Histone H1 Favors Folding and Parallel Fibrillar Aggregation of the 1

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Histone H1 favors folding and parallel fibrillar aggregation of the 1-42 amyloid-# peptide Alicia Roque, Rosalba Sortino, Salvador Ventura, Inma Ponte, and Pedro Suau Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504089g • Publication Date (Web): 29 May 2015 Downloaded from http://pubs.acs.org on June 3, 2015

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Histone H1 favors folding and parallel fibrillar aggregation of the 1-42 amyloid-β peptide. Alicia Roque1, Rosalba Sortino1, Salvador Ventura 1,2, Inma Ponte1 and Pedro Suau1*

1

Departamento de Bioquímica y Biología Molecular, Facultad de

Biociencias, Universidad Autónoma de Barcelona. 2

Instituto de Biotecnología y de Biomedicina. Universidad Autónoma de

Barcelona * corresponding author

ACS Paragon Plus Environment

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ABSTRACT Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-β peptide, Aβ(1-42), is believed to play an important role in the pathogenesis of AD. Histone H1 is found in the cytoplasm of neurons in AD, and it has been shown to interact with aggregated amyloid-β peptides and with amyloid fibrils. We have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism spectroscopy (CD), co-precipitation and transmission electron microscopy (TEM) to study the interaction of histone H1 with Aβ(1-42). Both freshly prepared (monomeric) Aβ(1-42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aβ(1-42) and histone H1 led to the loss of the random coil, which was replaced mostly by β-structure. Therefore, both Aβ(1-42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aβ(142) and histone H1. The interaction was confirmed by co-precipitation followed by SDS-PAGE. Mutual structure induction was also observed with the H1 terminal domains. Incubation of Aβ(1-42) for one week in the presence of histone H1 lead to the formation of laminar aggregates and thick bundles, characterized by the parallel association of large numbers of fibrils. The aggregates were particularly large and ordered with the H1 subtype H1.2. Further ageing of the complexes led to tight compaction of fibril bundles and to fiber growth. Stabilization of fibril-fibril interactions appeared to be determined by the C-terminal domain of histone H1. In summary, these observations indicate that histone H1 has at least two effects: it helps the folding of Aβ monomers and stabilizes the parallel association of fibrils.


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INTRODUCTION Many degenerative diseases result from the presence of protein molecules that are misfolded. A number of relevant degenerative pathologies, referred to as amyloidosis1, are characterized by the aggregation of the misfolded proteins into oligomers and polymers. The latter eventually ends in the appearance of ordered fibrils that become deposited intra- or extracellularly, forming what is commonly known as amyloid. Amyloid diseases include conditions of such clinical relevance as Alzheimer’s (AD) and Parkinson’s (PD) diseases, and type II diabetes mellitus1,2. AD is characterized by the presence of amyloid plaques surrounded by dead and dying neurons in the brain. The major component of the plaque is Aβ, a 39-43 residue peptide, resulting from the proteolytic processing of the much larger amyloid precursor protein (APP)3. We have studied the interaction of histone H1 and the 1-42 amyloidβ peptide (Aβ(1-42)), which is the most aggregation-prone of the various Aβ peptides. Aβ(1-42) monomers can evolve into oligomers, protofibrils and fibrils by a nucleation-elongation pathway3. Amyloid fibrils appear long, straight and unbranched, 6-10 nm in diameter and usually consist of 3-5 protofibrils rich in β-structure. Protofibrils are ~2.0 nm in diameter and often are twisted around each other4. Histone H1 binds to linker DNA and nucleosomes and is involved in chromatin higher-order structure and gene regulation. H1 stoichiometry varies between ~0.5 and ~1.3 H1 molecules per nucleosome in neurons and chicken erythrocytes, respectively5,6. Rat liver chromatin is reported to have 0.8 molecules of H1 per nucleosome, on average, while glial cell nuclei have a full complement of one H1 molecule per nucleosome6. Histone H1 contains three distinct domains: a short N-terminal domain (NTD) (20-35 amino acids), a stably-folded central globular domain (GD) (~80 amino acids) and a long carboxy-terminal domain


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(CTD) (~100 amino acids). In aqueous solution, the terminal domains are mostly unstructured, but they fold cooperatively upon interaction with DNA or hydrophobic ligands7-10. The histone H1 terminal domains, in particular the CTD, thus behave as intrinsically disordered proteins undergoing coupled binding and folding. Although the primary role of histone H1 is binding to DNA in cell nuclei, H1, and in particular the H1.2 subtype, can also be found in the cytoplasm and on cellular membranes of neurons and astrocytes in prion diseases and AD11. Histone H1.2 accumulation in the cytoplasm may play a role in apoptotic signaling through the release of cytochrome c from the mitochondria12,13. Histone H1 is phosphorylated by cyclin-dependent protein kinase Cdk2 and the neuron-specific Cdk5 at S/T-P-X-K/R consensus sequences. In diseased areas, Cdk5 is upregulated, which may lead to increased levels of phosphorylation of nuclear histone H114,15. Phosphorylation decreases the affinity of H1 for the DNA and, hence, the residence time of DNAbound H1. Phosphorylation may thus favor exportation of histone H1, in particular H1.2, to the cytoplasm, where it may interact with intracellular forms of amyloid-β. More than 20 different proteins are found in AD plaques16. Using pull-down assays with total proteins from cells or brain homogenates, Duce et al. 16 showed that the strongest interacting protein with the amyloidmotifs of amyloid-β, α-synuclein and lysozyme was histone H1. The interaction of histone H1 with amyloid plaques was confirmed by immunohistochemistry in Alzheimer’s models16. It would be of interest to know whether histone H1 contributes to the structure and stability of amyloid fibrils and fibers. Here, we have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism (CD), co-precipitation and transmission electron microscopy (TEM) to study the interaction of Aβ(142) with H1.2 and H1.0 histone H1 subtypes and their fully phosphorylated


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species (three phosphate groups in the C-terminal domain). H1.2 was chosen because of its role in apoptotic signaling11 and high turnover rate17, while H1.0 was examined because it accumulates in terminally differentiated neurons18. Both monomeric Aβ(1-42) and histone H1 contain large regions having the properties of intrinsically disordered or natively unfolded proteins. Aβ(1-42) and H1 readily interact in solution and undergo mutual binding and folding. The complexes of Aβ(1-42) and histone H1 formed aggregates characterized by the parallel association of a huge number of fibrils. The aggregates were particularly large and ordered in the presence of H1.2. Ageing increased the length and compaction of the complexes. Stabilization of fibril-fibril interactions appeared to be determined by the C-terminal domain of histone H1.


