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This research was originally published in the Journal of Biological Chemistry. Masayuki Adachi, Masatomo So, Kazumasa Sakura, József Kardos and Yuji Goto. Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation. J. Biol. Chem. 2015; 290, 18134-18145. © the American Society for Biochemistry and Molecular Biology, Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.

The roles of non-native α-helices frequently observed in the initial folding stage of β-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine β-lactoglobulin (βLG) in quenched-flow pH-pulse labeling experiments. βLG assumes a collapsed intermediate with a non-native α-helical structure (I0) in the early stage of folding, although its native form is predominantly composed of β-structures. The protection profile in I0 of pseudo-wild type (WT*) βLG was found to deviate from the pattern of the “average area buried upon folding” (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I0 of E44L, the mutant with an increased β-sheet propensity, was distinct from that of WT* and resembled the AABU...

The larvae of a water scavenger beetle, Hydrophilus acuminatus, have strongly asymmetric mandibles; the right one is long and slender, whereas the left one is short and stout. The fine structure and embryonic development of the head capsule and mandibles of this species were examined using light and scanning electron microscopy, and asymmetries in shape were detected in these structures applying an elliptic Fourier analysis. The larval mandibles are asymmetric in the following aspects: whole length, the number, structure and arrangement of retinacula (inner teeth), and size and shape of both the molar and incisor regions. The larval head is also asymmetric; the left half of the head capsule is larger than the right, and the left adductor muscle of the mandible is much thicker than the right. The origin and developmental process of asymmetric mandibles were traced in developing embryos whose developmental period is about 270 h and divided into 10 stages. Mandibular asymmetries are produced by the cumulative effects of six stepwise modifications that occur from about 36% of the total developmental time onward. The significance of these modifications was discussed with respect to the functional advantages of asymmetries and the phylogeny of members of the Hydrophilidae.

Oligomer species of amyloid β (Aβ) peptides are intensively investigated because of their relevance to Alzheimer's disease (AD), and a stable oligomer will be a cause of AD. In this article, we investigate the structural stability of two representative Aβ

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

This research was originally published in the Journal of Biological Chemistry. Yoko Akazawa-Ogawa, Mizuki Takashima, Young-Ho Lee, Takahisa Ikegami, Yuji Goto, Koichi Uegaki and Yoshihisa Hagihara. Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications. J. Biol. Chem. 2014; 289, 15666–15679. © the American Society for Biochemistry and Molecular Biology, The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.

The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains. Gibbs energy changes strongly correlated with the number of hydrogen bond acceptors of ligand. Thus, the broad binding capability of L-PGDS for ligands should be viewed as hydrophilic interactions delicately tuned by enthalpy–entropy compensation using combined effects of hydrophilic and hydrophobic interactions.

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.

本研究では、-yoに前接する形態素の最終音節の末子音が、n挿入にどのような影響を与えるのか、ソウルを含む京畿道方言話者3名、全羅道方言話者4名そして慶尚道方言話者3名を対象とした認知実験を行った。実験結果によれば、n挿入の選好度について形態素最終音節の末子音の影響は小さく、n挿入を好む比率が方言ごとに異なることが明らかとなった。さらに、先行研究では言及されてこなかった男女間での違いも確認された。このような-yoに見られるn挿入の選好度が地域方言や性別により異なるという事実は、言語変異研究の重要性を示唆する。

In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0-phase (G0-SCNT group) or early G1-phase (eG1-SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0-phase and eG1-phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake-off method). The fusion rate in the G0-SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1-SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1-SCNT groups (12.7%) per recipient cow was higher than that in G0-SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0-SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1-SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1-phase using the shake-off method improved the overall production efficiency of the clone offspring per transferred embryo.

High-frequency (~ 55 MHz) wireless quartz-crystal microbalance biosensor was used for studying heterogeneous deposition behavior of Aβ(1-40) peptide on Aβ(1-42) nuclei, which were grown under the stirring agitation and 200-kHz ultrasonication at pH 2.2, 4.6, and 7.4. The deposition reaction was monitored over 40 h, and the deposition rate was deduced. Among the agitation nuclei, the maximum deposition rate was observed on the nucleus grown at pH 4.6. However, ultrasonication nucleus grown at pH 7.4 produced much larger deposition rate, despite the same β-sheet concentration. This result indicates that local structural modulation is caused in the nucleus by ultrasonication, which adsorbs the Aβ peptide more actively than other nuclei. The resultant deposits clearly show oligomeric structure.

