Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement.

Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross ß-sheets. We solve a 3 Å cryo-EM structure of jR2R3-P301L fibrils with a pseudo 2 1 screw symmetry in which each half of the fibril's cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance Statement: This study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L-among the widest used mutations in cell and animal models of Alzheimer's Disease-destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimer's Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

Effect of Cosolutes on the Aggregation of a Tau Fragment: A Combined Experimental and Simulation Approach.

The intrinsically disordered protein Tau represents the main component of neurofibrillary tangles that are a hallmark of Alzheimer's disease. A small fragment of Tau, known as paired helical filament 6 (PHF6), is considered to be important for the formation of the ß-structure core of the fibrils. Here we study the aggregation of this fragment in the presence of different cosolutes, including urea, TMAO, sucrose and 2-hydroxypropyl-ß-cyclodextrin (2-HPßCD), using both experiments and molecular dynamics simulations. A novel implicit solvation approach (MIST - Model with Implicit Solvation Thermodynamics) is used, where an energetic contribution based on the concept of transfer free energies describes the effect of the cosolutes. The simulation predictions are compared to thioflavin-T and atomic force microscopy results, and the good agreement observed confirms the predictive ability of the computational approach herein proposed. Both simulations and experiments indicate that PHF6 aggregation is inhibited in the presence of urea and 2-HPßCD, while TMAO and sucrose stabilize associated conformations. The remarkable ability of HPßCD to inhibit aggregation represents an extremely promising result for future applications, especially considering the widespread use of this molecule as a drug carrier to the brain and as a solubilizer/excipient in pharmaceutical formulations.

Tachykinin Neuropeptides and Amyloid ß (25-35) Assembly: Friend or Foe?

Amyloid ß (Aß) protein is responsible for Alzheimer's disease, and one of its important fragments, Aß(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aß(25-35)'s toxicity in cells even though they share similar primary structures with Aß(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aß(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aß(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aß(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aß(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aß(25-35), NK, Kassinin, and Substance P do not form hexamers with a ß-sheet structure like Aß(25-35). The hexamer structure of Aß(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aß(25-35) are discussed.

Cosolvent Exclusion Drives Protein Stability in Trimethylamine N-Oxide and Betaine Solutions.

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.

A Transfer Free Energy Based Implicit Solvent Model for Protein Simulations in Solvent Mixtures: Urea-Induced Denaturation as a Case Study.

We developed a method for implicit solvent molecular dynamics simulations of proteins in solvent mixtures (model with implicit solvation thermodynamics, MIST). The MIST method introduces experimental group transfer free energies to the generalized Born formulation for generating molecular trajectories without the need for developing rigorous explicit-solvent force fields for multicomponent solutions. As a test case, we studied the urea-induced denaturation of the Trp-cage miniprotein in water. We demonstrate that our method allows efficient exploration of the conformational space of the protein in only a few hundreds of nanoseconds of all-atom unbiased simulations. Furthermore, selective implementation of the transfer free energies of specific peptide groups, backbone, and side chains enables us to decouple their specific energetic contributions to the conformational changes of the protein. The approach herein developed can readily be extended to the investigation of complex matrices as well as to the characterization of protein aggregation. The MIST method is implemented in Plumed (ver. 2.8) as a separate module called SASA.

Persistence and Exposure Assessment of Insecticide Indoxacarb Residues in Vegetables.

Indoxacarb, a promising new generation insecticide, is gaining popularity among vegetable growers in West Bengal, India, for controlling a large number of insects. However, it may simultaneously also increase the risk of contamination in the edible portions of the vegetables. This study was planned to analyze the persistence behavior of indoxacarb in cabbages, tomatoes, and soil. Moreover, indoxacarb residue contents were estimated to assess both the dietary and soil ecological risks associated with the application of the same. The experimental location was important because West Bengal is the leading vegetables producing state in India. Indoxacarb was found to dissipate quickly with a half-life ranging between 1.55 and 2.76 days, irrespective of the vegetable, dose, and season, and the safe waiting period was very less. The findings indicate that both vegetables can be safely consumed 1 day after the final spray. However, the risk to soil algae is predicted to be unacceptably high, which needs to be studied extensively.

Development of a Modified QuEChERS Method Coupled with LC-MS/MS for Determination of Spinetoram Residue in Soybean (Glycine max) and Cotton (Gossypium hirsutum).

