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Lipid interaction with α-synuclein (αSyn) has been long implicated in the pathogenesis of Parkinson’s disease (PD). However, it has not been fully determined which lipids are involved in the initiation of αSyn aggregation in PD. Here exploiting genetic understanding associating the loss-of-function mutation in Synaptojanin 1 (SYNJ1), a phosphoinositide phosphatase, with familial PD and analysis of postmortem PD brains, we identified a novel lipid molecule involved in the toxic conversion of αSyn and its relation to PD. We first established a SYNJ1 knockout cell model and found SYNJ1 depletion increases the accumulation of pathological αSyn. Lipidomic analysis revealed SYNJ1 depletion elevates the level of its substrate phosphatidylinositol-3,4,5-trisphosphate (PIP3). We then employed Caenorhabditis elegans model to examine the effect of SYNJ1 defect on the neurotoxicity of αSyn. Mutations in SYNJ1 accelerated the accumulation of αSyn aggregation and induced locomotory defects in the nematodes. These results indicate that functional loss of SYNJ1 promotes the pathological aggregation of αSyn via the dysregulation of its substrate PIP3, leading to the aggravation of αSyn-mediated neurodegeneration. Treatment of cultured cell line and primary neurons with PIP3 itself or with PIP3 phosphatase inhibitor resulted in intracellular formation of αSyn inclusions. Indeed, in vitro protein–lipid overlay assay validated that phosphoinositides, especially PIP3, strongly interact with αSyn. Furthermore, the aggregation assay revealed that PIP3 not only accelerates the fibrillation of αSyn, but also induces the formation of fibrils sharing conformational and biochemical characteristics similar to the fibrils amplified from the brains of PD patients. Notably, the immunohistochemical and lipidomic analyses on postmortem brain of patients with sporadic PD showed increased PIP3 level and its colocalization with αSyn. Taken together, PIP3 dysregulation promotes the pathological aggregation of αSyn and increases the risk of developing PD, and PIP3 represents a potent target for intervention in PD.

In the Japanese hagfish,Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n=52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n=36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome ofE. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1–6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes ofE. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7–10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1–10) by fluorescencein situhybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2–10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

Lipocalin-type prostaglandin D synthase (L-PGDS) binds various hydrophobic small molecules. Since we aim to use human L-PGDS as a carrier in a drug delivery system (DDS) for poorly water-soluble drugs, quality control of the protein is indispensable. In this study, we investigated the thermodynamic stability of human L-PGDS under various pH conditions. Differential scanning calorimetry revealed that the thermal unfolding of L-PGDS was an almost-reversible two-state transition between the native and unfolded states over the pH range from 2.5 to 7.4. The linear relationship of ΔH(Tm) to Tm in this pH range gave a heat capacity change (ΔCp) of 4.76 kJ/(K·mol), which was small compared to those commonly found in globular proteins. The temperature-dependent free energy of unfolding, ΔG(T), specified by Tm, ΔH(Tm) and ΔCp, showed a pH dependence with the highest value at pH 7.4 closest to the isoelectric point of 8.3. The small value of Cp resulted in a large value of ΔG(T), which contributed to the stability of the protein. Taken together, these results demonstrated that human L-PGDS is sufficiently thermostable for storage and practical use and can be useful as a delivery vehicle of protein-based DDS.

Protein aggregation causes intractable diseases such as Alzheimer’s disease. In these diseases, crystal-like protein aggregates, so-called amyloid fibrils, form in vivo and induce severe malfunction of biological tissues. Amyloid fibril formation comprises primary nucleation from a supersaturated solution of protein monomers and subsequent fibril growth. Within them, primary nucleation possesses an extremely high energy barrier, resulting in a long induction time for nucleation, typically over a decade in vivo and several days even in a test tube. Our previous study (1) revealed that ultrasonic irradiation to supersaturated protein solution drastically accelerates amyloid formation because of the effects of ultrasonic cavitation, which is applicable to clinical diagnosis (2). However, the degree of acceleration has not been controlled yet. Furthermore, the reproducibility of the induction time for nucleation highly depends on ultrasonic-irradiation conditions and the degree of supersaturation of initial protein solutions. Herein, we systematically investigate the optimized condition to accurately control amyloid formation using an originally developed sonoreactor (3) and discuss the underlying mechanism governing ultrasonic induction of amyloid formation. (1) K. Nakajima et al., Ultrason. Sonochem. 36, 206-211 (2017). (2) K. Nakajima et al., Nat. Commun. 13, 5689 (2022). (3) K. Nakajima et al., Ultrason. Sonochem. 73 105508 (2022).

