pathways. These observations indicated that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-ß symbol (Aß) peptide but with aggregated species. However, these pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here, we employed a native MS-based approach to directly detect oligomeric forms of Aß formed in solution. We found that WT DNAJB6 considerably reduces the signals from the various forms of Aß (1-40) oligomers, whereas a mutational DNAJB6 variant in which the S/T residues have been substituted with alanines does not. We also detected signals that appeared to represent DNAJB6 dimers and trimers to which varying amounts of Aß are bound. These data provide direct experimental evidence that it is the oligomeric forms of Aß that are captured by DNAJB6 in a manner which depends on the S/T residues. We conclude that, in agreement with the previously observed decrease in primary nucleation rate, strong binding of Aß oligomers to DNAJB6 inhibits the formation of amyloid nuclei.

Metal ion coordination delays amyloid-ß peptide self-assembly by forming an aggregation-inert complex.

A detailed understanding of the molecular pathways for amyloid-ß (Aß) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer's disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aß peptide and efficiently modulates Aß fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal-ion binding to Aß monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that substoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aß, forming a dynamic, partially compact complex. The metal-ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aß pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, because the bound state is significantly less stable than the amyloid state, Aß peptides are only transiently redirected from fibril formation, and eventually almost all Aß monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aß aggregation via weak metal-ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aß to their effects on bulk aggregation.

Effects of in vivo conditions on amyloid aggregation.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.

Native Ion Mobility-Mass Spectrometry Reveals the Formation of ß-Barrel Shaped Amyloid-ß Hexamers in a Membrane-Mimicking Environment.

The mechanisms behind the Amyloid-ß (Aß) peptide neurotoxicity in Alzheimer's disease are intensely studied and under debate. One suggested mechanism is that the peptides assemble in biological membranes to form ß-barrel shaped oligomeric pores that induce cell leakage. Direct detection of such putative assemblies and their exact oligomeric states is however complicated by a high level of heterogeneity. The theory consequently remains controversial, and the actual formation of pore structures is disputed. We herein overcome the heterogeneity problem by employing a native mass spectrometry approach and demonstrate that Aß(1-42) peptides form coclusters with membrane mimetic detergent micelles. The coclusters are gently ionized using nanoelectrospray and transferred into the mass spectrometer where the detergent molecules are stripped away using collisional activation. We show that Aß(1-42) indeed oligomerizes over time in the micellar environment, forming hexamers with collision cross sections in agreement with a general ß-barrel structure. We also show that such oligomers are maintained and even stabilized by addition of lipids. Aß(1-40) on the other hand form significantly lower amounts of oligomers, which are also of lower oligomeric state compared to Aß(1-42) oligomers. Our results thus support the oligomeric pore hypothesis as one important cell toxicity mechanism in Alzheimer's disease. The presented native mass spectrometry approach is a promising way to study such potentially very neurotoxic species and how they could be stabilized or destabilized by molecules of cellular or therapeutic relevance.

Copper ions induce dityrosine-linked dimers in human but not in murine islet amyloid polypeptide (IAPP/amylin).

Dysregulation and aggregation of the peptide hormone IAPP (islet amyloid polypeptide, a.k.a. amylin) into soluble oligomers that appear to be cell-toxic is a known aspect of diabetes mellitus (DM) Type 2 pathology. IAPP aggregation is influenced by several factors including interactions with metal ions such as Cu(II). Because Cu(II) ions are redox-active they may contribute to metal-catalyzed formation of oxidative tyrosyl radicals, which can generate dityrosine cross-links. Here, we show that such a process, which involves Cu(II) ions bound to the IAPP peptide together with H2O2, can induce formation of large amounts of IAPP dimers connected by covalent dityrosine cross-links. This cross-linking is less pronounced at low pH and for murine IAPP, likely due to less efficient Cu(II) binding. Whether IAPP can carry out its hormonal function as a cross-linked dimer is unknown. As dityrosine concentrations are higher in blood plasma of DM Type 2 patients - arguably due to disease-related oxidative stress - and as dimer formation is the first step in protein aggregation, generation of dityrosine-linked dimers may be an important factor in IAPP aggregation and thus relevant for DM Type 2 progression.

Metal binding to the amyloid-ß peptides in the presence of biomembranes: potential mechanisms of cell toxicity.

The amyloid-ß (Aß) peptides are key molecules in Alzheimer's disease (AD) pathology. They interact with cellular membranes, and can bind metal ions outside the membrane. Certain oligomeric Aß aggregates are known to induce membrane perturbations and the structure of these oligomers-and their membrane-perturbing effects-can be modulated by metal ion binding. If the bound metal ions are redox active, as e.g., Cu and Fe ions are, they will generate harmful reactive oxygen species (ROS) just outside the membrane surface. Thus, the membrane damage incurred by toxic Aß oligomers is likely aggravated when redox-active metal ions are present. The combined interactions between Aß oligomers, metal ions, and biomembranes may be responsible for at least some of the neuronal death in AD patients.

