Helical Filaments Research Articles

PI-2620 Lead Optimization Highlights the Importance of Off-Target Assays to Develop a PET Tracer for the Detection of Pathological Aggregated Tau in Alzheimer's Disease and Other Tauopathies.

The first candidate PI-2014 was tested in healthy controls and subjects with Alzheimer's disease (AD). As PI-2014 displayed off-target binding to monoamine oxidase A (MAO-A), a new lead with improved binding to Tau and decreased MAO-A binding was required. For compound optimization, Tau binding assays based on both human AD brain homogenate and Tau-paired helical filaments were employed. Furthermore, two MAO-A screening assays based on (1) human-recombinant MAO-A and (2) displacement of 2-fluoro-ethyl-harmine from mouse brain homogenate were employed. Removing the N-methyl group from the tricyclic core resulted in compounds displaying improved Tau binding. For the final round of optimization, the cyclic amine substituents were replaced by pyridine derivatives. PI-2620 (2-(2-fluoropyridin-4-yl)-9H-pyrrolo[2,3-b:4,5-c']dipyridine) emerged as a best candidate displaying high Tau binding, low MAO-A binding, high brain uptake, and fast and complete brain washout. Furthermore, PI-2620 showed Tau binding on brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease.

Molecular dynamics simulations reveal the disruption mechanism of a 2,4-thiazolidinedione derivative C30 against tau hexapeptide (PHF6) oligomer.

Derivatives of 2,4-thiazolidinedione have been reported to inhibit the aggregation of tau protein, in which compound 30 (C30) not only inhibit 80% of paired helical filament 6 (PHF6) aggregation, but also inhibit K18 and full-length tau aggregation. However, its inhibitory mechanism is unclear. In this study, to investigate the effect of C30 on tau protein, all-atom molecular dynamics simulation was performed on the PHF6 oligomer with and without C30. The results show that C30 can cause significant conformational changes in the PHF6 oligomer. The nematic order parameter P2 and secondary structure analyses show that C30 destroys the ordered structure of PHF6 oligomer, reduces the content of β-sheet structure, and transforms β-sheet into random coil structure. By clustering analysis, it was found that C30 has four possible binding sites on the PFH6 oligomer, and the binding ability order is S1 > S2 > S4 > S3. Following a more in-depth analyses of each site, it was determined that the S1 site is the most possible binding site mainly located between layers of L1 and L3. The hydrophobic interaction is the driving force for the binding of C30 to PHF6 oligomer. In addition, L1P4_Y310, L1P5_Y310, L3P1_V309, and L3P2_V309 are key residues for C30 binding to oligomer. Moreover, π-π interaction formed by L1P4_Y310 and L1P5_Y310 with C30 and the hydrogen bonding interaction formed by C30 with L3P3_Q307 are beneficial to the combination of C30 and oligomer. The fully understanding disrupt the mechanism of 2,4-thiazolidinedione derivative on PHF6 oligomer and the identification of binding sites will help design and discover new AD inhibitors in the future.

Prospects for the Mechanism of Spiroplasma Swimming.

Spiroplasma are helical bacteria that lack a peptidoglycan layer. They are widespread globally as parasites of arthropods and plants. Their infectious processes and survival are most likely supported by their unique swimming system, which is unrelated to well-known bacterial motility systems such as flagella and pili. Spiroplasma swims by switching the left- and right-handed helical cell body alternately from the cell front. The kinks generated by the helicity shift travel down along the cell axis and rotate the cell body posterior to the kink position like a screw, pushing the water backward and propelling the cell body forward. An internal structure called the “ribbon” has been focused to elucidate the mechanisms for the cell helicity formation and swimming. The ribbon is composed of Spiroplasma-specific fibril protein and a bacterial actin, MreB. Here, we propose a model for helicity-switching swimming focusing on the ribbon, in which MreBs generate a force like a bimetallic strip based on ATP energy and switch the handedness of helical fibril filaments. Cooperative changes of these filaments cause helicity to shift down the cell axis. Interestingly, unlike other motility systems, the fibril protein and Spiroplasma MreBs can be traced back to their ancestors. The fibril protein has evolved from methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase, which is essential for growth, and MreBs, which function as a scaffold for peptidoglycan synthesis in walled bacteria.

Tau Protein Interaction Partners and Their Roles in Alzheimer's Disease and Other Tauopathies.

