A DISC1 point mutation promotes oligomerization and impairs information processing in a mouse model of schizophrenia.

Disrupted-in-schizophrenia 1 (DISC1) is strongly associated with schizophrenia, but it remains elusive how the modification of the intermolecular interaction of DISC1 affects the information processing in brain. We show that a DISC1 point mutation alters intermolecular cohesiveness promoting the phase separation, and disrupts sensorimotor gating monitored by the prepulse inhibition in a mouse model of schizophrenia. Although the conformation of DISC1 partial peptide with the schizophrenia-related mutation L607F in human or the corresponding L604F in mouse was essentially indistinguishable from the wild type (WT) as long as monitored by fluorescence, circular dichroism, ultracentrifugation, dynamic light scattering and nuclear magnetic resonance, the atomic force microscopy was able to detect their morphological distinctions. The WT peptides were round and well dispersed, while mutants were inhomogeneous and disrupted to form dimer to trimer that aligned along one direction without apparent aggregate formation. Homozygous L604F mutant mice created by CRISPR exhibited the significant decrease in DISC1 level in the immunohistopathology at the hippocampal region compared to the WTs. The ratio of prepulse inhibition of the homozygous mutant mice was significantly impaired compared to WTs. Altered DISC1 distribution or function caused by aberrant intermolecular interactions may contribute to information processing characteristics in schizophrenia.

A valine-to-lysine substitution at position 210 induces structural conversion of prion protein into a ß-sheet rich oligomer.

Prion diseases are fatal neurodegenerative diseases associated with structural conversion of α-helical prion protein (PrP) into its ß-sheet rich isoform (PrPSc). Previous genetic analyses have indicated that several amino acid residues involved in the hydrophobic core of PrP (such as V180, F198, and V210) play a critical role in the development of prion diseases. To understand how these hydrophobic residues would contribute to the α-to-ß conversion process of PrP, we substituted the V210 residue with bulkier (V210F, V210I, and V210L), smaller (V210A), and charged amino acids (V210K) and characterized its effects. Interestingly, although most of the mutations had little or no effect on the biochemical properties of PrP, the V210K mutation induced structural conversion of PrP into a ß-structure. The ß-inducing effect was prominent and observed even under a physiological condition (i.e., in the absence of denaturant, acidic pH, reducing agent, and high temperature) in contrast to the disease-associated mutations in the PrP gene. We also examined structural features of V210K PrP using guanidine-hydrochloride unfolding, dynamic light scattering, 8-anilino-1-naphthalene sulfonate fluorescence, and electron microscopy, and revealed that V210K PrP assembles into a non-fibrillar ß-rich oligomer. Thus, the α-to-ß conversion can be induced by introduction of a charged residue into the hydrophobic core, which provide novel insight into the structural dynamics of PrP.

Poly-L-histidine inhibits prion propagation in a prion-infected cell line.

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GTFK), we identified poly-L-His as a novel anti-prion compound with an IC50 value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrPC and sterically blocks the structural conversion toward PrPSc. Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words).

Acceleration of nucleation of prion protein during continuous ultrasonication.

Although pulsatile irradiation of ultrasonication is frequently used for generating amyloid fibrils in vitro, the potential for inducing amyloid fibrillation of proteins during continuous ultrasonication is unknown. In this study, we implemented a continuous irradiation system and measured far-ultraviolet circular dichroism in a real-time manner. During the continuous ultrasonication, the conformation of full-length mouse prion protein (mPrP) was rapidly altered without a lag time and electron microscopy revealed that distorted fibrils, ß-oligomers and amorphous aggregates were formed at pH 2.2, 4.0 and 9.1, respectively. Similarly, hen egg white lysozyme formed distorted fibrils and small and large amorphous aggregates at pH 2.2 and 7.1 and 11.9, respectively, without a lag time. The concentration dependencies of the initial rates were different between the two systems. The aggregate formation of mPrP followed a first-order reaction, whereas that of lysozyme followed the zeroth-order reaction. Importantly, the reactions were immediately stopped by switching off ultrasonication, and restarted instantaneously when ultrasonication was restarted. Thus, the continuous ultrasonication significantly accelerates the nucleations of mPrP and lysozyme aggregates by the interaction between monomer and cavitation bubble. These cavitation bubbles may act as catalysts that decrease the activation free energy for nucleation, which is low in mPrP and high in lysozyme.

