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Elucidating the link between amyloid fibril formation and liquid–liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective

Amyloid fibril formation is involved in various human diseases, such as amyotrophic lateral sclerosis and Parkinson’s disease. Many amyloidogenic proteins undergo liquid–liquid phase separation (LLPS), forming liquid-like droplets of condensed proteins. Although these droplets represent potential amyloid nucleation sites, the kinetics of amyloid nucleation in droplets is poorly understood owing to the lack of quantitative analytical methods. This study aimed to develop a measurement method for the determination of amyloid droplet nucleation rate based on image analysis. Here, the prion determinant domain of the yeast prion protein Sup35 (Sup35NM), which is known as a model protein of prion diseases, was used for demonstration. Droplets (1–20 µm in size) of Sup35NM were fixed in agarose gels, and amyloid nucleation in each droplet was counted via confocal microscopy. From the comparison of the amyloid nucleation rates in the droplets and aqueous solutions, we found a critical concentration (C*) of Sup35NM: amyloid nucleation is suppressed by LLPS above C*, whereas it is enhanced below C*. These results highlight that droplet formation can suppress amyloid nucleation, contrary to current hypotheses in which the amyloid nucleation is enhanced by droplet formation. The proposed quantitative measurement method can provide accurate assessment of the role of LLPS for amyloidosis-related proteins from pathological perspectives.

Self-propagating β-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Amyloid fibrils are supramolecular protein assemblies represented by a cross-β structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of β-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several β-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the β-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.

Soluble prion proteins contingently encounter foreign prion aggregates, leading to cross-species prion transmission. However, how its efficiency is regulated by structural fluctuation of the host soluble prion protein remains unsolved. In the present study, through the use of two distantly related yeast prion Sup35 proteins, we found that a specific conformation of a short disordered segment governs interspecies prion transmissibility. Using a multidisciplinary approach including high-resolution NMR and molecular dynamics simulation, we identified critical residues within this segment that allow interspecies prion transmission in vitro and in vivo, by locally altering dynamics and conformation of soluble prion proteins. Remarkably, subtle conformational differences caused by a methylene group between asparagine and glutamine sufficed to change the short segment structure and substantially modulate the cross-seeding activity. Thus, our findings uncover how conformational dynamics of the short segment in the host prion protein impacts cross-species prion transmission. More broadly, our study provides mechanistic insights into cross-seeding between heterologous proteins. The side chain interaction within the short disordered segment of yeast prion protein Sup35 could affect the conformation of the main chain, alter the transmission barrier between species and regulate its cross-seeding activity.

This research was originally published in the Journal of Biological Chemistry. Yumiko Ohhashi, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Optimum Amyloid Fibril Formation of a Peptide Fragment Suggests the Amyloidogenic Preference of β2-Microglobulin under Physiological Conditions. J. Biol. Chem. 2004; 279, 10814-10821. © the American Society for Biochemistry and Molecular Biology, β2-Microglobulin (β2m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length β2m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of β2m, Ser20–Lys41, obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length β2m. This suggests that once the rigid native-fold of β2m is unfolded and additional factors triggering the nucleation process are provided, full-length β2m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys25. The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length β2m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.

Theophylline is one of the most frequently administered drug in children with bronchial asthma. But, accumulation of serum theophylline during viral infections or some febril disease has been described.We studied alteration of serum theophylline concentration, theophylline clearance and urinary excretion of theophylline metabolites during influenza virus infection. The results were as follows: 1) During influenza virus infection, the first febrile day, serum theophylline concentrations were two times as much as those in a convalescent state. For the first time, serum theophylline concentrations were normal at the third day since to alleviate fever.2) The first febrile day, theophylline clearances were half as much as those in a convalescent state.3) The urinary excretion of the theophylline metabolites, 1, 3-demethyluric acid, 1-methyluric acid, 3-methylxantine, were decreased during influenza virus infection.The ratios of theophylline metabolites to theophylline in urine were decreased, too.Our study indicates that serum concentrations of theophylline are higher in children with influenza virus infection than in those in a convalescent state. The higher concentrations are due to decreased theophylline clearance. We concluded that it may be the result of depression of the metabolic activity of hepatic cytochrome P-450 enzyme system during influenza virus infection.

A remarkable feature of prion biology is that the same prion protein can misfold into more than one infectious conformation, and these conformations in turn lead to distinct heritable prion strains with different phenotypes. The yeast prion [PSI(+)] is a powerful system for studying how changes in strain conformation affect cross-species transmission. We have previously established that a chimera of the Saccharomyces cerevisiae (SC) and Candida albicans (CA) Sup35 prion domains can cross the SC/CA species barrier in a strain-dependent manner. In vitro, the conversion of the monomeric chimera into the prion (amyloid) form can be seeded by either SC or CA Sup35 amyloid fibers, resulting in two strains: Chim[SC] and Chim[CA]. These strains have a "molecular memory" of their originating species in that Chim[SC] preferentially seeds the conversion of SC Sup35, and vice versa. To investigate how this species specificity is conformationally encoded, we used amide exchange and limited proteolysis to probe the structures of these two strains. We found that the amyloid cores of Chim[SC] and Chim[CA] are predominantly confined to the SC-derived and CA-derived residues, respectively. In addition, the chimera is able to propagate the Chim[CA] conformation even when the SC residues comprising the Chim[SC] core were deleted. Thus, the two strains have non-overlapping and modular amyloid cores that determine whether SC or CA residues are presented on the growing face of the prion seed. These observations establish how conformations determine the specificity of prion transmission and demonstrate a remarkable plasticity to amyloid misfolding.

