Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer''s disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.     

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Str?ussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6']bibenzothiazolyl-2,2'-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.     

Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH and a proteolysis mechanism leading to the formation of fibrils.

Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions.     

1H, 13C, and 15N resonance assignments for human regenerating family Iα protein

Human regenerating (Reg) genes belong to the C-type lectin superfamily and express secretory proteins in various tissues. Reg Iα, also named lithostathine, has multiple roles in numerous biological events such as cytokines, anti-apoptotic factors and the calcium carbonate crystals inhibitor. Under physiological pH, Reg Iα becomes largely insoluble after a self-proteolysis process, and the N-terminally truncated form readily polymerizes into fibrils, which leads to neurodegenerative diseases. Reg Iα may form protofibril via lateral hydrophobic interactions with a native-like conformation. The structural basis from the native to fibril form, as well as the carbohydrate binding sites on Reg Iα, remain unknown. Here we present the NMR backbone and side-chain assignments of Reg Iα for use in further NMR investigations.     

Sarcomeric proteins of the titin family form amyloids

It was shown for the first time that skeletal muscle sarcomeric proteins of the titin family (X-, C- and H-proteins) are able to form in vitro amyloid aggregates of different types: granular aggregates, protofibrils, helically twisted ribbons, linear fibrils, and bundles of linear fibrils. Their amyloid nature was confirmed by electron, polarization, and fluorescence microscopy and by spectral methods. As opposed to other amyloidogenic proteins, X-, C-, and H-proteins easily form amyloids under mild conditions close to physiological ones (pH, ionic strength, temperature). Like amyloid fibrils of Abeta-peptide and tau protein in Alzheimer's disease, amyloid aggregates formed by X-, C-, and H-proteins are destroyed by the antibiotic tetracycline. Thus, new proteins-precursors of amyloids and possible participants of amyloidoses in muscles were discovered. Further study of in vitro amyloidogenesis of these proteins would help to find approaches to controlling this process in organs and tissues.     

Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion

Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.     

Lipid-induced beta-amyloid peptide assemblage fragmentation

Alzheimer's disease is the most common cause of dementia and is widely believed to be due to the accumulation of beta-amyloid peptides (Abeta) and their interaction with the cell membrane. Abetas are hydrophobic peptides derived from the amyloid precursor proteins by proteolytic cleavage. After cleavage, these peptides are involved in a self-assembly-triggered conformational change. They are transformed into structures that bind to the cell membrane, causing cellular degeneration. However, it is not clear how these peptide assemblages disrupt the structural and functional integrity of the membrane. Membrane fluidity is one of the important parameters involved in pathophysiology of disease-affected cells. Probing the Abeta aggregate-lipid interactions will help us understand these processes with structural detail. Here we show that a fluid lipid monolayer develop immobile domains upon interaction with Abeta aggregates. Atomic force microscopy and transmission electron microscopy data indicate that peptide fibrils are fragmented into smaller nano-assemblages when interacting with the membrane lipids. Our findings could initiate reappraisal of the interactions between lipid assemblages and Abeta aggregates involved in Alzheimer's disease.     

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high beta-content, and proteins with significant native alpha-helical structure show increased beta-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.     

Inhibition of beta-amyloid peptide aggregation and neurotoxicity by alpha-d-mannosylglycerate, a natural extremolyte

The aggregation of soluble beta-amyloid (Abeta) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. alpha-d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 degrees C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Abeta aggregation and neurotoxicity to human neuroblastoma cells. According to our study--carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay--MG had significant inhibitory effect against Abeta amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as alpha-d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Abeta aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.     

A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.     

Alpha-helix structure in Alzheimer's disease aggregates of tau-protein

The discovery of beta-sheet structure in Alzheimer's amyloid fibrils, and then in many other disease-related protein fibrils, has led to the widely believed view that beta-sheet formation is the general mechanism of aberrant protein aggregation leading to disease. This notion is further reinforced by recent findings, which indicate that normal proteins can be induced to form beta-sheet fibrils in vitro. Alzheimer's disease, a paradigm proteopathy, is accompanied by the formation of two distinct aggregates, amyloid fibrils and paired helical filaments (PHFs). Electron microscope images of PHFs show pairs of twisted ribbons with 80 nm periodicity. However, there is little information of the molecular structure of PHFs, as previous studies have failed to identify signs of regular structure. Using far-UV circular dichroism and Fourier-transformed infrared spectroscopy, we find that PHFs are comprised of alpha-helices. This is remarkable as tau-protein, PHF's primary constituent, has a high abundance of helix-breaking amino acids and is unstructured in solution. We also find that PHFs are very stable, as judged by their high melting temperature and resistance to protease digestion. PHFs are the first example of pathological aggregation associated to the formation of alpha-helix.     

Aggregation of truncated GST-HD exon 1 fusion proteins containing normal range and expanded glutamine repeats.

We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al. (1997) Cell 90, 549-558). Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca. 25 nm. In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment.     

Correlations among morphology, beta-sheet stability, and molecular structure in prion peptide aggregates

The misfolding of proteins into beta-sheets and the subsequent aggregation of these sheets into fibrous networks underlies many diseases. In this paper, the role of peptide structure in determining the ordering of beta-sheet aggregates and the morphology of fibrils and protofibrils is dissected. Using a series of peptides based on residues 109-122 of the Syrian hamster prion protein (H1) with a range of substitutions at position 117, the link between side chain interactions and beta-sheet thermal stability has been investigated. The thermal stability of beta-sheets is associated with the peptides' ability to adopt the same alignment as wild-type H1, with residue 117 in register across all beta-strands [Silva, R. A. G. D., Barber-Armstrong, W., and Decatur, S. M. (2003) J. Am. Chem. Soc. 125, 13674-13675]. These aligned strands are capable of forming long, rigid, and twisted fibrils (as visualized by atomic force microscopy) which are thermostable. Peptides which do not adopt this strand alignment aggregate to form thin, flexible, and smooth protofibrils. The ability to form ordered aggregates, and thus to form twisted fibrils, is modulated by the structure of the side chain of residue 117.     