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

The cluster structures of hydrated aminopyrazines, APz-(H2O)n=2-4, in supersonic jets have been investigated measuring the size-selected electronic and vibrational spectra and determined with the aid of quantum chemical calculations. The APz-(H2O)2 structure is assigned as a cyclic N1 type where a homodromic hydrogen-bond chain starts from the amino group and ends at the 1-position nitrogen atom of the pyrazine moiety, corresponding to 2-aminopyridine-(H2O)2. On the other hand, APz-(H2O)n=3,4 has a linear hydrogen-bond network ending at the 4-position one (N4), which resembles 3-aminopyridine-(H2O)n=3,4. The hydrogen-bond network switching from the N1 type to the N4 one provides the accompanying red shifts of the S1-S0 electronic transition that are entirely consistent with those of the corresponding 2-aminopyridine and 3-aminopyridine clusters and also shows the drastically strengthened fluorescence intensity of origin bands in the electronic spectrum. The significant change in the excited-state dynamics is explored by the fluorescence lifetime measurement and the time-dependent density functional theory (TD-DFT) calculation. It is suggested that the drastic elongation of fluorescence lifetimes is due to the change in the electronic structure of the first excited state from nπ* to ππ*, resulting in the decreasing spin-orbit coupling to T1 (ππ*).

α-Synuclein inclusions are a pathological hallmark of several neurodegenerative diseases. Although it has been demonstrated a relationship between fibril polymorphism and different pathologies, the molecular origins of polymorphism are not understood. Employing biophysical approaches, we revealed that the conformational state of the monomeric αSyn is responsible for fibril polymorphism: αSyn adopts specific conformations at high NaCl that produce rod fibrils, and different conformations at low NaCl that generate twisted fibrils. Using NMR, we found that the high NaCl conformations establish a polar interaction between the initial part of the NAC region and a wide section of the C-terminus domain. These high NaCl conformations can be commonly promoted by changes in the chemical environment, like NaCl, the presence of Ca2+or cellular components, like endotoxins, that alter the interaction NAC/C-terminus domain. Our results provide mechanistic insights that explain how the behavior of the C-terminus domain imparts polymorphism during the fibril formation.Significance StatementThe accumulation of the protein α-Synuclein into amyloid aggregates in the brain is a key characteristic of neurodegenerative disorders like Parkinson’s disease and multiple system atrophy. Intensive research has demonstrated that structurally different amyloid fibrils are related to the development of different diseases; however, the molecular mechanisms that originate such fibril diversity from the same protein remain unknown. In this work, we discovered that the conformational state of the monomeric αSyn, regulated by an intramolecular polar interaction NAC region/C-terminus domain, is crucial for the generation of different fibrils. Our results represent the monomeric molecular events behind the diversity of fibrils and open the conformational state of αSyn as a target to understand how the fibrils get formed in the brain.

Till date, there had been no reported case of dialysis-related amyloidosis (DRA) associated with a β2-microglobulin variant. We report here a 41-year-old haemodialysis patient with systemic amyloidosis, exhibiting macroglossia and swelling salivary glands, uncommon clinical manifestations for DRA. Molecular analysis showed that the patient had a new variant of β2-microglobulin (V27M). Extracted amyloid protein was predominantly composed of variant β2-microglobulin. In vitro analysis revealed that this variant β2-microglobulin had a strong amyloidogenic propensity, probably owing to the decreased stability caused by a bulky methionine residue. Our data clearly show that V27M variant is amyloidogenic and this mutation results in unusual clinical manifestations. To date, only one amyloidogenic β2-microglobulin variant (D76N) has been reported in non-dialysis patients. It is noteworthy that the V27M and D76N variants show substantial differences in both clinical phenotypes and pathomechanical features. This is the first case of DRA associated with a naturally occurring β2-microglobulin variant.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1β, and interleukin-6, in macrophages, and the effect was polyP dose– and chain length–dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.