The inhibition of fibril formation of amyloid β (Aβ) and the disaggregation of Aβ fibrils are the promising approaches for a medical treatment of Alzheimer's disease (AD) therapy. In this study, we investigated the effects of liposomes on dopamine-induced disaggregation of Aβ fibrils by using the variety of liposomes. The used liposomes were normal liposomes, raft-forming liposomes, charged liposomes and oxidized liposomes. Those liposome could accelerate the disaggregation rate of fibrils. From the comparison of normal and charged liposomes, a certain contribution of dopamine via an electrostatic interaction to the disaggregation was confirmed. From raft-forming and oxidized liposomes, we revealed a significant contribution of bound water to liposomes, which could assist the formation of the quinine-form of dopamine by a removal of its proton. It is, therefore, concluded that the membrane surface of liposomes is considered to be an adequate environment for the dopamine-induced disaggregation of fibrils.

The kinetic folding of β2-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s− 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s− 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).

Amyloid fibrils with an ordered cross-β structure are one form of protein aberrant aggregates. Fibrils themselves and on-pathway small aggregates are involved in many neurodegenerative diseases and amylodoses. Over the past decade, much has been learned about the conformation of amyloid fibrils by using various biochemical and biophysical approaches. Amyloid fibrils accommodate rigid core structures composed of regular intra- and intermolecular non-covalent bonds such as hydrogen bonds, and disordered flexible regions exposed to solvents. In contrast to the improved understanding of fibril structures, few studies have investigated the short-living monomeric intermediates which interact with amyloid fibrils for elongation and the self-associated intermediates in the course of amyloidogenesis at the residue level. To study static fibrillar structures and kinetic intermediates, hydrogen/deuterium exchange (HD(ex)) coupled with solution-state NMR spectroscopy is one of the most powerful methods with a high time and atomic resolution. Here, we review studies on the structural properties of amyloid fibrils based on a combination of dimethylsulfoxide-quenched HD(ex) and NMR spectroscopy. Recent studies on transient kinetic intermediates during fibril growth by means of pulse-labeling HD(ex) aided by a quenched-flow apparatus and NMR spectroscopy are focused on.

The aggregation of proteins into amyloid fibrils is a topic that has attracted great interest because the process is associated with the pathology of numerous human diseases. Despite considerable progress in the elucidation of the structure of amyloid fibrils and the kinetic mechanism of their formation, knowledge on the thermodynamic aspects underlying the formation and stability of amyloid fibrils is limited. In this review, we summarize recent calorimetric studies of amyloid fibril formation, with the goal of obtaining a better understanding of the causal factors that thermally induce proteins to aggregate into amyloid fibrils. Calorimetric data show that differential scanning calorimetry is a useful technique to study the causative factors that thermally trigger the conversion to the amyloid structure and highlight the physics related to the thermal fluctuation of proteins during this conversion.

Katsuya Araki, Naoto Yagi, Rie Nakatani, Hiroshi Sekiguchi, Masatomo So, Hisashi Yagi, Noboru Ohta, Yoshitaka Nagai, Yuji Goto & Hideki Mochizuki "A small-angle X-ray scattering study of alpha-synuclein from human red blood cells", Scientific Reports, volume 6, Article number: 30473, Springer Nature, 2016, α-synuclein (α-syn) is the main component of Lewy bodies, which are neuropathological hallmarks of patients with Parkinson’s disease. As it has been controversial whether human α-syn from erythrocytes exists as a tetramer under physiological conditions, we tried solving this issue by the small-angle X-ray solution scattering method. Under two different conditions (high ionic strength with a Tris buffer and low ionic strength with an ammonium acetate buffer), no evidence was found for the presence of tetramer. When comparing erythrocyte and recombinant α-syn molecules, we found no significant difference of the molecular weight and the secondary structure although the buffer conditions strongly affect the radius of gyration of the protein. The results indicate that, even though a stable tetramer may not be formed, conformation of α-syn depends much on its environment, which may be the reason for its tendency to aggregate in cells.

Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation.

Kichitaro Nakajima, Hirotsugu Ogi, Kanta Adachi, Kentaro Noi, Masahiko Hirao, Hisashi Yagi & Yuji Goto "Nucleus factory on cavitation bubble for amyloid β fibril", Scientific Reports, volume 6, Article number: 22015, Springer Nature, 2016, Structural evolution from monomer to fibril of amyloid β peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability.