Tau protein plays a critical role in the assembly, stabilization, and modulation of microtubules, which are important for the normal function of neurons and the brain. In diseased conditions, several pathological modifications of tau protein manifest. These changes lead to tau protein aggregation and the formation of paired helical filaments (PHF) and neurofibrillary tangles (NFT), which are common hallmarks of Alzheimer’s disease and other tauopathies. The accumulation of PHFs and NFTs results in impairment of physiological functions, apoptosis, and neuronal loss, which is reflected as cognitive impairment, and in the late stages of the disease, leads to death. The causes of this pathological transformation of tau protein haven’t been fully understood yet. In both physiological and pathological conditions, tau interacts with several proteins which maintain their proper function or can participate in their pathological modifications. Interaction partners of tau protein and associated molecular pathways can either initiate and drive the tau pathology or can act neuroprotective, by reducing pathological tau proteins or inflammation. In this review, we focus on the tau as a multifunctional protein and its known interacting partners active in regulations of different processes and the roles of these proteins in Alzheimer’s disease and tauopathies.

In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite’s apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.

Numerical investigation of the effect of towers and nacelles on the near wake of a horizontal-axis wind turbine model

Near wake flow has a great effect on the technology of horizontal-axis wind turbines. In this paper, the effect of the nacelle and tower on the near wake of a small-scale horizontal-axis wind turbine is studied by a numerical method. A large eddy simulation is used to obtain the time-varying wake flow. There are tip vortices, hub vortices, and blade and tower shedding vortices active in the near wake. Dynamic mode decomposition (DMD) is employed to analyse the features of these unsteady vorticities in both an absolute frame and a frame static relative to the rotor. The DMD results in the absolute frame illustrate the downstream motion of the tip vortices. In the relative frame, the results indicate that the disturbances distort the helical vortex filament and finally lead to a breakdown of the tip vortices in the case without a nacelle and tower. However, considering the effect of the nacelle and tower, the shedding vortices of the nacelle and tower directly break the tip vortices. The interaction between the tip vortices and the tower shedding vortices not only dominates the breakdown of the tip vortices but also promotes the instability of the tip vortices. This condition leads to an earlier wake recovery and influences the energy extraction and fatigue loads of the downstream turbines.

Target site selection in CAST systems

CRISPR Biology Exciting genomic engineering possibilities exist for natural integration systems called transposons, which have coopted CRISPR/Cas systems. An unexplained feature of these systems involves how they direct insertions in a single orientation at a precise distance from the programmed target sequence. Park et al. show that orientation information is communicated to the transposase TnsB using the unidirectional growth of a helical filament made up of an AAA+ protein, TnsC. ATP hydrolysis trims the filament to a minimal unit that is marked by TniQ and defined by the Cas12k protein to provide spacing information. This finding may help future engineering of these systems for therapeutic applications. Science , abi8976, this issue p. [768][1] [1]: /lookup/doi/10.1126/science.abi8976

Suppression of MHD modes with active phase-control of probe-injected currents

Active phase-control of probe-injected current is shown to both suppress and amplify long-wavelength rotating magnetohydrodynamic instabilities in the HBT-EP tokamak. Four probes are connected in quadrature and energized to drive non-axisymmetric currents through the edge of the tokamak, creating magnetic perturbations comparable to previously-studied saturated kink modes or resonant magnetic perturbations that are generated by an external control coil array. Measurements of the magnetic perturbations from the probe-injected currents determine a set of current-carrying helical filaments used to model active feedback control of resistive wall modes. These experiments suggest current-injection feedback may be an effective alternative to external control coils for control of RWMs and other long-wavelength kink-like modes at the edge of tokamaks.

Nonlinear dynamics of two helical vortices: A dynamical system approach

Two interwoven helical vortices of the same circulation and pitch display a rich variety of dynamics. Using simplified models of straight vortices, ring vortices, and helical filaments, we introduce dynamical systems of a few degrees of freedom which we analyze in terms of orbits in phase spaces structured by hyperbolic and elliptic points. Possible inviscid motions, namely leapfrogging, overtaking and fluttering, are systematically described as a function of vortex pitch, core size, and initial configuration.