Amyloid formation in vivo occurs under complicated conditions in which various amyloidogenic and non-amyloidogenic components coexist, often under crowding. Controversy surrounds the role of additional components under complicated conditions. They have been suggested to accelerate amyloid formation because molecular crowding or interactions with additives increase effective concentrations and, thus, break the supersaturation of amyloidogenic proteins. On the other hand, cellular crowding conditions with various heterogeneous components may retard or prevent amyloid formation because they impede homologous amyloidogenic associations. To elucidate the roles of these additional components, we examined the amyloid formation of β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, with a simplified model system in which intact β2m and its proteolytic peptides coexist. Among the nine proteolytic peptides of β2m produced in vitro with lysyl endopeptidase, the 22-residue K3 peptide is highly amyloidogenic. The amyloid formation of the K3 peptide, which occurred with a lag time of 1 h at pH 2 and 37 °C, was significantly retarded by the coexistence of β2m or a mixture of the proteolytic digests. To identify the sites of inhibitory interactions, we performed paramagnetic relaxation enhancement measurements using spin-labeled K3 and uniformly 15N-labeled β2m with nuclear magnetic resonance detection. The results revealed that K3 interacted weakly with a broad cluster of the hydrophobic residues of β2m, which accommodated the residues located in some distant sequence, leading to competitive inhibition. The results showed that relatively weak and broad interactions formed a nonproductive complex, implying a role for heterogeneous interactions under complicated conditions.

For agricultural purposes, the drainage and deforestation of Southeast Asian peatland resulted in high greenhouse gases’ (GHGs, e.g., CO2, N2O and CH4) emission. A peatland regenerating initiative, by rewetting and vegetation restoration, reflects evidence of subsequent forest recovery. In this study, we compared GHG emissions from three Malaysian tropical peatland systems under the following different land-use conditions: (i) drained oil palm plantation (OP), (ii) rewetting-restored forest (RF) and (iii) undrained natural forest (NF). Biweekly temporal measurements of CO2, CH4 and N2O fluxes were conducted using a closed-chamber method from July 2017 to December 2018, along with the continuous measurement of environmental variables and a one-time measurement of the soil physicochemical properties. The biweekly emission data were integrated to provide cumulative fluxes using the trapezoidal rule. Our results indicated that the changes in environmental conditions resulting from draining (OP) or rewetting historically drained peatland (RF) affected CH4 and N2O emissions more than CO2 emissions. The cumulative CH4 emission was significantly higher in the forested sites (RF and NF), which was linked to their significantly higher water table (WT) level (p < 0.05). Similarly, the high cumulative CO2 emission trends at the RF and OP sites indicated that the RF rewetting-restored peatland system continued to have high decomposition rates despite having a significantly higher WT than the OP (p < 0.05). The highest cumulative N2O emission at the drained-fertilized OP and rewetting-restored RF sites was linked to the available substrates for high decomposition (low C/N ratio) together with soil organic matter mineralization that provided inorganic nitrogen (N), enabling ideal conditions for microbial mediated N2O emissions. Overall, the measured peat properties did not vary significantly among the different land uses. However, the lower C/N ratio at the OP and the RF sites indicated higher decomposition rates in the drained and historically drained peat than the undrained natural peat (NF), which was associated with high cumulative CO2 and N2O emissions in our study.

Amyloid fibrils are formed from various proteins, some of which cause the corresponding neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. It has been reported that many compounds inhibit the formation of amyloid fibrils. Anthocyanins are flavonoid pigments present in fruits and vegetables, which are known to suppress symptoms related with Alzheimer's disease. However, the influence of anthocyanins on the amyloid fibril remains unclear. Here, we succeeded in the direct monitoring of the disaggregation reaction of single amyloid β (Aβ) fibrils by anthocyanins using total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM). It is found that the disassembly activity to the Aβ fibrils depends on the number of hydroxyl groups in six-membered ring B of anthocyanin, and only delphinidin-3-galactoside, possessing three hydroxyl groups there, shows high disassembly activity. Our results show the importance of the number of hydroxyl groups and demonstrate the usefulness of TIRFM-QCM as a powerful tool in studying interactions between amyloid fibrils and compounds.

This study aimed to investigate the effects of high and low levels of energy intake during the entire gestation period on the skeletal muscle development, organ development, and adipose tissue accumulation in fetuses of Wagyu (Japanese Black) cows, a breed with highly marbled beef. Cows were allocated to a high-nutrition (n = 6) group (fed 120% of the nutritional requirement) or low-nutrition (n = 6) group (fed 60% of the nutritional requirement). The cows were artificially inseminated with semen from the same sire, and the fetuses were removed by cesarean section at 260 ± 8.3 days of fetal age and slaughtered. The whole-body, total muscle, adipose, and bone masses of the fetal half-carcasses were significantly higher in the high-nutrition group than the low-nutrition group (p = 0.0018, 0.009, 0.0004, and 0.0362, respectively). Fifteen of 20 individual muscles, five of six fat depots, nine of 17 organs, and seven of 12 bones that were investigated had significantly higher masses in the high-nutrition group than the low-nutrition group. The crude components and amino acid composition of the longissimus muscle significantly differed between the low- and high-nutrition groups. These data indicate that maternal nutrition during gestation has a marked effect on the muscle, bone, and adipose tissue development of Wagyu cattle fetuses.