Conformational Conversion May Precede or Follow Aggregate Elongation on Alternative Pathways of Amyloid Protofibril Formation

A major goal in the study of protein aggregation is to understand how the conformational heterogeneity characteristic of the process leads to structurally distinct amyloid fibrils. The small protein barstar is known to form amyloid protofibrils in multiple steps at low pH: a small oligomer, the A-form, first transforms into a larger spherical higher oligomeric intermediate (HOI), which then self-associates to form the elongated protofibril. To determine how the conformational conversion reaction during aggregation is coupled to the process of protofibril formation, cysteine-scanning mutagenesis was first used to identify specific residue positions in the protein sequence, which are important in defining the nature of the aggregation process. Two classes of mutant proteins, which are distinguished by their kinetics of aggregation at high protein concentration, have been identified: Class I mutant proteins undergo conformational conversion, as measured by an increase in thioflavin T binding ability and an increase in circular dichroism at 216 nm, significantly faster than Class II mutant proteins. At low protein concentration, the rates of conformational conversion are, however, identical for both classes of mutant proteins. At high protein concentration, the two classes of mutant proteins can be further distinguished on the basis of their rates of protofibril growth, as determined from dynamic light-scattering measurements. For Class I mutant proteins, protofibril elongation occurs at the same, or slightly faster, rate than conformational conversion. For Class II mutant proteins, protofibril elongation is significantly slower than conformational conversion. Dynamic light scattering measurements and atomic force microscopy imaging indicate that for the Class I mutant proteins, conformational conversion occurs concurrently with the self-association of prefibrillar HOIs into protofibrils. On the other hand, for the Class II mutant proteins, the prefibrillar HOI first undergoes conformational conversion, and the conformationally converted HOIs then self-associate to form protofibrils. The two classes of mutant proteins appear, therefore, to use structurally distinct pathways to form amyloid protofibrils. On one pathway, conformational conversion occurs along with, or after, elongation of the oligomers; on the other pathway, conformational conversion precedes elongation of the oligomers. Single mutations in the protein can cause aggregation to switch from one pathway to the other. Importantly, the protofibrils formed by the two classes of mutant proteins have significantly different diameters and different internal structures.     

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.     

Role of oxidative carbonylation in protein quality control and senescence

Proteins can become modified by a large number of reactions involving reactive oxygen species. Among these reactions, carbonylation has attracted a great deal of attention due to its irreversible and unrepairable nature. Carbonylated proteins are marked for proteolysis by the proteasome and the Lon protease but can escape degradation and form high-molecular-weight aggregates that accumulate with age. Such carbonylated aggregates can become cytotoxic and have been associated with a large number of age-related disorders, including Parkinson's disease, Alzheimer's disease, and cancer. This review focuses on the generation of and defence against protein carbonyls and speculates on the potential role of carbonylation in protein quality control, cellular deterioration, and senescence.     

Protofibril assemblies of the arctic, Dutch, and Flemish mutants of the Alzheimer's Abeta1-40 peptide

Using a coarse-grained model of the Aβ peptide, we analyze the Arctic (E22G), Dutch (E22Q), and Flemish (A21G) familial Alzheimer's disease (FAD) mutants for any changes in the stability of amyloid assemblies with respect to the wild-type (WT) sequence. Based on a structural reference state of two protofilaments aligned to create the “agitated” protofibril as determined by solid-state NMR, we determine free energy trends for Aβ assemblies for the WT and FAD familial sequences. We find that the structural characteristics and oligomer size of the critical nucleus vary dramatically among the hereditary mutants. The Arctic mutant's disorder in the turn region introduces new stabilizing interactions that better align the two protofilaments, yielding a well-defined protofibril axis at relatively small oligomer sizes with respect to WT. By contrast, the critical nucleus for the Flemish mutant is beyond the 20 chains characterized in this study, thereby showing a strong shift in the equilibrium toward monomers with respect to larger protofibril assemblies. The Dutch mutant forms more ordered protofilaments than WT, but exhibits greater disorder in protofibril structure that includes an alternative polymorph of the WT fibril. An important conclusion of this work is that the Dutch mutant does not support the agitated protofibril assembly. We discuss the implications of the structural ensembles and free energy profiles for the FAD mutants in regards to interpretation of the kinetics of fibril assembly using chromatography and dye-binding experiments.     

Physiochemical characterization of the Alzheimer's disease-related peptides A beta 1-42Arctic and A beta 1-42wt

The amyloid beta peptide (A beta) is crucial for the pathogenesis of Alzheimer's disease. Aggregation of monomeric A beta into insoluble amyloid fibrils proceeds through several soluble A beta intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimer's disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimer's disease, and synthetic A beta 1-40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887-893]. To increase our understanding of the mechanisms involved in A beta aggregation, especially A beta monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of A beta 1-42wt and A beta 1-42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both A beta 1-42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes - monomer oligomerization and protofibril fibrillization - were affected differently by changes in the micro-environment and that the Arctic mutation alters the peptide response to such changes.