The PHENIX experiment at the Relativistic Heavy Ion Collider has measured φ meson production and its nuclear modification in asymmetric Cu + Au heavy

Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β-lactoglobulin (βLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻⁴ M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions.

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.

Alzheimer’s disease is the most common form of senile dementia. This neurodegenerative disorder is characterized by an amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, amyloid β (Aβ). The modeling of a senile plaque formation on a model neuronal membrane under the physiological condition is an attractive issue. In this study, we used anionic liposomes to model the senile plaque formation by Aβ. The growth behavior of amyloid Aβ fibrils was directly observed, revealing that the induction of the spherulitic Aβ aggregates could result from the growth of seeds in the presence of anionic liposomes. The seeds of Aβ fibrils strongly interacted with negatively charged liposome and the subsequent association of the seeds were induced to form the seed cluster with many growth ends, which is advantageous for the formation of spherulitic Aβ aggregates. Therefore, anionic liposomes mediated not only fibril growth but also the aggregation process. These results imply that anionic liposome membranes would affect the aggregate form of Aβ fibrils. The modeling of senile plaque reported here is considered to have great potential for study on the amyloidosis.

A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, "flow-and-stop" control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily and a secretory lipid-transporter protein, which binds a wide variety of hydrophobic small molecules. Here we show the feasibility of a novel drug delivery system (DDS), utilizing L-PGDS, for poorly water-soluble compounds such as diazepam (DZP), a major benzodiazepine anxiolytic drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and anticonvulsant. Calorimetric experiments revealed for both compounds that each L-PGDS held three molecules with high binding affinities. By mass spectrometry, the 1:3 complex of L-PGDS and NBQX was observed. L-PGDS of 500 μM increased the solubility of DZP and NBQX 7- and 2-fold, respectively, compared to PBS alone. To validate the potential of L-PGDS as a drug delivery vehicle in vivo, we have proved the prospective effects of these compounds via two separate delivery strategies. First, the oral administration of a DZP/L-PGDS complex in mice revealed an increased duration of pentobarbital-induced loss of righting reflex. Second, the intravenous treatment of ischemic gerbils with NBQX/L-PGDS complex showed a protective effect on delayed neuronal cell death at the hippocampal CA1 region. We propose that our novel DDS could facilitate pharmaceutical development and clinical usage of various water-insoluble compounds.

Y. Matsushita, H. Sekiguchi, K. Ichiyanagi, N. Ohta, K. Ikezaki, Y. Goto & Y. C. Sasaki "Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks", Scientific Reports, volume 5, Article number: 17647, Springer Nature, 2015, Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 μs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Components of cherry trees have been used as traditional herbal remedies for various diseases. These components are known to possess antioxidative effects. However, the mechanisms underlying cherry tree component-mediated antioxidative effects remain largely unknown. This study focused on cherry leaves extract (CLE) and examined the mechanism underlying the effect of CLE on tert-butyl hydroperoxide (t-BOOH)-induced melanocytic cell death with DNA damage. Interestingly, CLE prevented t-BOOH-induced cell death with reduction in DNA damage, p38 kinase activation, and reactive oxygen species (ROS) production. CLE-mediated suppression of cell death with reduction of DNA damage, p38 kinase activity and ROS production was prevented by a thioredoxin (Trx) system inhibitor but not by a glutathione (GSH) system inhibitor. Finally, data showed that CLE prevented t-BOOH-induced reduction of Trx2 but not Trx1 and Trx reductases (TrxR1 and TrxR2) protein expression. Thus, our results suggest that CLE prevents t-BOOH-induced reduction in Trx2 expression, promotion of ROS production, activation of p38 kinase, and increase in DNA damage and that it protects against cell death.

To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HD(ex)). Fd-FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd-FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HD(ex) rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP(+) (H), were quenched in the closed form, thereby facilitating exit of NADP(+) (H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP(+) (H) via FNR.

This research was originally published in the Journal of Biological Chemistry. Kotaro Yanagi, Mizue Ashizaki, Hisashi Yagi, Kazumasa Sakurai, Young-Ho Lee and Yuji Goto. Hexafluoroisopropanol Induces Amyloid Fibrils of Islet Amyloid Polypeptide by Enhancing Both Hydrophobic and Electrostatic Interactions. J. Biol. Chem. 2011; 286, 23959-23966. © the American Society for Biochemistry and Molecular Biology, Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ̅o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.

