Short disordered protein segment regulates cross-species transmission of a yeast prion.

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

Molecular basis for diversification of yeast prion strain conformation.

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

α-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein.

Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as α-synuclein (αS) in Parkinson's disease, amyloid-ß in Alzheimer's disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show αS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric αS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between αS and monomeric PrP nor fibrillar PrP. These results suggested that αS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric αS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of αS against PrP amyloidogenesis, suggesting a possible involvement of αS in the pathology of TSEs.

Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

Amyloid fibrils are filamentous protein aggregates associated with the pathogenesis of a wide variety of human diseases. The formation of such aggregates typically follows nucleation-dependent kinetics, wherein the assembly and structural conversion of amyloidogenic proteins into oligomeric aggregates (nuclei) is the rate-limiting step of the overall reaction. In this study, we sought to gain structural insights into the oligomeric nuclei of the human prion protein (PrP) by preparing a series of deletion mutants lacking 14-44 of the C-terminal 107 residues of PrP and examined the kinetics and thermodynamics of these mutants in amyloid formation. An analysis of the experimental data using the concepts of the Φ-value analysis indicated that the helix 2 region (residues 168-196) acquires an amyloid-like ß-sheet during nucleation, whereas the other regions preserves a relatively disordered structure in the nuclei. This finding suggests that the helix 2 region serves as the nucleation site for the assembly of amyloid fibrils.-Honda, R., Kuwata, K. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

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.

Logical design of an anti-cancer agent targeting the plant homeodomain in Pygopus2.

Pygopus2 (Pygo2) is a component of the Wnt signaling pathway, which is required for ß-catenin mediated transcription. Plant homeodomain (PHD) finger in Pygo2 intercalates the methylated histone 3 (H3K4me) tail and HD1 domain of BCL9 that binds to ß-catenin. Thus, PHD finger may be a potential target for the logical design of an anti-cancer drug. Here, we found that Spiro[2H-naphthol[1,2-b]pyran-2,4'-piperidine]-1'ethanol,3,4-dihydro-4-hydroxy-α-(6-methyl-1H-indol-3-yl)) termed JBC117 interacts with D339, A348, R356, V376 and A378 in PHD corresponding to the binding sites with H3K4me and/or HD1, and has strong anti-cancer effects. For colon (HCT116) and lung (A549) cancer cell lines, IC50 values were 2.6 ± 0.16 and 3.3 ± 0.14 µM, respectively, while 33.80 ± 0.15 µM for the normal human fibroblast cells. JBC117 potently antagonized the cellular effects of ß-catenin-dependent activity and also inhibited the migration and invasion of cancer cells. In vivo studies showed that the survival time of mice was significantly prolonged by the subcutaneous injection of JBC117 (10 mg/kg/day). In conclusion, JBC117 is a novel anti-cancer lead compound targeting the PHD finger of Pygo2 and has a therapeutic effect against colon and lung cancer.

Logical design of anti-prion agents using NAGARA.

To accelerate the logical drug design procedure, we created the program "NAGARA," a plugin for PyMOL, and applied it to the discovery of small compounds called medical chaperones (MCs) that stabilize the cellular form of a prion protein (PrP(C)). In NAGARA, we constructed a single platform to unify the docking simulation (DS), free energy calculation by molecular dynamics (MD) simulation, and interfragment interaction energy (IFIE) calculation by quantum chemistry (QC) calculation. NAGARA also enables large-scale parallel computing via a convenient graphical user interface. Here, we demonstrated its performance and its broad applicability from drug discovery to lead optimization with full compatibility with various experimental methods including Western blotting (WB) analysis, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) measurements. Combining DS and WB, we discovered anti-prion activities for two compounds and tegobuvir (TGV), a non-nucleoside non-structural protein NS5B polymerase inhibitor showing activity against hepatitis C virus genotype 1. Binding profiles predicted by MD and QC are consistent with those obtained by SPR and NMR. Free energy analyses showed that these compounds stabilize the PrP(C) conformation by decreasing the conformational fluctuation of the PrP(C). Because TGV has been already approved as a medicine, its extension to prion diseases is straightforward. Finally, we evaluated the affinities of the fragmented regions of TGV using QC and found a clue for its further optimization. By repeating WB, MD, and QC recursively, we were able to obtain the optimum lead structure.

