An acetylation mimicking mutation, K274Q, in tau imparts neurotoxicity by enhancing tau aggregation and inhibiting tubulin polymerization

2019 ◽  
Vol 476 (10) ◽  
pp. 1401-1417 ◽  
Author(s):  
Jitendra S. Rane ◽  
Anuradha Kumari ◽  
Dulal Panda
Keyword(s):  
Conformational Changes ◽  
Critical Concentration ◽  
Neuroblastoma Cells ◽  
Memory Loss ◽  
Tryptophan Fluorescence ◽  
Dot Blot ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Tau Aggregation ◽  
Dot Blot Analysis

Abstract In Alzheimer's disease, tau is predominantly acetylated at K174, K274, K280, and K281 residues. The acetylation of K274-tau is linked with memory loss and dementia. In this study, we have examined the molecular mechanism of the toxicity of acetylated K274-tau. We incorporated an acetylation mimicking mutation at K274 (K→Q) residue of tau. The mutation (K274Q) strongly reduced the ability of tau to bind to tubulin and also to polymerize tubulin while K274R mutation did not reduce the ability of tau either to bind or polymerize tubulin. In addition, K274Q-tau displayed a higher aggregation propensity than wild-type tau as evident from thioflavin S fluorescence, tryptophan fluorescence, and electron microscopic images. Furthermore, dynamic light scattering, atomic force microscopy, and dot blot analysis using an oligomer-specific antibody suggested that K274Q mutation enhanced the oligomerization of tau. The K274Q mutation also strongly decreased the critical concentration for the liquid–liquid phase separation of tau. The oligomeric forms of K274Q-tau were found to be more toxic than wild tau to neuroblastoma cells. Using circular dichroism and fluorescence spectroscopy, we provide evidence indicating that the acetylation mimicking mutation (K274Q) induced conformational changes in tau. The results suggested that the acetylation of tau at 274 residues can increase tau aggregation and enhance the cytotoxicity of tau oligomers.

scholarly journals Human Polymerase δ-Interacting Protein 2 (PolDIP2) Inhibits the Formation of Human Tau Oligomers and Fibrils

2021 ◽  
Vol 22 (11) ◽  
pp. 5768
Author(s):  
Kazutoshi Kasho ◽  
Lukas Krasauskas ◽  
Vytautas Smirnovas ◽  
Gorazd Stojkovič ◽  
Ludmilla A. Morozova-Roche ◽  
...  
Keyword(s):  
Dot Blot ◽  
Interacting Protein ◽  
Force Microscopy ◽  
Tau Oligomers ◽  
Atomic Force ◽  
Central Characteristic ◽  
Tau Aggregation ◽  
Dot Blot Analysis ◽  
The Brain

A central characteristic of Alzheimer’s disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.

scholarly journals Baicalein inhibits heparin-induced Tau aggregation by initializing non-toxic Tau oligomer formation

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shweta Kishor Sonawane ◽  
Vladimir N. Uversky ◽  
Subashchandrabose Chinnathambi
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Senile Plaques ◽  
Neuroblastoma Cells ◽  
Memory Loss ◽  
Disordered Proteins ◽  
Protein Accumulation ◽  
Scutellaria Baicalensis Georgi ◽  
Intrinsically Disordered ◽  
Tau Aggregation

Abstract Background Amyloid aggregate deposition is the key feature of Alzheimer’s disease. The proteinaceous aggregates found in the afflicted brain are the intra-neuronal neurofibrillary tangles formed by the microtubule-associated protein Tau and extracellular deposits, senile plaques, of amyloid beta (Aβ) peptide proteolytically derived from the amyloid precursor protein. Accumulation of these aggregates has manifestations in the later stages of the disease, such as memory loss and cognitive inabilities originating from the neuronal dysfunction, neurodegeneration, and brain atrophy. Treatment of this disease at the late stages is difficult, and many clinical trials have failed. Hence, the goal is to find means capable of preventing the aggregation of these intrinsically disordered proteins by inhibiting the early stages of their pathological transformations. Polyphenols are known to be neuroprotective agents with the noticeable potential against many neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Prion diseases. Methods We analyzed the capability of Baicalein to inhibit aggregation of human Tau protein by a multifactorial analysis that included several biophysical and biochemical techniques. Results The potency of Baicalein, a polyphenol from the Scutellaria baicalensis Georgi, against in vitro Tau aggregation and PHF dissolution has been screened and validated. ThS fluorescence assay revealed the potent inhibitory activity of Baicalein, whereas ANS revealed its mechanism of Tau inhibition viz. by oligomer capture and dissociation. In addition, Baicalein dissolved the preformed mature fibrils of Tau thereby possessing a dual target action. Tau oligomers formed by Baicalein were non-toxic to neuronal cells, highlighting its role as a potent molecule to be screened against AD. Conclusion In conclusion, Baicalein inhibits aggregation of hTau40 by enhancing the formation of SDS-stable oligomers and preventing fibril formation. Baicalein-induced oligomers do not affect the viability of the neuroblastoma cells. Therefore, Baicalein can be considered as a lead molecule against Tau pathology in AD.