Atomic Level Investigations of Early Aggregation of Tau43 in Water I. Conformational Propensity of Monomeric Tau43

Recent studies in Alzheimer's disease (AD) investigated the precise mechanisms responsible for neurofibrillary tangles (NFT) and senile plaques formation. NFTs, the aggregated Tau protein isoforms, are one of the primary factors behind AD. The corresponding smallest variant Tau43, as paired helical filaments (PHFs), self‐assemble into pathological diseased aggregates. However, the molecular details in rationalizing the aggregation propensity of Tau43 remain elusive. Herein, using molecular dynamics simulations on aqueous Tau43, we identify the molecular factors responsible for early behavior of Tau43 aggregation propensity in water. The variant is intrinsically unstructured yet compact in nature, in agreement with previous studies. The PHF6 (11VQIVYK16) segment is relatively less fluctuating, yet most extended and hydrophobic, thereby shielded from aqueous environment by intermolecular polar interactions between terminal residues. We also compared structural propensities of Tau43 and oppositely charged Aβ42 peptide, providing a comparative understanding of early AD pathway, leading to corresponding drug designing avenues.

The structural, functional, and dynamic effect of Tau tubulin kinase1 upon a mutation: A neuro-degenerative hotspot.

Alzheimer's disease (AD) is a progressive disorder that causes brain cells to degenerate and die. AD is one of the common causes of dementia that leads to a decline in thinking, behavioral and social skills that disrupts a person's ability to function independently. Tau-tubulin kinase1 (TTBK1) is a crucial disease regulating AD protein, which is majorly responsible for the phosphorylation and accumulation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy. TTBK1 involvement in many diseases and the restricted expression of TTBK1 to the central nervous system (CNS) makes TTBK1 an attractive therapeutic target for tauopathies. The genetic variations in TTBK1 are primarily involved in the TTBK1 pathogenesis. This study highlighted the destabilizing, damaging and deleterious effect of the mutation R142Q on TTBK1 structure through computational predictions and molecular dynamics simulations. The protein deviation, fluctuations, conformational dynamics, solvent accessibility, hydrogen bonding, and the residue-residue mapping confirmed the mutant effect to cause structural aberrations, suggesting overall destabilization due to the protein mutation. The presence of well-defined free energy minima was observed in TTBK1-wild type, as opposed to that in the R142Q mutant, reflecting structural deterioration. The overall findings from the study reveal that the presence of R142Q mutation on TTBK1 is responsible for the structural instability, leading to disruption of its biological functions. The mutation could be used as future diagnostic markers in treating AD.

Hydrodynamics and direction change of tumbling bacteria.

The bacterium Escherichia coli (E. coli) swims in viscous fluids by rotating several helical flagellar filaments, which are gathered in a bundle behind the cell during 'runs' wherein the cell moves steadily forward. In between runs, the cell undergoes quick 'tumble' events, during which at least one flagellum reverses its rotation direction and separates from the bundle, resulting in erratic motion in place and a random reorientation of the cell. Alternating between runs and tumbles allows cells to sample space by stochastically changing their propulsion direction after each tumble. The change of direction during a tumble is not uniformly distributed but is skewed towards smaller angles with an average of about 62°-68°, as first measured by Berg and Brown (1972). Here we develop a theoretical approach to model the angular distribution of swimming E. coli cells during tumbles. We first use past experimental imaging results to construct a kinematic description of the dynamics of the flagellar filaments during a tumble. We then employ low-Reynolds number hydrodynamics to compute the consequences of the kinematic model on the force and torque balance of the cell and to deduce the overall change in orientation. The results of our model are in good agreement with experimental observations. We find that the main change of direction occurs during the 'bundling' part of the process wherein, at the end of a tumble, the dispersed flagellar filaments are brought back together in the helical bundle, which we confirm using a simplified forced-sphere model.

Structural basis for target site selection in RNA-guided DNA transposition systems.

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

Chemical-mediated translocation in protocell-based microactuators

Artificial cell-like communities participate in diverse modes of chemical interaction but exhibit minimal interfacing with their local environment. Here we develop an interactive microsystem based on the immobilization of a population of enzyme-active semipermeable proteinosomes within a helical hydrogel filament to implement signal-induced movement. We attach large single-polynucleotide/peptide microcapsules at one or both ends of the helical protocell filament to produce free-standing soft microactuators that sense and process chemical signals to perform mechanical work. Different modes of translocation are achieved by synergistic or antagonistic enzyme reactions located within the helical connector or inside the attached microcapsule loads. Mounting the microactuators on a ratchet-like surface produces a directional push-pull movement. Our methodology opens up a route to protocell-based chemical systems capable of utilizing mechanical work and provides a step towards the engineering of soft microscale objects with increased levels of operational autonomy.