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.

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.

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to 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.

Structure-based discovery of anti-influenza virus A compounds among medicines.

BACKGROUND: Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme. METHODS: We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay. RESULTS: We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase. CONCLUSIONS: Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase. GENERAL SIGNIFICANCE: Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.

Protein-specific force field derived from the fragment molecular orbital method can improve protein-ligand binding interactions.

Accurate computational estimate of the protein-ligand binding affinity is of central importance in rational drug design. To improve accuracy of the molecular mechanics (MM) force field (FF) for protein-ligand simulations, we use a protein-specific FF derived by the fragment molecular orbital (FMO) method and by the restrained electrostatic potential (RESP) method. Applying this FMO-RESP method to two proteins, dodecin, and lysozyme, we found that protein-specific partial charges tend to differ more significantly from the standard AMBER charges for isolated charged atoms. We did not see the dependence of partial charges on the secondary structure. Computing the binding affinities of dodecin with five ligands by MM PBSA protocol with the FMO-RESP charge set as well as with the standard AMBER charges, we found that the former gives better correlation with experimental affinities than the latter. While, for lysozyme with five ligands, both charge sets gave similar and relatively accurate estimates of binding affinities.

Synthesis and structure-activity relationship of 2-phenyliminochromene derivatives as inhibitors for aldo-keto reductase (AKR) 1B10.

Inhibitors of a human member (AKR1B10) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of cancer. Recently, we have discovered (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) as the potent competitive inhibitor using the virtual screening approach, and proposed its 4-methoxy group on the 2-phenylimino moiety as an essential structural prerequisite for the inhibition. In this study, 18 derivatives of 1 were synthesized and their inhibitory potency against AKR1B10 evaluated. Among them, 7-hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (5n) was the most potent inhibitor showing a Ki value of 1.3nM. The structure-activity relationship of the derivatives indicated that the 7-hydroxyl group on the chromene ring, but not the 4-methoxy group, was absolutely required for inhibitory activity, The molecular docking of 5n in AKR1B10 and site-directed mutagenesis of the enzyme residues suggested that the hydrogen-bond interactions between the 7-hydroxyl group of 5n and the catalytic residues (Tyr49 and His111) of the enzyme, together with a π-stacking interaction of the benzylamide moiety of 5n with Trp220, are important for the potent inhibition.

A theoretical study of the two binding modes between lysozyme and tri-NAG with an explicit solvent model based on the fragment molecular orbital method.

To examine the stabilities and binding characteristics, fragment molecular orbital (FMO) calculations were performed for the two binding modes of hen egg-white lysozyme with tri-N-acetyl-D-glucosamine (tri-NAG). Solvent effects were considered using an explicit solvent model. For comparison with the computational results, we experimentally determined the enthalpic contribution of the binding free-energy. Our calculations showed that the binding mode observed by X-ray analysis was more stable than the other binding mode by -6.2 kcal mol(-1), where it was found that the interaction of protein with solvent molecules was crucial for this stability. The amplitude of this energy difference was of the same order as the experimental enthalpic contribution. Our detailed analysis using the energies divided into each residue was also consistent with a previous mutant study. In addition, the electron density analysis showed that the formal charge of the lysozyme (+8.0 e) was reduced to +5.16 e by charge transfer with solvent molecules.

3,4-Dicaffeoylquinic Acid, a Major Constituent of Brazilian Propolis, Increases TRAIL Expression and Extends the Lifetimes of Mice Infected with the Influenza A Virus.

Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus (IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities of these compounds in vivo. PWE or PEE (Brazilian green propolis ethanol extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were significantly extended compared to the untreated mice. Moreover, oral administration of 3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique mechanism of anti-influenza viral activity, that is, enhancing viral clearance by increasing TRAIL.