Ogi H., Fukunishi Y., Yanagida T., et al. "Seed-dependent deposition behavior of Aβ peptides studied with wireless quartz-crystal-microbalance biosensor", Analytical Chemistry, 83(12), 4982-4988, May 11, 2011. Copyright © 2011 American Chemical Society. https://doi.org/10.1021/ac2007703., Real-time monitoring of the deposition processes of Aβ1-40 and Aβ1-42 peptides on various seeds has been performed using a 55 MHz wireless quartz-crystal microbalance (QCM) over long-time periods (∼40 h). Dissolved peptide solutions were stirred for nucleation and growth of seeds at pH = 7.4 and 4.6, which were immobilized on the sensor chips. The isolated Aβ peptides were then flowed at the neutral pH, focusing on the interaction between the seeds and the monomers (or small multimers), excluding other interactions among seeds and other aggregates. The thioflavin-T fluorescence assay and atomic-force microscopy were used for evaluating structures of the seeds and deposited aggregates. The deposition rate, determined by the frequency decrease, is about 100 monomers/nm2/year in the case of fibril formation. The notable deposition behavior was observed in the deposition of Aβ1-40 peptide on Aβ1-42 seeds grown at the lower pH, which can be an important model for Alzheimer's disease. © 2011 American Chemical Society.

This research was originally published in the Journal of Biological Chemistry. Daisaku Ozawa, Yuichi Kaji, Hisashi Yagi, Kazumasa Sakurai, Toru Kawakami, Hironobu Naiki, and Yuji Goto. Destruction of Amyloid Fibrils of Keratoepithelin Peptides byLaser Irradiation Coupled with Amyloid-specific Thioflavin T. J. Biol. Chem. 2011; 286, 10856-10863. © the American Society for Biochemistry and Molecular Biology, Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β2-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D phys , and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration ( R g ) of the guanidine unfolded state (U) is 26.9 ± 0.7 A, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R g value extrapolated from kinetic R g data to time zero, R g,0 , is 24.3 ± 0.1 A, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 A). After the burst-phase change, a single-exponential reduction in R g 2 was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R g of the minor component of D phys containing the non-native cis proline isomer (D phys,cis ) to be 25.7 ± 0.6 A. Moreover, R g of the major component of D phys containing the native proline isomer (D phys,tra ) was estimated as 23.9 ± 0.2 A based on R g,0 . Consequently, both components of the burst-phase intermediate of barnase (D phys,tra and D phys,cis ) are still largely expanded. It was inferred that D phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.

The cross section and xF dependence of AN of very forward neutron production in polarized p + p collisions at √s = 200 GeV were measured in the PHENIX experiment at RHIC. The measured cross sections were consistent with the xF scaling claimed by the ISR experiment. Significant negative AN was observed in the forward region, and no significant backward AN was observed. We also measured √s and pT dependence of AN of very forward neutron production at √s from 62.4 to 500 GeV.

alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.

The conversion of the soluble, nontoxic amyloid-beta (Abeta) peptide into an aggregated, toxic form rich in beta-sheets is considered a key step in the development of Alzheimer's disease. Whereas growing evidence indicates that the Abeta amyloid fibrils consist of in-register parallel beta-sheets, little is known about the structure of soluble oligomeric intermediates because of their transient nature. To understand the mechanism by which amyloid fibrils form, especially the initial development of the "nucleus" oligomeric intermediates, we prepared covalently linked dimeric Abeta peptides and analyzed the kinetics of the fibril-forming process. A covalent bond introduced between two Abeta molecules dramatically facilitated the spontaneous formation of aggregates with a beta-sheet structure and affinity for thioflavin T. Transmission electron microscopy revealed, however, that these aggregates differed in morphology from amyloid fibrils, more closely resembling protofibrils. The protofibril-like aggregates were not the most thermodynamically stable state but were a kinetically trapped state. The results emphasize the importance of the conformational flexibility of the Abeta molecule and a balance in the association and dissociation rate for the formation of rigid amyloid fibrils.