Formation and properties of amyloid fibrils of prion protein.

Amyloid fibrils formed from prion protein (PrP) are associated with prion diseases. In this review we discuss a number of extrinsic and intrinsic experimental factors related to the formation of PrP amyloid fibrils in vitro. We first examined the effects of ultrasonic power on the induction of amyloid fibrillation from PrP. The most important conclusion drawn from the results is that an applied ultrasonic power of approximately 2 W enhanced the nucleation of amyloid fibrils efficiently but that more powerful ultrasonication led to retardation of growth. We also reviewed evidence on the amyloidogenic regions of PrP based on peptide screening throughout the polypeptide sequence. These results showed that helix 2 (H2) peptides of PrP were capable of both the fibrillation and propagation of straight, long fibrils. Moreover, the conformation of preformed H2 fibrils changed reversibly depending on the pH of the solution, implying that interactions between side-chains modulated the conformation of amyloid fibrils. The evidence discussed in this review relates specifically to PrP but may be relevant to other amyloidogenic proteins.

A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein.

Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here, we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 µs, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways.

Synthesis of double-fluorescent labeled prion protein for FRET analysis.

An abnormal form of prion protein (PrP) is considered to be the pathogen in prion diseases. However, the structural details of this abnormal form are not known. To characterize the non-native structure of PrP, we synthesized position-specific double-fluorescent labeled PrP for a fluorescence resonance energy transfer (FRET) experiment. Using FRET, we observed a conformational change in the labeled PrP associated with amyloid fibril formation. The FRET analysis indicated that the distance between fluorescent labeled N- and C-terminal sites of PrP increased upon the formation of amyloid fibrils compared with that of the native state. This approach using FRET analysis is useful for elucidating the structure of abnormal PrP.

Acid-induced molten globule state of a prion protein: crucial role of Strand 1-Helix 1-Strand 2 segment.

The conversion of a cellular prion protein (PrP(C)) to its pathogenic isoform (PrP(Sc)) is a critical event in the pathogenesis of prion diseases. Pathogenic conversion is usually associated with the oligomerization process; therefore, the conformational characteristics of the pre-oligomer state may provide insights into the conversion process. Previous studies indicate that PrP(C) is prone to oligomer formation at low pH, but the conformation of the pre-oligomer state remains unknown. In this study, we systematically analyzed the acid-induced conformational changes of PrP(C) and discovered a unique acid-induced molten globule state at pH 2.0 termed the "A-state." We characterized the structure of the A-state using far/near-UV CD, 1-anilino-8-naphthalene sulfonate fluorescence, size exclusion chromatography, and NMR. Deuterium exchange experiments with NMR detection revealed its first unique structure ever reported thus far; i.e. the Strand 1-Helix 1-Strand 2 segment at the N terminus was preferentially unfolded, whereas the Helix 2-Helix 3 segment at the C terminus remained marginally stable. This conformational change could be triggered by the protonation of Asp(144), Asp(147), and Glu(196), followed by disruption of key salt bridges in PrP(C). Moreover, the initial population of the A-state at low pH (pH 2.0-5.0) was well correlated with the rate of the ß-rich oligomer formation, suggesting that the A-state is the pre-oligomer state. Thus, the specific conformation of the A-state would provide crucial insights into the mechanisms of oligomerization and further pathogenic conversion as well as facilitating the design of novel medical chaperones for treating prion diseases.