Respiratory and cardiovascular toxicity studies of a novel antiprion compound, GN8, in rats and dogs.

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders for which no effective curative or prophylactic method has been established. Recently, we discovered a novel antiprion compound, GN8. Administration of GN8 was found to prolong the survival of prion-infected mice. The aim of this study was to characterize the toxicological and pharmacological features of GN8 in rats and dogs treated via a single intravenous injection. Minimum lethal doses of GN8 were estimated to be approximately 60 and 40 mg/kg in rats and dogs, respectively. In the respiratory toxicity experiments, GN8 was administered to rats at doses of 0, 15.6, and 46.9 mg/kg, and rats were observed for consciousness, behavior, autonomic nervous symptoms, and body weights. GN8 was found to have little adverse effect on the rat respiratory system at a dose of 46.9 mg/kg. In the cardiovascular toxicity experiments, GN8 was administered to dogs at doses of 0, 7.8, and 31.3 mg/kg, and dogs were observed similarly. Although GN8 was found to have a slight effect on the cardiovascular system at a dose of 31.3 mg/kg, we did not find severe adverse effects of GN8 at doses sufficient for antiprion activity. This study would serve as a stepping stone to a clinical application of GN8 as an antiprion agent.

Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells.

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC(50) values of 0.51-0.83 µM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.

Caffeoylquinic acids are major constituents with potent anti-influenza effects in brazilian green propolis water extract.

Influenza A viral infections reached pandemic levels in 1918, 1957, 1968, and, most recently, in 2009 with the emergence of the swine-origin H1N1 influenza virus. The development of novel therapeutics or prophylactics for influenza virus infection is urgently needed. We examined the evaluation of the anti-influenza virus (A/WSN/33 (H1N1)) activity of Brazilian green propolis water extract (PWE) and its constituents by cell viability and real-time PCR assays. Our findings showed strong evidence that PWE has an anti-influenza effect and demonstrate that caffeoylquinic acids are the active anti-influenza components of PWE. Furthermore, we have found that the amount of viral RNA per cell remained unchanged even in the presence of PWE, suggesting that PWE has no direct impact on the influenza virus but may have a cytoprotective activity by affecting internal cellular process. These findings indicate that caffeoylquinic acids are the active anti-influenza components of PWE. Above findings might facilitate the prophylactic application of natural products and the realization of novel anti-influenza drugs based on caffeoylquinic acids, as well as further the understanding of cytoprotective intracellular mechanisms in influenza virus-infected cells.

Positioning of autoimmune TCR-Ob.2F3 and TCR-Ob.3D1 on the MBP85-99/HLA-DR2 complex.

Since the first determination of structure of the HLA-A2 complex, >200 MHC/peptide structures have been recorded, whereas the available T cell receptor (TCR)/peptide/MHC complex structures now are <20. Among these structures, only six are TCR/peptide/MHC Class II (MHCII) structures. The most recent of these structures, obtained by using TCR-Ob.1A12 from a multiple sclerosis patient and the MBP85-99/HLA-DR2 complex, was very unusual in that the TCR was located near the N-terminal end of the peptide-binding cleft of the MHCII protein and had an orthogonal angle on the peptide/MHC complex. The unusual structure suggested the possibility of a disturbance of its signaling capability that could be related to autoimmunity. Here, homology modeling and a new simulation method developed for TCR/peptide/MHC docking have been used to examine the positioning of the complex of two additional TCRs obtained from the same patient (TCR-Ob.2F3 or TCR-Ob.3D1 with MBP85-99/HLA-DR2). The structures obtained by this simulation are compatible with available data on peptide specificity of the TCR epitope. All three TCRs from patient Ob including that from the previously determined crystal structure show a counterclockwise rotation. Two of them are located near the N terminus of the peptide-binding cleft, whereas the third is near the center. These data are compatible with the hypothesis that the rotation of the TCRs may alter the downstream signaling.