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EXPERIMENTAL Expression and purification of histones H1.2 and H1.0 and its domains We have used murine subtypes H1.0 and H1.2, which share 95 % identity and 89 % identity, respectively, with the corresponding human subtypes (Fig. S1). Histone H1.0 from mouse was cloned in the pQE-60 vector and expressed in E.coli M15 as previously described10. Histone H1.2 was amplified by PCR from rat genomic DNA. The primers were 5’AGAGGCTCCATATGTCGGAAACTGCTCCTGCTGC 3’ and 5’GGAGACCTCGAGTTACTTCTTCTTGGCTGCAAC 3’. The amplification product was digested with NdeI and XhoI, cloned into pET21a and expressed in E.coli BL21(DE3) as described19. Both H1 subtypes and the CTD of H1.0 were purified by hydroxyapatite chromatography on a CHT-II cartridge (Bio-Rad) and desalted by gel filtration through Sephadex G-25 (Amersham Biosciences) as previously described9. The globular domain of H1.0 was expressed and purified as previously described20. The peptide corresponding to residues 1-20 of the N-terminal domain of H1.0, TENSTSAPAAKPKRAKASKK-NH2, was synthesized by standard methods by DiverDrugs, Barcelona, Spain.

In vitro phosphorylation assay Histones H1.0 and H1.2 were phosphorylated in vitro with Cdk2-cyclin A kinase, which recognizes the S/T-P-X-K/R consensus sequence. The reaction buffer was 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 20 mM dithiotreitol, pH 7.5, plus 200 µM ATP. One unit of Cdk2-cyclin A per 5 mg of protein was used. The mixture was incubated at 30 ºC for 1 h and the reaction buffer eliminated by gel filtration on Sephadex G-25. The extent of phosphorylation was evaluated by MALDI-TOF mass spectrometry (Fig. S2).


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Amyloid peptide preparation The sequence of the recombinant amyloid peptide Aβ(1-42) was DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (rPeptide; Georgia, USA). The peptide in the form of lyophilized powder was dissolved in 10 mM NH4OH and 2% DMSO at a concentration of 1 mg/ml (222 µM). The solution was sonicated twice for 15 seconds, separated by a 15 s interval, at 30 % power. Peptide aliquots were stored at -20 ºC. Before use, peptide aliquots were neutralized with 10 mM HCl and diluted with buffer, so that the final concentration of DMSO was 0.06%. Peptide samples were sonicated again as described above.

Thioflavin (ThT) fluorescence measurements Samples were prepared as previously described with a few modifications21. Briefly, aggregation reactions were performed in stirred, 1-ml fluorescence quartz cuvettes at 30 °C for 24 h. The reaction (1ml) contained the following final concentrations: 7.4 µM Aβ(1-42), 10 mM phosphate buffer, pH 7.4, 50 mM NaCl, and 50 µM thioflavin T. Mixtures of Aβ(1-42) and several histone H1 species (H1.0 and H1.2 and their triphosphorylated species) were examined. The molar ratio H1: Aβ(1-42) was 0.1 in all cases. The fluorescence emission at 482 nm was monitored with a Cary Eclipse fluorescence spectrometer with the Kinetics software, version 1.1. The excitation wavelength was 440 nm and the measurements were recorded every three min for 30 s, and averaged. To demonstrate differences in kinetics normalized ThT intensities were used, in which the data are shown with the same final fluorescence intensity (100 %). Three replicates were made for each condition and averaged. The control curves corresponding to the isolated H1 subtypes and their phosphorylated species are shown in Fig. S3.


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Circular dichroism Samples containing the β-amyloid peptide, histone H1 (H1.0, H1.2 and their fully phosphorylated species) and the β-amyloid peptide/H1 mixtures at a molar ratio H1:Aβ(1-42)=0.1 were analyzed by circular dichroism (CD). Samples were examined immediately after preparation, after 24 h and after one week. Protein concentration was 7.4 µM for the β-amyloid peptide and 0.74 µM for histone H1 species. The molar ratio H1:Aβ(1-42) per molecule was 0.1 and 0.5 when expressed per residue. The buffer was 10 mM phosphate buffer, pH 7.4, plus 50 mM NaCl. Samples were incubated at 30 ºC for 24 h or kept at room temperature for one week without shaking. Before each measurement, samples were thoroughly shaken and sonicated with two pulses of 15 s, separated by 15 s. Spectra were recorded on a Jasco J-715 spectrometer in 1-mm cells at room temperature, with a resolution of 0.5 nm in the interval from 190 nm to 250 nm. Each spectrum was the average of six scans. The results were analyzed with Standard Analysis software (JACSO) and expressed as mean residue molar ellipticity (MRE), [θ]. The isolated domains of H1.0 and poly-Llysine (15000-30000 Da, Sigma) and the corresponding mixtures with Aβ(1-42) were analyzed by CD as described for the entire protein.