This research was originally published in the Journal of Biological Chemistry. Hisashi Yagi, Daisaku Ozawa, Kazumasa Sakurai, Toru Kawakami, Hiroki Kuyama, Osamu Nishimura,Toshinori Shimanouchi, Ryoichi Kuboi, Hironobu Naiki, and Yuji Goto. Laser-induced Propagation and Destruction of Amyloid β Fibrils. J. Biol. Chem. 2010; 285, 19660-19667. © the American Society for Biochemistry and Molecular Biology, The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β2-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Amyloid fibril structures of peptides having the sequence of the #20–41 region of β2-microglobulin (β2m20–41 and β2m21–29) were investigated with FTIR microscope spectroscopy. pH dependence of the ...

The pathological process of Alzheimer’s disease is closely related to amyloid fibril formation by the causative protein, amyloid  (A). The growth behavior of A fibrils is predominated by the seeds-monomeric A interaction. In this study, the local hydrophobicity of seeds of A fibrils was investigated by the aqueous two-phase partitioning method to evaluate the hydrophobic interaction between the seed-monomeric A. The seeds showed a high local hydrophobicity relative to the monomer and fibrils. From the fibril growth experiment and the additive effect of Triton X-100, we could demonstrate the contribution of the hydrophobic seeds-monomer interaction to fibril formation.

Y. Matsushita, H. Sekiguchi, C. Jae Wong, M. Nishijima, K. Ikezaki, D. Hamada, Y. Goto & Y. C. Sasaki "Nanoscale Dynamics of Protein Assembly Networks in Supersaturated Solutions", Scientific Reports, Volume 7, Article number: 13883, Springer Nature, 2017, Proteins in solution are conventionally considered macromolecules. Dynamic microscopic structures in supersaturated protein solutions have received increasing attention in the study of protein crystallisation and the formation of misfolded aggregates. Here, we present a method for observing rotational dynamic structures that can detect the interaction of nanoscale lysozyme protein networks via diffracted X-ray tracking (DXT). Our DXT analysis demonstrated that the rearrangement behaviours of lysozyme networks or clusters, which are driven by local density and concentration fluctuations, generate force fields on the femtonewton to attonewton (fN – aN) scale. This quantitative parameter was previously observed in our experiments on supersaturated inorganic solutions. This commonality provides a way to clarify the solution structures of a variety of supersaturated solutions as well as to control nucleation and crystallisation in supersaturated solutions.

In mammals, the dosage compensation of sex chromosomes between males and females is achieved by transcriptional inactivation of one of the two X chromosomes in females. However, a number of genes escape X-inactivation in humans. It remains poorly understood how the transcriptional activity of these 'escape genes' is maintained despite the chromosome-wide heterochromatin formation. To address this question, we analyzed a putative chromatin boundary between the inactivated RBM10 and an escape gene, UBA1/UBE1. Chromatin immunoprecipitation revealed that trimethylated histone H3 lysine 9 and H4 lysine 20 were enriched in the last exon through the proximal downstream region of RBM10, but were remarkably diminished at approximately 2 kb upstream of the UBA1 transcription start site. Whereas RNA polymerase II was not loaded onto the intergenic region, CTCF (CCCTC binding factor) was enriched around the boundary, where some CpG sites were hypomethylated specifically on inactive X. These findings suggest that local DNA hypomethylation and CTCF binding are involved in the formation of a chromatin boundary, which protects the UBA1 escape gene against the chromosome-wide transcriptional silencing.

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteins to form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorter fibrils. Here, combining these 2 opposing effects on β 2 -microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of ≈1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis.

Bovine beta-lactoglobulin (beta LG) has been one of the most extensively studied proteins in the history of protein science mainly because its abundance in cow's milk makes it readily available to researchers. However, compared to other textbook proteins, progress in the study of beta LG has been slow because of obstacles such as a low reversibility from denaturation linked with thiol-disulfide exchange or monomer-dimer equilibrium preventing a detailed NMR analysis. Recently, the expression of various types of recombinant beta LGs combined with heteronuclear NMR analysis has significantly improved understanding of the physico-chemical properties of beta LG. In this review, we address several topics including pH-dependent structural dynamics, ligand binding, and the complex folding mechanism with non-native intermediates. These unique properties might be brought about by conformational frustration of the beta LG structure, partly attributed to the relatively large molecular size of beta LG. We expect studies with beta LG to continue to reveal various important findings, difficult to obtain with small globular proteins, leading to a more comprehensive understanding of the conformation, dynamics and folding of proteins.