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of β2-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, i.e., epigallocatechin gallate (EGCG) and kaempferol-7-O-glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α-synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols. This article is protected by copyright. All rights reserved.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. β2-Microglobulin (β2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of β2m in patients are observed at neutral pH, formation of β2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that β2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration–dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the β2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.

Significance Lewy bodies (LBs), which mainly consist of α-syn, are neuropathological hallmarks of patients with Parkinson’s disease (PD). Recently, it has been reported that aggregates of α-syn with cross-β structures are capable of propagating within the brain in a prionlike manner. However, there is still no evidence that such propagation occurs in the patient’s brain. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and confirmed that aggregates of α-syn with a cross-β structure exist in brains of PD patients. Our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation and propagation of amyloid fibrils of α-syn., Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson’s disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-β structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-β sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-β structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-β structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of β2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged β2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of β2m at several micromoles per liter, a similar concentration range to that in vivo. Since β2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with β2m electrostatically. PolyPs appear to dehydrate water molecules around β2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of β2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis.

The hydration structures of 3-aminopyridine (3AP)–(H2O)n (n = 2–4) in supersonic jets have been investigated by measuring the electronic and vibrational spectra with the aid of quantum chemical calculations. The S1–S0 electronic transition of 3AP–(H2O)2 is observed at a slightly red-shifted position from 3AP–(H2O)1, while further hydration induces drastic red shifts and complicated vibrational structures. We assign the cluster structures of 3AP–(H2O)2 as a cyclic structure composed of the homodromic hydrogen bond (H-bond) chain connecting the pyridyl CH bond acting as the proton donor toward a pyridyl nitrogen acceptor. For 3AP–(H2O)n (n = 3, 4), on the other hand, the initial donor site in the H-bond network changes from a pyridyl CH group to an amino group. The observed red shift derived from H-bond network switching can be reproduced very well with the S1–S0 origin band estimation obtained by applying geometry optimization and subsequent harmonic vibrational analysis of (TD-)DFT calculations to each el...

The formation of amyloid fibrils of various amyloidogenic proteins is dramatically enhanced by ultrasound irradiation. To apply this phenomenon to the study of protein aggregation science and diagnosis of neurodegenerative diseases, a multichannel ultrasound irradiation system with individually adjustable ultrasound irradiation conditions is necessary. Here, we develop a sonochemical reaction system, where an ultrasonic transducer is placed in each well of a 96-well microplate to perform ultrasonic irradiation of sample solutions under various conditions with high reproducibility, and applied it to study the amyloid fibril formation of amyloid β, α-synuclein, β2-microglobulin, and lysozyme. The results clearly show that our instrument is superior to the conventional shaking method in terms of the degree of acceleration and reproducibility of fibril formation reaction. The acceleration degree is controllable by controlling the driving voltage applied to each transducer. We have thus succeeded in developing a useful tool for the study of amyloid fibril formation in various proteins.

Protein-bound uremic toxins (PBUTs) are difficult to remove using conventional dialysis treatment owing to their high protein-binding affinity. As pH changes the conformation of proteins, it may be associated with the binding of uremic toxins. Albumin conformation at pH 2 to 13 was analyzed using circular dichroism. The protein binding behavior between indoxyl sulfate (IS) and albumin was examined using isothermal titration calorimetry. Albumin with IS, and serum with IS, p-cresyl sulfate, indole acetic acid or phenyl sulfate, as well as serum from hemodialysis patients, were adjusted pH of 3 to 11, and the concentration of the free PBUTs was measured using mass spectrometry. Albumin was unfolded at pH &lt, 4 or &gt, 12, and weakened interaction with IS occurred at pH &lt, 5 or &gt, 10. The concentration of free IS in the albumin solution was increased at pH 4.0 and pH 11.0. Addition of human serum to each toxin resulted in increased free forms at acidic and alkaline pH. The pH values of serums from patients undergoing hemodialysis adjusted to 3.4 and 11.3 resulted in increased concentrations of the free forms of PBUTs. In conclusion, acidic and alkaline pH conditions changed the albumin conformation and weakened the protein binding property of PBUTs in vitro.