Thermosetting supramolecular polymerization of compartmentalized DNA fibers with stereo sequence and length control

Thermosetting supramolecular polymerization of compartmentalized DNA fibers with stereo sequence and length control

Oral (−)-Epicatechin Inhibits Progressive Tau Pathology in rTg4510 Mice Independent of Direct Actions at GSK3β

Aggregation of the microtubule-associated protein tau into paired helical filaments (PHFs) and neurofibrillary tangles is a defining characteristic of Alzheimer’s Disease. Various plant polyphenols disrupt tau aggregation in vitro but display poor bioavailability and low potency, challenging their therapeutic translation. We previously reported that oral administration of the flavonoid (−)-epicatechin (EC) reduced Amyloid-β (Aβ) plaque pathology in APP/PS1 transgenic mice. Here, we investigated whether EC impacts on tau pathology, independent of actions on Aβ, using rTg4510 mice expressing P301L mutant tau. 4 and 6.5 months old rTg4510 mice received EC (∼18 mg/day) or vehicle (ethanol) via drinking water for 21 days and the levels of total and phosphorylated tau were assessed. At 4 months, tau appeared as two bands of ∼55 kDa, phosphorylated at Ser262 and Ser396 and was unaffected by exposure to EC. At 6.5 months an additional higher molecular weight form of tau was detected at ∼64 kDa which was phosphorylated at Ser262, Ser396 and additionally at the AT8 sites, indicative of the presence of PHFs. EC consumption reduced the levels of the ∼64 kDa tau species and inhibited phosphorylation at Ser262 and AT8 phosphoepitopes. Regulation of the key tau kinase glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9 was not altered by exposure to EC in mice or primary neurons. Furthermore, EC did not significantly inhibit GSK3β activity at physiologically-relevant concentrations in a cell free assay. Therefore, a 21-day intervention with EC inhibits or reverses the development of tau pathology in rTg4510 mice independently of direct inhibition of GSK3β.

Structure of Tau filaments in Prion protein amyloidoses

In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann–Sträussler–Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.

Numerical simulation of bundling of helical elastic rods in a viscous fluid

We introduce a numerical framework to study the fluid-structure interaction between two helical filaments rotating under low Reynolds number condition, motivated by the propulsion of bacteria using helical flagella. Our numerical framework couples the elasticity of the thin filaments, nonlocal hydrodynamic loading, and the contact between multiple elastic rods. Each of these three ingredients is respectively modeled by the Discrete Elastic Rods method (for a geometrically nonlinear description of soft filaments), Regularized Stokeslet Segments method (for the nonlocal drag force in a viscous fluid), and non-penetration condition between rod segments. Two helical rods rotating side by side attract each other and become closer because of their hydrodynamic interplay in a viscous environment. Depending on the initial distance between the two and their rotational frequency, the two filaments can come in physical contact. Exploiting the efficiency and robustness of the simulator, we perform a systematic parameter sweep to quantify the bundling behavior. The findings may shed light on the physics of the bio-locomotion of microorganisms and inspire the design of novel biomimetic soft robots.

Epigallocatechin-3-gallate modulates Tau Post-translational modifications and cytoskeletal network.

Background: Alzheimer’s disease is a type of dementia denoted by progressive neuronal death due to the accumulation of proteinaceous aggregates of Tau. Post-translational modifications like hyperphosphorylation, truncation, glycation, etc. play a pivotal role in Tau pathogenesis. Glycation of Tau aids in paired helical filament formation and abates its microtubule-binding function. The chemical modulators of Tau PTMs, such as kinase inhibitors and antibody-based therapeutics, have been developed, but natural compounds, as modulators of Tau PTMs are not much explored.Materials and Methods: We applied biophysical and biochemical techniques like fluorescence kinetics, oligomerization analysis and transmission electron microscopy to investigate the impact of EGCG on Tau glycation in vitro. The effect of glycation on cytoskeleton instability and its EGCG-mediated rescue were studied by immunofluorescence microscopy in neuroblastoma cells.Results: EGCG inhibited methyl glyoxal (MG)-induced Tau glycation in vitro. EGCG potently inhibited MG-induced advanced glycation endproducts formation in neuroblastoma cells as well modulated the localization of AT100 phosphorylated Tau in the cells. In addition to preventing the glycation, EGCG enhanced actin-rich neuritic extensions and rescued actin and tubulin cytoskeleton severely disrupted by MG. EGCG maintained the integrity of the Microtubule Organizing Center (MTOC) stabilized microtubules by Microtubule-associated protein RP/EB family member 1 (EB1).Conclusions: We report EGCG, a green tea polyphenol, as a modulator of in vitro methylglyoxal-induced Tau glycation and its impact on reducing advanced glycation end products in neuroblastoma cells. We unravel unprecedented function of EGCG in remodeling neuronal cytoskeletal integrity.