Nearly reversible conformational change of amyloid fibrils as revealed by pH-jump experiments.

pH-jump induced conformational transitions between substates of preformed amyloid fibrils made by a fragmented peptide of helix 2 (H2 peptide) of MoPrP were detected, and their kinetics were analyzed using a novel pH-jump apparatus specially designed for observing amyloids. Previously, we reported that H2 peptide formed ordered fibrils with a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils), but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5 (named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed, the CD spectrum changed instantly, but the finally observed ellipticities were clearly distinct from those of pH 7.5 fibrils. Thus, the finally observed state is termed 'pH 7.5-like fibrils'. However, pH 7.5-like fibrils reverted to the conformation very similar to that of the pH 2.9 fibrils when the pH of the solution was restored to 2.9. Then, we examined the kinetics of the nearly reversible conformational changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence stopped-flow, and we observed relatively fast phases (0.7-18 s(-1)). In contrast, the conversion between pH 7.5-like fibrils and pH 7.5 fibrils never occurred (<0.2 day(-1)). Thus, H2 fibrils can be switched readily between distinct conformations separated by a low energy barrier, while a large energy barrier clearly separated the different conformations. These conformational varieties of amyloid fibrils may explain the physical basis of the diversity in prion.

Characterizing antiprion compounds based on their binding properties to prion proteins: implications as medical chaperones.

A variety of antiprion compounds have been reported that are effective in ex vivo and in vivo treatment experiments. However, the molecular mechanisms for most of these compounds remain unknown. Here we classified antiprion mechanisms into four categories: I, specific conformational stabilization; II, nonspecific stabilization; III, aggregation; and IV, interaction with molecules other than PrP(C). To characterize antiprion compounds based on this classification, we determined their binding affinities to PrP(C) using surface plasmon resonance and their binding sites on PrP(C) using NMR spectroscopy. GN8 and GJP49 bound specifically to the hot spot in PrP(C), and acted as "medical chaperones" to stabilize the native conformation. Thus, mechanisms I was predominant. In contrast, quinacrine and epigallocathechin bound to PrP(C) rather nonspecifically; these may stabilize the PrP(C) conformation nonspecifically including the interference with the intermolecular interaction following mechanism II. Congo red and pentosan polysulfate bound to PrP(C) and caused aggregation and precipitation of PrP(C), thus reducing the effective concentration of prion protein. Thus, mechanism III was appropriate. Finally, CP-60, an edarabone derivative, did not bind to PrP(C). Thus these were classified into mechanism IV. However, their antiprion activities were not confirmed in the GT + FK system, whose details remain to be elucidated. This proposed antiprion mechanisms of diverse antiprion compounds could help to elucidate their antiprion activities and facilitate effective antiprion drug discovery.

Proper calibration of ultrasonic power enabled the quantitative analysis of the ultrasonication-induced amyloid formation process.

To elucidate the mechanisms of ultrasonication on the amyloid fibril formation, we quantitatively determined the ultrasonic power using both calorimetry and potassium iodide (KI) oxidation, and under the properly calibrated ultrasonic power, we investigated the ultasonication-induced amyloid formation process of the mouse prion protein (mPrP(23-231)). These methods revealed that the ultrasonic power in our system ranged from 0.3 to 2.7 W but entirely dependent on the positions of the ultrasonic stage. Intriguingly, the nucleation time of the amyloid fibrils was found to be shortened almost proportionally to the ultrasonic power, indicating that the probability of the occurrence of nucleus formation increases proportionally to the ultrasonic power. Moreover, mPrP(23-231) formed two types of aggregates: rigid fibrils and short fibrils with disordered aggregates, depending on the ultrasonic power. The nucleation of rigid fibrils required an ultrasonic power larger than 1.5 W. While at the strong ultrasonic power larger than 2.6 W, amyloid fibrils were formed early, but simultaneously fine fragmentation of fibrils occurred. Thus, an ultrasonic power of approximately 2.0 W would be suitable for the formation of rigid mPrP(23-231) fibrils under the conditions utilized (ultrasonication applied for 30 s every 9 min). As ultrasonication has been widely used to amplify the scrapie form of the prion protein, or other amyloids in vitro, the calorimetry and KI oxidation methods proposed here might help determining the adequate ultrasonic powers necessary to amplify them efficiently.

Critical region for amyloid fibril formation of mouse prion protein: unusual amyloidogenic properties of the helix 2 peptide.