Transmission electron microscopy Samples of Aβ(1-42) and of the mixtures of Aβ(1-42) and histones H1.0 and H1.2 and their fully phosphorylated species were prepared as described in the ThT assay and incubated at 30 ºC for 24 h or kept at room temperature for one or two weeks without shaking. Then, the samples were diluted in 1 mM triethanolamine (TEA) to a final concentration of Aβ(142) of 45 µg/ml. Carbon-coated grids were used as sample support. Ten µl


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of sample were deposited over the grid for five minutes. Then, excess sample was removed and a drop of 0.5 % uranyl acetate was added and immediately removed. Sample grids were air-dried and visualized in a JEM-1400 TEM. Control samples of H1 species without Aβ(1-42) were prepared. They are shown in Fig.S4. The isolated domains of H1.0 and poly-L-lysine and the corresponding mixtures with Aβ(1-42) were analyzed by TEM as described for the entire protein.

Co-precipitation of Aβ(1-42) and histone H1 or its C-terminal domain Samples containing the β-amyloid peptide (2 µg) and histone H1 (H1.0) or its CTD at a molar ratio H1/CTD:Aβ(1-42)=0.1 were incubated at room temperature for 24 h and then centrifuged at 16000 x g for 30 min. The pellet was dissociated in Tris-Tricine sample buffer containing 8% SDS, 8M urea and boiled for 10 min as described by MacLean et al22. The supernatants and pellets were analyzed by 16% Tris-Tricine SDS-PAGE. Samples of H1.0 and its CTD were prepared as described above and analyzed by 15% SDS-PAGE. Proteins were visualized by Coomassie-Blue staining.


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RESULTS

Kinetics of Aβ(1-42) aggregation in the absence and in the presence of histone H1 and its fully phosphorylated species. A ThT fluorescence assay was performed to evaluate the effects of histone H1 on the time-course of Aβ(1-42) aggregation in vitro. ThT has a certain specificity for β-structure, which is the main secondary structure motif of amyloid fibrils. The method is limited to the quantitative determination of β-containing structures, i.e., mainly fibrils, but also protofibrils, high molecular weight oligomers and soluble oligomers23. Transient spherical oligomers, which appear in an early phase of aggregation, yield a negligible fluorescence signal in the ThT assay because β-structure has not yet developed. Late processes, involving longitudinal and lateral aggregation of fibrils once the plateau phase has been reached, may imply great changes in the gross structure of the fibrillar aggregates, but cannot be followed with ThT because they do not involve an increase in β-structure2,23. The time-course of ThT fluorescence enhancement was examined in the presence of the H1.2 and H1.0 histone H1 subtypes and their fully phosphorylated species (phosphorylated in the three S/T-P-X-K/R motifs of the carboxy-terminal domain) (Fig. 1). Aβ(1-42) yields a characteristic sigmoidal profile with an initial lag phase, corresponding mostly to unordered conformation, followed by an exponential growth phase, reflecting the increase in β-structure, and finally a plateau2. Under our conditions, the whole process was completed in about 20 h. Amyloid fibril formation typically follows nucleation-polymerization kinetics. It can be seen in Fig. 1 that the unphosphorylated species of H1.2 and H1.0 suppress the lag phase. This could be due to fast nucleation in the presence of histones, which would effectively “seed” the growth phase, and, more generally, it indicates that insoluble nuclei are formed faster in the case of


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unphosphorylated H1. In contrast, the phosphorylated species did not affect the lag phase, and slightly slowed the rate of fluorescence enhancement.

Circular dichroism of the complexes of Aβ(1-42) and histone H1. The interaction of Aβ(1-42) with unphosphorylated and phosphorylated histone H1 was examined by circular dichroism (CD). It is known that of Aβ(1-42) contains a C-terminal β-hairpin in an otherwise flexible structure24. Accordingly, we found that freshly dissolved of Aβ(1-42), i.e., at the beginning of the aggregation process, the peptide showed little, but significant, ellipticity in the region of β-structure, but had an intense negative peak at 197 nm, which is typical of the random coil (Fig. 2A). The spectrum evolved over time, indicating an increase in β-structure. After 24 h, the spectrum was characteristic of an all-β conformation, with a maximum at 197 nm and a minimum at 217 nm, which is the canonical position of the β-structure (Fig. 2A). A similar spectrum was obtained after one week of incubation (Fig. 2A). Histone H1 and its phosphorylated species were also dominated by the random coil, with a negative peak at ~200 nm, although, α-helical components belonging to the stably folded globular domain were also present. No changes were observed at 24 h, but after one week of incubation the histone H1 species also acquired a significant amount of secondary structure (α helix + β-structure), concomitant with a decrease of the random coil (Fig. 2B, S5B, S6B and S7B). To examine the effects of histone H1 on the conformation of Aβ(142), histone H1 was added to freshly dissolved Aβ(1-42) (at a molar ratio of H1:Aβ of 0.1). The mixing of Aβ(1-42) and histone H1 led to the substitution of the random coil bands in both Aβ(1-42) and histone H1 by a strong negative band at 224-226 nm, which was absent in the amyloid peptide alone. This band was attributed mainly to β-structure and could


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also contain some contribution of α-helix. The spectral change occurred immediately after mixing (Fig. 2C, S5C, S6C and S7C), indicating that Aβ(1-42) becomes structured upon interaction with histone H1. Monomeric Aβ(1-42) thus behaves as an intrinsically disordered protein with coupled binding and folding. The absence of random coil bands in the CD spectra from the mixtures of Aβ(1-42) and histone H1 shows that the unstructured regions of the latter, corresponding basically to the N- and C-terminal domains, also become structured upon interaction with Aβ(1-42). Therefore, starting with freshly dissolved Aβ(1-42) and histone H1, which are both unstructured to a large extent, we are dealing with a process of mutual binding and folding. The interaction of Aβ(1-42) and histone H1 was confirmed by coprecipitation followed by denaturing gel electrophoresis (Fig. S8). Dissociated precipitates contained the monomeric and dimeric forms of Aβ(1-42) and histone H1. Little amounts of the amyloid β peptide were found in the supernanats. The ~227 nm band is shifted by up to 10 nm, relative to the classic 217 nm β-sheet minimum25,26. The same shift has been observed in fungal prion and bacterial inclusion bodies of β-amyloid peptides. The ~227 nm peak is thought to arise from a superposition of the aromatic CD band of Aβ amyloid and the classic β-sheet spectrum, following changes in the stacking of the polypeptide aromatic side-chains. In our case, the negative band at ~227 nm was only observed in freshly prepared Aβ+H1 mixtures. Later on, the band shifted to shorter wavelengths (Fig. 2C, S5C, S6C and S7C), indicating the restructuring of the aromatics. After one week of incubation, the peak was at ~217 nm, which is the canonical position of βstructure.