An analytical method for the quantitative determination of the insecticide spinetoram in cotton and soybean was established and validated using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spinetoram is the mixture of two spinosyns, 3'-O-ethyl-5,6-dihydro spinosyn J and 3'-O-ethyl spinosyn L. The method involves extraction with ethyl acetate followed by dispersive solid phase extraction (dSPE) clean-up with primary secondary amine (PSA), C18 and graphitised carbon black (GCB). The final quantitation of spinetoram was done by using LC-MS/MS with positive electrospray ionization. The method was reproducible (Horwitz ratio (HorRat) < 0.5 at 25 ng g−1) and validated by the analysis of samples spiked at 25, 50 and 100 ng g−1 in soybean, cotton and soil. The recoveries of spinosyns were found to be more than 85% when spiked at different levels. The identities of spinosyns were confirmed by using the ion ratio. A field dissipation study was conducted in soybean and cotton to find out the environmental fate of spinetoram, and samples were analysed following the proposed analytical method. Both isomers were found to be dissipated quickly. The pre-harvest interval of spinetoram was calculated in different substrates.

Photodegradation of Flucetosulfuron, a Sulfonylurea-Based Herbicide in the Aqueous Media Is Influenced by Ultraviolet Irradiation.

Photodegradation (photolysis) causes the breakdown of organic pesticides molecules by direct or indirect solar radiation energy. Flucetosulfuron herbicide often encounters water bodies. For this reason, it is important to know the behavior of the compound under these stressed conditions. In this context, photodegradation of flucetosulfuron, a sulfonylurea-based herbicide, has been assessed in aqueous media in the presence of photocatalyst TiO2 and photosensitizers (i.e., H2O2, humic acid, and KNO3) under the influence of ultraviolet (UV) irradiation. The influence of different water systems was also assessed during the photodegradation study. The photodegradation followed the first-order reaction kinetics in each case. The metabolites after photolysis were isolated in pure form by column chromatographic method and characterized using the different spectral data (i.e., XRD, IR, NMR, UV-VIS, and mass spectrometry). The structures of these metabolites were identified based on the spectral data and the plausible photodegradation pathways of flucetosulfuron were suggested. Based on the findings, photocatalyst TiO2 with the presence of ultraviolet irradiation was found effective for the photodegradation of toxic flucetosulfuron residues under aqueous conditions.

Catalytic Cross Talk between Key Peptide Fragments That Couple Alzheimer's Disease with Amyotrophic Lateral Sclerosis.

Protein aggregation is a common feature in prominent neurodegenerative diseases, usually thought to be due to the assembly of a single peptide or protein. Recent studies have challenged this notion and suggested several proteins may be involved in promoting and amplifying disease. For example, the TDP-43 protein associated with Amyotrophic Lateral Sclerosis has been found in the brain along with Aß assemblies associated with Alzheimer's disease, and those patients that show the presence of TDP-43 are 10 times more likely to demonstrate cognitive impairment compared to TDP-43-negative Alzheimer's patients. Here we examine the interactions between the amyloidogenic core of TDP-43, TDP-43307-319, and a neurotoxic physiologically observed fragment of Aß, Aß25-35. Utilizing ion mobility mass spectrometry in concert with atomic force microscopy and molecular dynamics simulations, we investigate which oligomers are involved in seeding aggregation across these two different protein systems and gain insight into which structures initiate and result from these interactions. Studies were conducted by mixing Aß25-35 with the toxic wild type TDP-43307-319 peptide and with the nontoxic synthetic TDP-43307-319 mutant, G314V. Our findings identify a strong catalytic effect of TDP-43307-319 WT monomer in the acceleration of Aß25-35 aggregation to its toxic cylindrin and ß barrel forms. This observation is unprecedented in both its speed and specificity. Interestingly, the nontoxic G314V mutant of TDP-43307-319 and dimers or higher order oligomers of WT TDP-43307-319 do not promote aggregation of Aß25-35 but rather dissociate preformed toxic higher order oligomers of Aß25-35. Reasons for these very different behaviors are reported.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

ADD Force Field for Sugars and Polyols: Predicting the Additivity of Protein-Osmolyte Interaction.

The protein-osmolyte interaction has been shown experimentally to follow an additive construct, where the individual osmolyte-backbone and osmolyte-side-chain interactions contribute to the overall conformational stability of proteins. Here, we computationally reconstruct this additive relation using molecular dynamics simulations, focusing on sugars and polyols, including sucrose and sorbitol, as model osmolytes. A new set of parameters (ADD) is developed for this purpose, using the individual Kirkwood-Buff integrals for sugar-backbone and sugar-side-chain interactions as target experimental data. We show that the ADD parameters can reproduce the additivity of protein-sugar interactions and correctly predict sucrose and sorbitol self-association and their interaction with water. The accurate description of the separate osmolyte-backbone and osmolyte-side-chain contributions also automatically translates into a good prediction of preferential exclusion from the surface of ribonuclease A and α-chymotrypsinogen A. The description of sugar polarity is improved compared to previous force fields, resulting in closer agreement with the experimental data and better compatibility with charged groups, such as the guanidinium moiety. The ADD parameters are developed in combination with the CHARMM36m force field for proteins, but good compatibility is also observed with the AMBER 99SB-ILDN and the OPLS-AA force fields. Overall, exploiting the additivity of protein-osmolyte interactions is a promising approach for the development of new force fields.