To gain insight into the structural mechanism of the conformational conversion process of prion, we examined the potential amyloidogenic property of each secondary structural element in a mouse prion protein (mPrP) and discriminated their relative significance for the formation of amyloid fibrils. Although peptides corresponding to alpha-helix 2 and alpha-helix 3 (named H2 peptide and H3 peptide, respectively) formed the amyloid-like fibrils, their structures were quite different. H2 fibrils formed the ordered beta-sheet with the beta-turn conformation, and the resultant fibrils were long and straight. In contrast, H3 fibrils consisted of the beta-sheet with the random conformation, and the resultant fibrils were short and flexible. These properties are basically consistent with their hydrophobicity and beta-strand propensity profiles. To examine the cross reactivity between peptide fragments and full-length mPrP, we then carried out seeding experiments. While H2 seeds induced the formation of fibrils of full-length mPrP as quickly as full-length mPrP seeds, H3 seeds exhibited a long lag time. This implies that the region of alpha-helix 2 rather than alpha-helix 3 in mPrP has great potential for initiating fibril formation. As a whole, the alpha-helix 2 region would be crucial for the nucleation-dependent replication process of the prion protein.

Dimethylsulfoxide-quenched hydrogen/deuterium exchange method to study amyloid fibril structure.

A general method to analyze the structure of a supramolecular complex of amyloid fibrils at amino acid residue resolution has been developed. This method combines the NMR-detected hydrogen/deuterium (H/D) exchange technique to detect hydrogen-bonded amide groups and the ability of the aprotic organic solvent dimethylsulfoxide (DMSO) to dissolve amyloid fibrils into NMR-observable, monomeric components while suppressing the undesired H/D exchange reaction. Moreover, this method can be generally applied to amyloid fibrils to elucidate the distribution of hydrogen-bonded amino acid residues in the three-dimensional molecular organization in the amyloid fibrils. In this study, we describe theoretical considerations in the H/D exchange method to obtain the structural information of proteins, and the DMSO-quenched H/D exchange method to study a supramolecular complex of amyloid fibrils. A possible application of this method to study the interaction of a protein/peptide with phospholipid membrane is also discussed.

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism.

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols.

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.

Seeding-dependent propagation and maturation of amyloid fibril conformation.

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.

Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid {beta} peptide.

Although metal ions such as Cu(2+), Zn(2+), and Fe(3+) are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu(2+) and Zn(2+) but not Fe(3+) renders the amyloid beta peptide, Abeta(1-40), nonfibrillogenic in nature. However, preformed fibrils of Abeta(1-40) were stable when treated with these metal ions. Consequently, fibril growth of Abeta(1-40) could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu(2+)-suppressed but not the Zn(2+)-suppressed fibril growth of Abeta(1-40). The observed synergistic effect of clioquinol and Zn(2+) suggests that Zn(2+)-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Abeta(1-40) through metal chelation, it further retards the fibril growth along with Zn(2+). These results indicate the mechanism of metal ions in suppressing Abeta amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.

Stereospecific amyloid-like fibril formation by a peptide fragment of beta2-microglobulin.

Understanding the role of the L/D-stereospecificity of amino acids is important in obtaining further insight into the mechanism of the formation of amyloid fibrils. Beta(2)-microglobulin is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. A 22-residue peptide of beta(2)-microglobulin, Ser20-Lys41 (L-K3 peptide), obtained by digestion with Acromobacter protease I, formed amyloid-like fibrils in 50% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl at 25 degrees C, as confirmed by thioflavin T fluorescence, circular dichroism spectra, and atomic force microscopy images. A synthetic K3 peptide composed of D-amino acids (D-K3 peptide) formed similar fibrils but with opposite chirality as indicated by circular dichroism spectra. A mixture of L-K3 and D-K3 peptides also formed fibrils, although the L- and D-amino acid composition of each fibril is unknown. To examine the possible cross-reactivity between L- and D-enantiomers, we carried out seeding experiments in which preformed seeds were extended by monomers. The results revealed that only the homologous extensions proceed smoothly, i.e., the growth of L-seeds by L-monomers or D-seeds by D-monomers. The results suggest that, while the fibrils derived from L- and D-peptides form in a similar manner but with opposite stereochemistry, a cross-reaction between them is prevented because the geometry of the mixed sheet cannot satisfy dominant factors for beta-sheet stabilization.

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange.

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.