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Circular dichoism of Aβ(1-42) in the presence of the isolated histone H1 domains and polylysine. Histone H1 has three domains differing in size, composition and structure. We have studied the effects of the isolated domains on the folding of Aβ(1-42). Polylysine has also been examined, in view of its polycationic character, which makes it comparable to the H1 C-terminal domain (~40% lysine). The spectra of the highly basic N- and C-terminal domains were dominated by the random coil, as shown by the strong negative peak near 200 nm (Fig. 3A and 3C). As in the entire H1, the spectra of the mixtures of Aβ(1-42) and the H1 terminal domains lacked the random coil peak, which was replaced by bands mainly from βstructure (Fig. 3A, and 3C) . The spectral changes occurred immediately after mixing and manifested the interaction of the isolated terminal domains with Aβ(1-42). The interaction of Aβ(1-42) and the CTD was confirmed by co-precipitation followed by gel electrophoresis (Fig. S8). The CTD, like the entire protein, was found in the pellet with monomeric and dimeric Aβ(1-42). The spectrum of the globular domain reflected its high content of α-helix. The globular domain apparently slowed down the folding of Aβ(1-42), as the spectrum observed immediately after mixing was still dominated by the random coil, although eventually (after 24h) the spectrum evolved towards a mixture of β-structure and α-helix (Fig. 3B). Polylysine behaved very much as the H1 globular domain as it impaired the folding of Aβ(1-42). Again, at 24h the complexes of polylysine and Aβ(142) were fully structured (Fig. 3D).

Electron microscopy of the complexes of Aβ(1-42) and histone H1. We examined the morphology of the complexes of Aβ(1-42) with the H1 subtypes H1.2 and H1.0 and their phosphorylated species by TEM.


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The study of the morphology of Aβ peptide aggregation is made difficult by intrinsic structural polymorphism23 and by the great effects of small changes in the experimental conditions. In the presence of histone H1, these effects were smaller, likely because histone H1 favors fibrillation pathways and fiber stability. Several conclusions can be drawn from the morphology of the fibers. After 24 h of incubation, Aβ(1-42) formed fibrils of 6-10 nm without a defined pattern of mutual interactions (Fig. 4A) In the presence of H1.0, abundant short and rigid protofilaments of a cross section diameter of ~3 nm were observed (Fig. 4B). Some aggregates of protofilaments were present that could be the precursors of the large aggregates observed after one week of ageing. In the presence of H1.2, Aβ(1-42) formed longer and thicker protofibrils than with H1.0; small laminar aggregates were also present (Fig. 4C). Ageing for one week in the presence of histone H1 led to the formation of large and ordered aggregates. With H1.0, thick bundles with cross sections of 50-200 nm were observed (Fig. 5B). These kinds of aggregates were characterized by the parallel assembly of a large number of fibrils of a diameter of 6-10 nm. In contrast, without H1 the aggregates were formed by 4-5 fibrils (Fig. 5A). With phosphorylated H1.0 the results were similar to those of the unphosphorylated species (Fig. 5C). The complexes of Aβ(1-42) with H1.2 had an even higher propensity to form large and ordered aggregates than did the complexes with H1.0. After one week of incubation in the presence of H1.2, Aβ(1-42) formed aggregates consisting of 30 or more fibrils of 6-10 nm laterally assembled (Fig. 5D and 5E). The aggregates often had a laminar appearance. This is clearly seen in Fig. 5D thanks to the local twist of the lamina. Laminae could also be arranged in successively stacked layers (Fig. 5E). Complexes with phosphorylated H1.2 usually formed thick bundles (Fig. 5F and 5G).


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Fibril-fibril interactions, leading to either laminae or thick bundles, may be determined by electrostatic and hydrophobic interactions between Aβ(1-42) and histone H1. These interactions could explain the extensive growth of fibril aggregates. Ageing of the complexes for more than one week led to the compaction of fibril bundles. Compaction could be so tight as to mask the fibrils on the surface of the fibers. At the same time, fibers became very long. Figure 6 shows one of these compact fibers. A frayed end makes possible the observation of the constituent individual fibrils.

Electron microscopy of Aβ(1-42) in the presence of the isolated histone H1 domains and polylysine. We examined the morphology of Aβ(1-42) in the presence of the isolated H1 domains and polylysine by TEM (Fig. 7). In the presence of the CTD, Aβ(1-42) formed large aggregates of fibrils parallely arranged, similar to those observed with the entire H1. Fibril bundles were formed by 30 or more fibrils. The capacity of histone H1 to stabilize the parallel assembly of large numbers of fibrils was thus already fully manifested by the CTD. The NTD favored the formation of fibrils and laminar structures, but the characteristic parallel arrangement of fibrils in the presence of the entire H1 and the CTD was not observed. The GD interfered with fibrillation, leading to amorphous aggregates. In the presence of polylysine Aβ(1-42) formed fibrils of ~6 nm, often with granular appearance.