The Neuronal Tau Protein Blocks in Vitro Fibrillation of the Amyloid-ß (Aß) Peptide at the Oligomeric Stage.

In Alzheimer's disease, amyloid-ß (Aß) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aß and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aß40 peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aß40 peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aß40 peptides. The Aß monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aß fibrillation.

Co-aggregation of pro-inflammatory S100A9 with α-synuclein in Parkinson's disease: ex vivo and in vitro studies.

BACKGROUND: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of α-synuclein (α-syn) and S100A9 both in vitro and ex vivo in PD brain. METHODS: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and α-syn location and aggregation. In vitro studies revealing S100A9 and α-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods. RESULTS: Co-localized and co-aggregated S100A9 and α-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 µm outer diameter, with S100A9 and α-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and α-syn were shown to interact with each other via the α-syn C-terminus with an apparent dissociation constant of ca. 5 µM. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of α-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers. CONCLUSIONS: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving α-syn and S100A9 and leading to PD, similar to the effect of S100A9 and Aß co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

Zinc as chaperone-mimicking agent for retardation of amyloid ß peptide fibril formation.

Metal ions have emerged to play a key role in the aggregation process of amyloid ß (Aß) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aß-Zn(2+) binding features as well as how Zn(2+) modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn(2+) effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aß40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn(2+), the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn(2+) transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn(2+) ion, forming a marginally stable, short-lived folded Aß40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aß40-Zn(2+) complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aß40.

Cross-interactions between the Alzheimer Disease Amyloid-ß Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis.

Many protein folding diseases are intimately associated with accumulation of amyloid aggregates. The amyloid materials formed by different proteins/peptides share many structural similarities, despite sometimes large amino acid sequence differences. Some amyloid diseases constitute risk factors for others, and the progression of one amyloid disease may affect the progression of another. These connections are arguably related to amyloid aggregates of one protein being able to directly nucleate amyloid formation of another, different protein: the amyloid cross-interaction. Here, we discuss such cross-interactions between the Alzheimer disease amyloid-ß (Aß) peptide and other amyloid proteins in the context of what is known from in vitro and in vivo experiments, and of what might be learned from clinical studies. The aim is to clarify potential molecular associations between different amyloid diseases. We argue that the amyloid cascade hypothesis in Alzheimer disease should be expanded to include cross-interactions between Aß and other amyloid proteins.

Ionic Strength Modulation of the Free Energy Landscape of Aß40 Peptide Fibril Formation.

Protein misfolding and formation of cross-ß structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid ß peptide (Aß40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates Aß40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different ß-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fß*) and a mature stable state (Fß), where salt favors formation of the Fß fibril morphology. Addition of salt to preformed Fß* accelerates transition to Fß, underlining the dynamic nature of Aß40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aß40 folding to the Fß state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

Self-Assembled Cyclic d,l-α-Peptides as Generic Conformational Inhibitors of the α-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies.

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-ß component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein.

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn(II) < Fe(II) < Ni(II) < Co(II) < Cu(II) > Zn(II)). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn(II) and Fe(II) in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe(II) over Mn(II) as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

Heterogeneity and Turnover of Intermediates during Amyloid-ß (Aß) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy.

Self-assembly of amyloid ß (Aß) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among Aß molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. Aß self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding Aß aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying Aß aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of Aß peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 × 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during Aß aggregation. The effect of different Aß concentrations, Aß:ThT ratios, differences between the 40 (Aß40) and 42 (Aß42) residue long variants of Aß, and the effect of stirring were also examined.

Non-chaperone proteins can inhibit aggregation and cytotoxicity of Alzheimer amyloid ß peptide.

Many factors are known to influence the oligomerization, fibrillation, and amyloid formation of the Aß peptide that is associated with Alzheimer disease. Other proteins that are present when Aß peptides deposit in vivo are likely to have an effect on these aggregation processes. To separate specific versus broad spectrum effects of proteins on Aß aggregation, we tested a series of proteins not reported to have chaperone activity: catalase, pyruvate kinase, albumin, lysozyme, α-lactalbumin, and ß-lactoglobulin. All tested proteins suppressed the fibrillation of Alzheimer Aß(1-40) peptide at substoichiometric ratios, albeit some more effectively than others. All proteins bound non-specifically to Aß, stabilized its random coils, and reduced its cytotoxicity. Surprisingly, pyruvate kinase and catalase were at least as effective as known chaperones in inhibiting Aß aggregation. We propose general mechanisms for the broad-spectrum inhibition Aß fibrillation by proteins. The mechanisms we discuss are significant for prognostics and perhaps even for prevention and treatment of Alzheimer disease.