Abeta2M (beta(2)-microglobulin-related) amyloidosis is a frequent and serious complication in patients on long-term dialysis. Partial unfolding of beta2-m (beta(2)-microglobulin) may be essential to its assembly into Abeta2M amyloid fibrils in vivo. Although SDS around the critical micelle concentration induces partial unfolding of beta2-m to an alpha-helix-containing aggregation-prone amyloidogenic conformer and subsequent amyloid fibril formation in vitro, the biological molecules with similar activity under near-physiological conditions are still unknown. The effect of various NEFAs (non-esterified fatty acids), which are representative anionic amphipathic compounds in the circulation, on the growth of Abeta2M amyloid fibrils at a neutral pH was examined using fluorescence spectroscopy with thioflavin T, CD spectroscopy, and electron microscopy. Physiologically relevant concentrations of laurate, myristate, oleate, linoleate, and mixtures of palmitate, stearate, oleate and linoleate, induced the growth of fibrils at a neutral pH by partially unfolding the compact structure of beta2-m to an aggregation-prone amyloidogenic conformer. In the presence of human serum albumin, these NEFAs also induced the growth of fibrils when their concentrations exceeded the binding capacity of albumin, indicating that the unbound NEFAs rather than albumin-bound NEFAs induce the fibril growth reaction in vitro. These results suggest the involvement of NEFAs in the development of Abeta2M amyloidosis, and in the pathogenesis of Abeta2M amyloidosis.

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Low concentrations of sodium dodecyl sulfate induce partial unfolding of beta2-m to an amyloidogenic conformer and subsequent amyloid fibril formation in vitro, but the biological molecules that induce them under near-physiological conditions have not been determined.We investigated the effect of some lysophospholipids on the nucleation, extension and stabilization of Abeta2M amyloid fibrils at a neutral pH, using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy and electron microscopy. We also measured plasma concentrations of lysophospholipids in 103 haemodialysis patients and 14 healthy subjects and examined the effect of uraemic and normal plasmas on the stabilization of Abeta2M amyloid fibrils at a neutral pH.Some lysophospholipids, especially lysophosphatidic acid (LPA), induced not only the extension of Abeta2M amyloid fibrils but also the formation of Abeta2M amyloid fibrils from the beta2-m monomer at a neutral pH, by partially unfolding the compact structure of beta2-m to an amyloidogenic conformer as well as stabilizing the extended fibrils. Haemodialysis patients had significantly higher plasma concentrations of LPA than healthy subjects. Furthermore, uraemic plasmas with the highest ranking LPA concentrations stabilized Abeta2M amyloid fibrils significantly more potently than normal plasmas. On the other hand, simple addition of LPA to normal plasma did not enhance the fibril stabilizing activity.These results suggest a possible role of lysophospholipids in the development of Abeta2M amyloidosis.

How various anti-amyloidogenic compounds inhibit the formation of Alzheimer's beta-amyloid fibrils (fAbeta) from amyloid beta-peptide (Abeta) and destabilize fAbeta remains poorly understood. Using spectrophotometry, spectrofluorometry, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and surface plasmon resonance (SPR), we investigated the anti-amyloidogenic effects of five flavonoids on fAbeta in vitro. Oxidized flavonoids generally inhibited fAbeta(1-40) formation significantly more potently than fresh compounds. Characterization of the novel fluorescence of myricetin (Myr) emitted at 575 nm with an excitation maximum at 430 nm in the presence of fAbeta(1-40) revealed the specific binding of Myr to fAbeta(1-40). By SPR analysis, distinct association and dissociation reactions of Myr with fAbeta(1-40) were observed, in contrast to the very weak binding to the Abeta monomer. A significant decrease in the rate of fibril extension was observed when0.5 microM Myr was injected into the SPR experimental system. These findings suggest that flavonoids, especially Myr, exert an anti-amyloidogenic effect in vitro by preferentially and reversibly binding to the amyloid fibril structure of fAbeta, rather than to Abeta monomers.

This research was originally published in the Journal of Biological Chemistry. Yumiko Ohhashi, Miho Kihara, Hironobu Naiki and Yuji Goto. Ultrasonication-induced Amyloid Fibril Formation of β2-Microglobulin. J. Biol. Chem. 2005; 280, 32843-32848. © the American Society for Biochemistry and Molecular Biology, To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on β2 -microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded β2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1–2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native β2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of β2-m, producing fibrils adapted to the respective pH.

Although native beta(2)-microglobulin (beta2-m), the light chain of the major histocompatibility complex class I antigen, assumes an immunoglobulin domain fold, it is also found as a major component of dialysis-related amyloid fibrils. In the amyloid fibrils, the conformation of beta2-m is considered to be largely different from that of the native state, and a monomeric denatured form is likely to be a precursor to the amyloid fibril. To obtain insight into the conformational dynamics of beta2-m leading to the formation of amyloid fibrils, we studied the reduction and reoxidation of the disulfide bond by reduced and oxidized dithiothreitol, respectively, and the effects on the reduction of the chaperonin GroEL, a model protein that might destabilize the native state of beta2-m. We show that beta2-m occasionally unfolds into a denatured form even under physiological conditions and that this transition is promoted upon interaction with GroEL. The results imply that in vivo interactions of beta2-m with other proteins or membrane components could destabilize its native structure, thus stabilizing the amyloid precursor.

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.