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DISCUSSION Aβ(1-42) is a particular type of intrinsically disordered protein in that, although the peptide is largely disordered in freshly prepared aqueous solutions, over time it acquires a predominant β-structure conformation through a self-interaction process27,28. In our conditions, the acquisition of the maximal amount of β-structure takes approximately 20 h, as shown by the kinetics of ThT fluorescence enhancement. Further rearrangements not affecting the amount of β-structure occur over a period of days to weeks and involve fibril and fiber organization. Histone H1 has a three domain structure, the central globular domain is stably folded, but the highly basic N- and C-terminal domains behave as intrinsically disordered proteins that fold upon interaction with DNA or lipids7,10. In buffer solution, the H1 CD spectrum is dominated by the random coil. However, H1 can also spontaneously acquire a substantial amount of secondary structure, as shown by the CD spectrum after one week of incubation. It is worth noting that histone H1 itself can form amyloid fibers in the presence of SDS10. We have used CD to show that monomeric Aβ(1-42) and histone H1 interact with each other. The random coil bands, typical of the freshly disolved Aβ(1-42) and histone H1 were replaced by bands corresponding mostly to β-structure. Each molecular species thus behaves as the ligand of the other in a mutual process of coupled binding and folding. The interaction of Aβ(1-42) and histone H1 was confirmed by co-precipitation followed by gel electrophoresis. The plateau in ThT fluorescence enhancement assays with Aβ(1-42) was reached in about 20 h for all H1 species, either phosphorylated or unphosphorylated. The main difference among subtypes was the absence of a lag phase in the presence of the unphosphorylated species of H1. In analogy with the “seeding” effect of pre-formed fibrils that suppresses the lag phase, this feature could be explained by fast nucleation in the presence


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of the unphosphorylated H1 species. The TEM images were consistent with fast nucleation of protofilaments. Phosphorylation decreases the net positive charge of histone H1 and thus may lower the affinity for anionic species such as Aβ(1-42). The higher affinity of unphosphorylated H1 may explain the faster kinetics in the initial part of the process of fibrillation. Phosphorylation also induces a major structural change in the CTD, characterized by the induction of β-structure. The gain of ordered structure in the CTD could slow down the mutual structure induction. Histone H1 had deep effects on fibril-fibril interactions and on the size of amyloid fibers, which were apparent after ~1 week of ageing. Histone H1 favored the parallel aggregation of large numbers of fibrils, leading to the formation of laminae and bundles. The high numbers of fibrils arranged in parallel (>25) in the complexes with H1 are to be compared with the 3-4 fibrils in the absence of H1. The parallel arrangement of fibrils may be energetically favorable, as it allows for fibrilfibril interactions all along their length29. H1 could behave, thanks to its capacity to interact with both cationic and hydrophobic ligands, as a crosslinker of Aβ(1-42) fibrils. Fiber formation in the presence of H1 may consist of an equilibrium process driven by the optimization of fibril-fibril parallel contacts. An argument in favor of a free-energy component in fibril contacts is the observation that with longer ageing time, fibers became more compact so as to make the surface details no longer visible. The formation of ordered, large parallel complexes in the presence of histone H1 may not be a general property of amyloids. In contrast to Aβ(142), the incorporation of histone H1 into α-synuclein fibrils in Parkinson’s disease was not accompanied by either a significant change in morphology or by an enhanced frequency of fibril contacts, which were irregular and sparse, and not affected by the presence of H130, indicating that fibril lateral


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interaction was no favored. Our results suggest that histone H1 facilitates ordered Aβ(1-42) aggregation through the stabilization of the parallel alignment of fibrils. A schematic representation of the interaction among fibrils in the presence and in the absence of histone H1 is shown in Fig. 8. In addition to entire histone H1 molecules, we examined the interaction of the isolated H1 domains and polylysine with Aβ(1-42) with the purpose of studying the specificity of the interaction of histone H1 and Aβ(1-42). Mixtures of Aβ(1-42) and the H1 terminal domains undergo mutual structure induction immediately after mixing, while the GD appeared to have no interaction with the amyloid β peptide. In contrast, polylysine did not induce the immediate folding of Aβ(1-42), but after 24 h both proteins were completely structured. These results suggest that the electrostatic interactions are not enough to induce the immediate folding of Aβ(1-42). Although both NTD and CTD induced the folding of Aβ(1-42), only the CTD stabilized fibril lateral aggregation as observed by TEM. The CTD is the main determinant of histone H1 binding to chromatin31. Preferential binding of histone H1 to scaffold-associated regions20 and activation of apoptotic nuclease32 also appear to be determined by the CTD. The main conclusion to be drawn from the study of the isolated domains is that the interaction of histone H1 with Aβ(1-42) is determined by the CTD. In particular, the CTD appears to have the same capacity as the entire histone H1 to stabilize fibril lateral aggregation. It seems that in diseased zones of the brain there is an overexpression of Cdk5, a neural tissue-specific kinase14,15. Cdk5 activity would hyperphosphorylate H1, weakening its binding to chromatin and thus favoring its translocation from cell nuclei to cytoplasm12,33. This effect would lead mainly to the translocation of H1.2, because this subtype already has the lowest affinity for DNA among the H1 subtypes in mature neurons and also has the highest synthesis rate17. Cytoplasmic H1.2 of


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nuclear origin would readily interact with the different forms of Aβ, facilitating monomer folding and fibrillation. In this hypothetical pathway, the most upstream identified event in the formation of amyloid deposits would be Cdk5 upregulation, which would lead to the increase of the cytoplasmic pool of phosphorylated H1.234. Phosphorylation of histone H1 may determine some aspects of amyloid morphology, such as the preference for bundles over laminae, but, presumably, its main role may be to facilitate the translocation of H1.2 from cell nuclei to cytoplasm. The state of phosphorylation of H1 associated with amyloid plaques has not been investigated thus far. Previous results showed that the main effect of phosphorylation in the presence of DNA or detergents was a decrease in the α-helix content and an increase in that of the β-structure. Conditions can be found in which the CTD becomes an all-β protein. Such a conformational change in the phosphorylated H1 CTD could also occur upon interaction with Aβ(1-42). In the H1 CTD, most Lys residues are in doublets, where the Lys residues point to opposite directions35. This may explain why phosphorylated H1 could be a better crosslinker than unphophorylated H1. Until recently, mature amyloid fibrils were considered cytotoxic, since they were the kind of aggregate commonly detected in pathological deposits. However, an increasing number of reports indicate that prefibrillar assemblies, preceding the appearance of the mature fibrils, are the main cytotoxic species36,37. In this view, hindering the formation of mature fibrils and fibers, could be disadvantageous rather than beneficial. However, it remains to be seen to what extent the presence of large intracellular amyloid aggregates can be disruptive for the cells, especially for cell and mitochondrial membranes.