Terminal Capping of an Amyloidogenic Tau Fragment Modulates Its Fibrillation Propensity.

Aberrant protein folding leading to the formation of characteristic cross-ß-sheet-rich amyloid structures is well known for its association with a variety of debilitating human diseases. Often, depending upon amino acid composition, only a small segment of a large protein participates in amyloid formation and is in fact capable of self-assembling into amyloid, independent of the rest of the protein. Therefore, such peptide fragments serve as useful model systems for understanding the process of amyloid formation. An important factor that has often been overlooked while using peptides to mimic full-length protein is the charge on the termini of these peptides. Here, we show the influence of terminal charges on the aggregation of an amyloidogenic peptide from microtubule-associated protein Tau, implicated in Alzheimer's disease and tauopathies. We found that modification of terminal charges by capping the peptide at one or both of the termini drastically modulates the fibrillation of the hexapeptide sequence paired helical filament 6 (PHF6) from repeat 3 of Tau, both with and without heparin. Without heparin, the PHF6 peptide capped at both termini and PHF6 capped only at the N-terminus self-assembled to form amyloid fibrils. With heparin, all capping variants of PHF6, except for PHF6 with both termini free, formed typical amyloid fibrils. However, the rate and extent of aggregation both with and without heparin as well as the morphology of aggregates were found to be highly dependent on the terminal charges. Our molecular dynamics simulations on PHF6 capping variants corroborated our experiments and provided critical insights into the mechanism of PHF6 self-assembly. Overall, our results emphasize the importance of terminal modifications in fibrillation of small peptide fragments and provide significant insights into the aggregation of a small Tau fragment, which is considered essential for Tau filament assembly.

Protein Stability in TMAO and Mixed Urea-TMAO Solutions.

Osmolytes are essential for cellular function under ubiquitous osmotic stress. Trimethylamine N-oxide (TMAO) is one such osmolyte that has gained remarkable attention due to its protein-protective ability against urea. This Review aims at providing a detailed account of recent theoretical and experimental developments in characterizing the structural changes and thermodynamic stability of proteins in the presence of TMAO and urea. New vapor pressure osmometry and molecular dynamics simulation results on urea-TMAO solutions are presented, and a unified molecular mechanism of TMAO counteraction of urea-induced protein denaturation is introduced. In addition, a detailed technical assessment of molecular dynamics force fields for TMAO and for urea-TMAO solutions is presented. The force field analysis highlights how many of the commonly used force field models are in fact incompatible with solvation thermodynamics and can lead to misleading conclusions. A new optimized force field for TMAO (Shea(m)) is presented, and a recently optimized force field for TMAO-urea (Netz(m)) that best reproduces experimental data is highlighted.

Persistence behaviour and safety risk evaluation of pyridalyl in tomato and cabbage.

A field experiment was set up to evaluate persistence behaviour of pyridalyl in tomato, cabbage and cultivated field soil over two consecutive seasons. An analytical method was developed to analyze pyridalyl residues in different matrices and duly validated, based on single laboratory method validation criteria. Pyridalyl residues were detected and quantified using a gas chromatograph equipped with an electron capture detector. The compound exhibited low persistence in tomato, cabbage and soil. A safe waiting period of 17-18 d after final insecticide application needs to be maintained before harvesting the crop. Both dietary and soil ecological risk were assessed and it was found that the harvested vegetables were toxicologically safe for consumption. However, there was concern about insecticidal toxicity against the algal population of soil which needs to be reconfirmed by further studies.

Distinct and Nonadditive Effects of Urea and Guanidinium Chloride on Peptide Solvation.

Using enhanced-sampling replica exchange fully atomistic molecular dynamics simulations, we show that, individually, urea and guanidinium chloride (GdmCl) denature the Trpcage protein, but remarkably, the helical segment 1NLYIQWL7 of the protein is stabilized in mixed denaturant solutions. GdmCl induces protein denaturation via a combination of direct and indirect effects involving dehydration of the protein and destabilization of stabilizing salt bridges. In contrast, urea denatures the protein through favorable protein-urea preferential interactions, with peptide-specific indirect effects of urea on the water structure around the protein. In the case of the helical segment of Trpcage, urea "oversolvates" the peptide backbone by reorganizing water molecules from the peptide side chains to the peptide backbone. An intricate nonadditive thermodynamic balance between GdmCl-induced dehydration of the peptide and the urea-induced changes in solvation structure triggers partial counteraction to urea denaturation and stabilization of the helix.

Persistence behavior of metamifop and its metabolite in rice ecosystem.