CONCLUSIONS


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The aggregation of the amyloid-β peptide, Aβ(1-42), is believed to play an important role in the pathogenesis of Alzheimer’s disease. It has been shown by immunohistochemistry that histone H1 is present in amyloid plaques in Alzheimer models. Aβ(1-42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Interaction of monomeric Aβ(1-42) and histone H1 has been demonstrated by mutual structure induction using CD and confirmed by co-precipitation. In the presence of histone H1, Aβ(1-42) formed laminar aggregates and thick bundles, characterized by the lateral association of large numbers of fibrils (>25), while in the absence of histone H1 the aggregates contained 3-4 fibrils. The parallel arrangement of fibrils may be energetically favorable, as it allows for fibril-fibril interactions all along their length. Stabilization of fibril-fibril interactions appeared to be determined by the CTD of histone H1. These observations indicate that histone H1 fulfills at least two roles: it helps the folding of Aβ(1-42) monomers and promotes the parallel association of a large number of fibrils.

FUNDING This work was supported by the Ministerio de Ciencia e Innovación (BFU2008-00460 to PS) and (BFU2013-44763 to SV). SV has been granted an ICREA ACADEMIA award.

SUPPORTING INFORMATION Protein sequence alignments of H1 subtypes. MALDI-TOF spectra of phosphorylated and unphosphorylated H1 subtypes. Electron micrographs of the histone H1 subtypes and their phosphorylated species. Circular dichroism spectra of Aβ(1-42), three H1 species (H1.0, H1.2, H1.0p) and their corresponding mixtures. Co-precipitation of Aβ(1-42) and histone H1


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or its C-terminal domain. This material is available free of charge via Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS We thank Dr. S. Villegas for the gift of samples of amyloid β peptide used in preliminary experiments and P. Castro and N. Castell for their help in the TEM experiments.


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CAPTIONS FOR FIGURES Figure 1. Effect of histone H1 on the kinetics of fibrillation of Aβ(1-42). Histone H1 subtypes H1.0 and H1.2 and their phosphorylated species were examined. Aggregation reactions were performed in 1-ml fluorescence quartz cuvettes at 37 ºC for 24 h. The reactions (1 ml) contained the following final concentrations: 7.4 µM Aβ(1-42), 10 mM phosphate buffer, pH 7.4, 50 mM NaCl and 50 µM thioflavin T. The histone H1:Aβ(1-42): molar ratio was 0.1. Fluorescence excitation was at 440 nm. The fluorescence emission at 482 nm was recorded every three min for 30 s. In green Aβ(1-42), in red and in blue the mixtures of Aβ(1-42) and unphosphorylated H1.0 and H1.2, respectively. In yellow and purple the mixtures of Aβ(1-42) and phosphorylated H1.0 and H1.2, respectively. The curves are the average of three measurements. Non-normalized ThT intensities were used and the error bars correspond to the standard deviation. The fibrillation controls of the isolated proteins are shown in Fig.S3.

Figure 2. Circular dichroism of Aβ(1-42) and phosphorylated H1.2. A, Aβ(1-42); B, phosphorylated H1.2 (H1.2p); C, Aβ(1-42)+H1.2p. In blue the samples immediately after mixing; in red and in green the samples incubated for 24 h and 1 week, respectively. Measurements were carried out at room temperature in the presence of 10 mM phosphate buffer, pH 7.4, plus 50 mM NaCl. Protein concentration was 7.4 µM for the β-amyloid peptide and 0.74 µM for histone H1. Before each measurement, samples were sonicated with two pulses of 15 s, separated by 15 s. Molar residue ellipticity (MRE), [θ] (deg cm2 dmol-1).

Figure 3. Circular dichroism of isolated domains of H1.0 and polylysine with or without Aβ(1-42). A, N-terminal domain; B, globular domain; C,


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C-terminal domain, and D, polylysine. The dashed lines correspond to the isolated proteins and the continuous line correspond to the mixtures with Aβ(1-42). In blue, samples immediately after mixing; in red and in green the samples incubated for 24 h and 1 week, respectively. Measurements were carried out at room temperature in the presence of 10 mM phosphate buffer, pH 7.4, plus 50 mM NaCl. Protein concentration was 7.4 µM for the β-amyloid peptide and 0.74 µM for each domain or polylysine. Before each measurement, samples were sonicated with two pulses of 15 s, separated by 15 s. MRE, [θ] (deg cm2 dmol-1).

Figure 4. Electron micrographs of fibrils and fibers formed by Aβ(1-42) in the presence of histone H1. Samples were incubated at 30 ºC for 24 h without stirring. A, Aβ(1-42); B, Aβ(1-42)+histone H1.0; C, Aβ(142)+histone H1.2. The bars correspond to 100 nm.

Figure 5. Electron micrographs of fibrils and fibers formed by Aβ(1-42) in the presence of histone H1. Samples were incubated at room-temperature for one week without stirring. A, Aβ(1-42); B, Aβ(1-42)+histone H1.0; C, Aβ(1-42)+phosphorylated H1.0; D and E, Aβ(1-42)+histone H1.2; F and G, Aβ(1-42)+phosphorylated H1.2. The bars correspond to 100 nm.

Figure 6. Electron micrograph of a fiber of Aβ(1-42) in the presence of histone H1.2. The sample was incubated for 15 days at room temperature.

Figure 7. Electron micrographs of the isolated domains of H1.0 and polylysine with Aβ(1-42). A, B, C, D, mixtures of Aβ(1-42) and the Nterminal domain, globular domain, C-terminal domain and polylysine, respectively. Samples were incubated at room-temperature for one week without stirring. The bars correspond to 100 nm.


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Figure 8. Schematic representation of the role of histone H1 in promoting parallel and ordered fibrillar aggregates of amyloid fibers. A, fibrils and fibers of Aβ(1-42). B, fibers of Aβ(1-42) in the presence of histone H1. The cylinders represent amyloid fibrils of 6-10 nm. Dashed lines indicate the interaction surface.


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(12) Konishi, A.; Shimizu, S.; Hirota, J.; Takao, T.; Fan, Y.; Matsuoka, Y.; Zhang, L.; Yoneda, Y.; Fujii, Y.; Skoultchi, A. I.; Tsujimoto, Y. Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks. Cell. 2003, 114, 673-88. (13) Tsoneva, I.; Nikolova, B.; Georgieva, M.; Guenova, M.; Tomov, T.; Rols, M. P.; Berger, M. R. Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells. Biochim. Biophys Acta. 2005, 1721, 55-64. (14) Cruz, J.C.; Tsai, L.H. Cdk5 deregulation in the pathogenesis of Alzheimer's disease. Trends Mol Med. 2004, 10, 452-8. (15) Arif, A. Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5. Biochem. Pharmacol. 2012, 84, 985-93. (16) Duce, J. A.; Smith, D. P.; Blake, R. E.; Crouch, P. J.; Li, Q. X.; Masters, C. L.; Trounce, I. A. Linker histone H1 binds to disease associated amyloid-like fibrils. J. Mol. Biol. 2006, 361, 493-505. (17) Domínguez, V.; Piña, B.; Suau, P. Histone H1 subtype synthesis in neurons and neuroblasts. Development. 1992, 115, 181-5. (18) Piña, B.; Martínez, P.; Simón, L.; Suau P. Differential kinetics of histone H1(0) accumulation in neuronal and glial cells from rat cerebral cortex during postnatal development. Biochem Biophys Res Commun. 1984, 123, 697-702. (19) Terme, J.M.; Millán-Ariño, L.; Mayor, R.; Luque, N.; IzquierdoBouldstridge, A.; Bustillos, A.; Sampaio, C.; Canes, J.; Font, I.; Sima, N.; Sancho, M.; Torrente, L.; Forcales, S.; Roque, A.; Suau, P.; Jordan, A. Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications. FEBS Lett. 2014, 588, 235362. (20) Roque, A.; Orrego, M.; Ponte, I.; Suau, P. The preferential binding of histone H1 to DNA scaffold-associated regions is determined by its Cterminal domain. Nucleic Acids Res. 2004, 32, 6111-9.


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(21) Finder, V.H.; Vodopivec, I.; Nitsch, R.M.; Glockshuber, R. The recombinant amyloid-beta peptide Abeta1-42 aggregates faster and is more neurotoxic than synthetic Abeta1-42. J. Mol. Biol. 2010, 396, 9-18. (22) McLean, C.A.; Cherny, R.A.; Fraser, F.W.; Fuller, S.J.; Smith, M.J.; Beyreuther, K.; Bush, A.I.; Masters, C.L. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol. 1999, 46, 860-6. (23) Bartolini, M.; Naldi, M.; Fiori, J.; Valle, F.; Biscarini, F.; Nicolau, D.V.; Andrisano, V. Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach. Anal Biochem. 2011, 414, 215-25. (24) Sgourakis, N.G.; Yan,Y.; McCallum, S.A.; Wang, C.; Garcia, A.E. The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD / NMR study. J Mol Biol. 2007, 368, 1448-57. (25) Nyrkova, I.A.; Semenov, A.N.; Aggeli, A.; Boden, N. Fibril stability in solutions of twisted β-sheet peptides: a new kind of micellization in chiral systems. Eur. Phys. J. B 2000, 17, 481-497. (26) Dasari, M.; Espargaro, A.; Sabate, R.; Lopez del Amo, J. M.; Fink, U.; Grelle, G.; Bieschke, J.; Ventura, S.; Reif, B. Bacterial inclusion bodies of Alzheimer's disease β-amyloid peptides can be employed to study nativelike aggregation intermediate states. Chembiochem. 2011, 12, 407-23. (27) Uversky, V.N.; Fink, A.L. Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim. Biophys Acta. 2004, 1698, 131-53. (28) Bartolini, M.; Bertucci, C; Bolognesi, M.L.; Cavalli, A.; Melchiorre, C.; Andrisano, V. Insight into the kinetic of amyloid beta (1-42) peptide self-aggregation: elucidation of inhibitors' mechanism of action. Chembiochem. 2007, 8, 2152-61. (29) Aggeli, A.; Nyrkova, I.A.; Bell, M.; Harding, R.; Carrick, L.; McLeish, T. C.B.; Semenov, A.N.; Boden, N. Hierarchical self-assembly of chiral rod-like molecules as a model for peptide β-sheet tapes, ribbons, fibrils and fibers. PNAS. 2001, 98, 11857-11862. (30) Goers, J.; Manning-Bog, A. B.; McCormack, A. L.; Millett, I. S.; Doniach, S.; Di Monte, D. A.; Uversky, V. N.; Fink, A. L. Nuclear