A field experiment was conducted to determine the persistence of metamifop in transplanted rice crop for two seasons. Metamifop 10% EC was applied at two doses: 100 g a.i. ha-1 and 200 g a.i. ha-1 at 2-3 leaf stage of Echinochloa crusgalli. The residues of metamifop along with its major metabolite, N-(2-fluorophenyl)-2-hydroxy-N-methylpropionamide (HFMPA), were estimated in rice plant, field water and soil using Liquid Chromatography Mass Spectrometry. Limit of detection and limit of quantification of the method for both the compounds were set at 0.003 µg g-1 and 0.010 µg g-1 respectively. Metamifop showed less persistence in field water and rice plant as compared to soil samples. Presence of HFMPA was recorded in rice plant and soil. Both the compounds were found below level of quantification in harvest samples of straw, grains, husk and soil. A safe waiting period of 52 d was suggested for harvesting of rice when metamifop was applied at 100 g a.i. ha-1 (recommended dose).

Dissipation kinetics and risk assessment of chlorfenapyr on tomato and cabbage.

A field experiment was conducted over two seasons to evaluate the dissipation kinetics and assess the risks of chlorfenapyr in tomato and cabbage following foliar application of chlorfenapyr 10% SC at 100 and 200 g a.i. ha-1. Samples of tomato, cabbage, and soil were analyzed and quantified by gas chromatography-electron capture detector (GC-ECD). The limit of detection (LOD) and limit of quantification (LOQ) of chlorfenapyr were found to be 0.01 and 0.03 mg kg-1, respectively, in tomato, cabbage, and soil. The dissipation of chlorfenapyr followed first-order kinetics. The compound showed less persistence in both the vegetables and soil as the calculated half-life values of chlorfenapyr ranged between 4.54 and 7.74 days considering two different doses and seasons. The residue was below detection limit in all the untreated plant and soil samples. The pre-harvest interval (PHI) of chlorfenapyr in both the vegetables was determined to be 9-14 days regardless of dose or season. The theoretical maximum residue contribution (TMRC) of chlorfenapyr was calculated for tomato and cabbage and was found to be lower than the maximum permissible intake (MPI) of the compound. Therefore, the application of chlorfenapyr at the recommended dose in tomato and cabbage for crop protection seems to be safe from both environmental contamination and consumer safety standpoints.

Trimethylamine N-oxide Counteracts Urea Denaturation by Inhibiting Protein-Urea Preferential Interaction.

Osmolytes are small organic molecules that can modulate the stability and function of cellular proteins by altering the chemical environment of the cell. Some of these osmolytes work in conjunction, via mechanisms that are poorly understood. An example is the naturally occurring protein-protective osmolyte trimethylamine N-oxide (TMAO) that stabilizes cellular proteins in marine organisms against the detrimental denaturing effects of another naturally occurring osmolyte, urea. From a computational standpoint, our understanding of this counteraction mechanism is hampered by the fact that existing force fields fail to capture the correct balance of TMAO and urea interactions in ternary solutions. Using molecular dynamics simulations and Kirkwood-Buff theory of solutions, we have developed an optimized force field that reproduces experimental Kirkwood-Buff integrals. We show through the study of two model systems, a 15-residue polyalanine chain and the R2-fragment (273GKVQIINKKLDL284) of the Tau protein, that TMAO can counteract the denaturing effects of urea by inhibiting protein-urea preferential interaction. The extent to which counteraction can occur is seen to depend heavily on the amino acid composition of the peptide.

Persistence of chlorfluazuron in cabbage under different agro-climatic conditions of India and its risk assessment.

A multilocational field trial was conducted at 4 locations in India-Rajasthan, Gujarat, Madhya Pradesh, and West Bengal-to determine the persistence in cabbage of chlorfluazuron applied twice at 75 and 150 g active ingredient ha-1 . Cabbage head samples were collected from each replicated plot on 0 (2 h after spraying), 1, 3, 5, 7, 10, and 15 d after final insecticide application, including an untreated control. Chlorfluazuron residue in cabbage and field soil was estimated by high-performance liquid chromatography using a photo diode array detector. The limit of determination and limit of quantification of the method were recorded as 0.05 and 0.10 µg g-1 , respectively. Results revealed that chlorfluazuron dissipated linearly with progress of time, following first-order kinetics. The mean (± standard deviation) half-life value of chlorfluazuron in cabbage was found to be 7.18 ± 0.71 d, considering different locations and treatments. The residue was below the level of quantification in the harvested cabbage and soil samples. Harvesting cabbage in the experimental location, at least on day 7, after 2 applications of chlorfluazuron at the recommended dose, may not pose any ill effect for Indian adults. Environ Toxicol Chem 2017;36:3028-3033. © 2017 SETAC.

Signature of an aggregation-prone conformation of tau.

The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended ß-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended ß-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.