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localization of alpha-synuclein and its interaction with histones. Biochemistry. 2003, 42, 8465-71. (31) Lu, X.; Hansen, J.C. Identification of specific functional subdomains within the linker histone H10 C-terminal domain. J Biol Chem. 2004, 279, 8701-7. (32) Widlak, P.; Kalinowska, M., Parseghian, M.H.; Lu, X.; Hansen, J.C.; Garrard. W.T. The histone H1 C-terminal domain binds to the apoptotic nuclease, DNA fragmentation factor (DFF40/CAD) and stimulates DNA cleavage. Biochemistry. 2005, 44, 7871-8. (33) Bleher, R.; Martin, R. Nucleo-cytoplasmic translocation of histone H1 during the HeLa cell cycle. Chromosoma. 1999, 108, 308-16. (34) Zlatanova, J. S.; Srebreva, L. N.; Banchev, T. B.; Tasheva, B. T.; Tsanev, R. G. Cytoplasmic pool of histone H1 in mammalian cells. J. Cell Sci. 1990, 96, 461-8. (35) Roque, A.; Ponte, I.; Suau. P. Role of charge neutralization in the folding of the carboxy-terminal domain of histone H1. J Phys Chem B. 2009, 113, 12061-6. (36) Marín-Argany, M.; Rivera-Hernández, G.; Martí, J.; Villegas, S. An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formationand cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway. Biochem. J. 2011, 437, 25-34. (37) Kreplak, L.; Aebi, U. From the polymorphism of amyloid fibrils to their assembly mechanism and cytotoxicity. Adv. Protein Chem. 2006, 73, 217-33.


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Figure 1. Effect of histone H1 on the kinetics of fibrillation of Aβ(1-42). Histone H1 subtypes H1.0 and H1.2 and their phosphorylated species were examined. Aggregation reactions were performed in 1-ml fluorescence quartz cuvettes at 37 ºC for 24 h. The reactions (1 ml) contained the following final concentrations: 7.4 µM Aβ(1-42), 10 mM phosphate buffer, pH 7.4, 50 mM NaCl and 50 µM thioflavin T. The histone H1:Aβ(1-42): molar ratio was 0.1. Fluorescence excitation was at 440 nm. The fluorescence emission at 482 nm was recorded every three min for 30 s. In green Aβ(1-42), in red and in blue the mixtures of Aβ(1-42) and unphosphorylated H1.0 and H1.2, respectively. In yellow and purple the mixtures of Aβ(1-42) and phosphorylated H1.0 and H1.2, respectively. The curves are the average of three measurements. Non-normalized ThT intensities were used and the error bars correspond to the standard deviation. The fibrillation controls of the isolated proteins are shown in Fig.S3. 81x48mm (150 x 150 DPI)


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Figure 2. Circular dichroism of Aβ(1-42) and phosphorylated H1.2. A, Aβ(1-42); B, phosphorylated H1.2 (H1.2p); C, Aβ(1-42)+H1.2p. In blue the samples immediately after mixing; in red and in green the samples incubated for 24 h and 1 week, respectively. Measurements were carried out at room temperature in the presence of 10 mM phosphate buffer, pH 7.4, plus 50 mM NaCl. Protein concentration was 7.4 µM for the βamyloid peptide and 0.74 µM for histone H1. Before each measurement, samples were sonicated with two pulses of 15 s, separated by 15 s. Molar residue ellipticity (MRE), [θ] (deg cm2 dmol-1). 125x235mm (150 x 150 DPI)


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Figure 3. Circular dichroism of isolated domains of H1.0 and polylysine with or without Aβ(1-42). A, Nterminal domain; B, globular domain; C, C-terminal domain, and D, polylysine. The dashed lines correspond to the isolated proteins and the continuous line correspond to the mixtures with Aβ(1-42). In blue, samples immediately after mixing; in red and in green the samples incubated for 24 h and 1 week, respectively. Measurements were carried out at room temperature in the presence of 10 mM phosphate buffer, pH 7.4, plus 50 mM NaCl. Protein concentration was 7.4 µM for the β-amyloid peptide and 0.74 µM for each domain or polylysine. Before each measurement, samples were sonicated with two pulses of 15 s, separated by 15 s. MRE, [θ] (deg cm2 dmol-1). 77x51mm (300 x 300 DPI)


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Figure 4. Electron micrographs of fibrils and fibers formed by Aβ(1-42) in the presence of histone H1. Samples were incubated at 30 ºC for 24 h without stirring. A, Aβ(1-42); B, Aβ(1-42)+histone H1.0; C, Aβ(142)+histone H1.2. The bars correspond to 100 nm. 71x187mm (300 x 300 DPI)


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Figure 5. Electron micrographs of fibrils and fibers formed by Aβ(1-42) in the presence of histone H1. Samples were incubated at room-temperature for one week without stirring. A, Aβ(1-42); B, Aβ(142)+histone H1.0; C, Aβ(1-42)+phosphorylated H1.0; D and E, Aβ(1-42)+histone H1.2; F and G, Aβ(142)+phosphorylated H1.2. The bars correspond to 100 nm. 48x72mm (300 x 300 DPI)


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Figure 6. Electron micrograph of a fiber of Aβ(1-42) in the presence of histone H1.2. The sample was incubated for 15 days at room temperature. 153x160mm (300 x 300 DPI)


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Figure 7. Electron micrographs of the isolated domains of H1.0 and polylysine with Aβ(1-42). A, B, C, D, mixtures of Aβ(1-42) and the N-terminal domain, globular domain, C-terminal domain and polylysine, respectively. Samples were incubated at room-temperature for one week without stirring. The bars correspond to 100 nm. 190x254mm (300 x 300 DPI)


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Figure 8. Schematic representation of the role of histone H1 in promoting parallel and ordered fibrillar aggregates of amyloid fibers. A, fibrils and fibers of Aβ(1-42). B, fibers of Aβ(1-42) in the presence of histone H1. The cylinders represent amyloid fibrils of 6-10 nm. Dashed lines indicate the interaction surface. 153x96mm (300 x 300 DPI)


Histone H1 Favors Folding and Parallel Fibrillar Aggregation of the 1

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