Interventional Thermal Injury of the Arterial Wall: Unfolding of von Willebrand Factor and Its Increased Binding to Collagen after 55° C Heating

1996 ◽  
Vol 75 (03) ◽  
pp. 515-519 ◽  
Author(s):  
Mark J Post ◽  
Anke N de Graaf-Bos ◽  
George Posthuma ◽  
Philip G de Groot ◽  
Jan J Sixma ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Arterial Wall ◽  
Platelet Adhesion ◽  
Type I ◽  
Temperature Dependent ◽  
Collagen Types ◽  
Electron Microscopic ◽  
Von Willebrand ◽  
Willebrand Factor

Summary Purpose. Thermal angioplasty alters the thrombogenicity of the arterial wall. In previous studies, platelet adhesion was found to increase after heating human subendothelium to 55° C and decrease after heating to 90° C. In the present electron microscopic study, the mechanism of this temperature-dependent platelet adhesion to the heated arterial wall is elucidated by investigating temperature-dependent conformational changes of von Willebrand factor (vWF) and collagen types I and III and the binding of vWF to heated collagen. Methods. Purified vWF and/or collagen was applied to electron microscopic grids and heated by floating on a salt-solution of 37° C, 55° C or 90° C for 15 s. After incubation with a polyclonal antibody against vWF and incubation with protein A/gold, the grids were examined by electron microscopy. Results. At 37° C, vWF was coiled. At 55° C, vWF unfolded, whereas heating at 90° C caused a reduction in antigenicity. Collagen fibers heated to 37° C were 60.3 ± 3.1 nm wide. Heating to 55° C resulted in the unwinding of the fibers, increasing the width to 87.5 ± 8.2 nm (p < 0.01). Heating to 90° C resulted in denatured fibers with an enlarged width of 85.1 ± 6.1 nm (p < 0.05). Heating of collagen to 55° C resulted in an increased vWF binding as compared to collagen heated to 37° C or to 90° C. Incubation of collagen with vWF, prior to heating, resulted in a vWF binding after heating to 55° C that was similar to the 37° C binding and a decreased binding after 90° C. Conclusions. After 55° C heating, the von Willebrand factor molecule unfolds and collagen types I and III exhibit an increased adhesiveness for von Willebrand factor. Heating to 90° C denatures von Willebrand factor and collagen. The conformation changes of von Willebrand factor and its altered binding to collagen type I and III may explain the increased and decreased platelet adhesion to subendothelium after 55° C and 90° C heating, respectively.

New insights into the structural basis of integrin activation

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1155-1159 ◽  
Author(s):  
Jian-Ping Xiong ◽  
Thilo Stehle ◽  
Simon L. Goodman ◽  
M. Amin Arnaout
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Basis ◽  
Integrin Activation ◽  
Cellular Functions ◽  
Electron Microscopic ◽  
The Past ◽  
Intracellular Signals ◽  
Bidirectional Signaling ◽  
New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

Investigation of surface morphology of thin metal films with magnetic layers Fe-Cr-Co

2021 ◽  
Author(s):  
V.S. Zayonchkovsky ◽  
Aung Kyaw Kyaw ◽  
A.V. Andreev
Keyword(s):  
Atomic Force Microscopy ◽  
Magnetron Sputtering ◽  
Electron Microscopic Examination ◽  
Nearest Neighbors ◽  
Substrate Plane ◽  
Large Area ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Atomic Force ◽  
Large Projection

Films containing layers of dispersion-hardening alloys (LDHA) based on the Fe-Cr-Co system were obtained by magnetron sputtering. LDHA acquire the properties of film permanent magnets after a single-stage «fast» high-vacuum annealing. Bulk materials acquire such properties only after many hours of multi-stage heat treatment. The film samples acquire these properties in tens of seconds. The morphology of their surface was studied to determine the origin of the coercive force of film samples. The surface morphology was studied using high resolution scanning electron microscopy and atomic force microscopy. We studied two compositions that, in bulk, have a different tendency to form many phases during crystallization. In magnetron sputtering, the alloy in which a multiphase state is easily formed is polycrystalline. The antipode alloy in magnetron sputtering is realized in an amorphous state. After annealing, both alloys are in a polycrystalline state. Electron microscopic examination showed that as a result of annealing, crystallites are formed with a large projection onto the substrate plane, which grow due to the nearest neighbors. Moreover, these crystallites have not only a large area, but also a height. After annealing, both alloys are in a polycrystalline state. Electron microscopic examination showed that as a result of annealing, crystallites are formed with a large projection onto the substrate plane, which grow due to the nearest neighbors. Moreover, these crystallites have not only a large area, but also a height. What is determined by atomic force microscopy. High crystallites are also faceted. This may indicate that the composition of these crystallites differs from the composition of the surrounding layer, which may be the reason for the increase in coercive force as a result of annealing.

Dot Blot Analysis for Measuring Global N6-Methyladenosine Modification of RNA

2018 ◽  
pp. 263-271 ◽  
Author(s):  
Arvindhan Nagarajan ◽  
Radoslav Janostiak ◽  
Narendra Wajapeyee
Keyword(s):  
Blot Analysis ◽  
Dot Blot ◽  
Dot Blot Analysis

scholarly journals Light-Induced Conformational Changes of S. aurantiaca Bacteriophytochromes as Revealed by Atomic Force Microscopy

2017 ◽  
Vol 112 (3) ◽  
pp. 191a
Author(s):  
Rima Rebiai ◽  
Stefan Tsonchev ◽  
Kenneth T. Nicholson ◽  
Emina A. Stojković
Keyword(s):  
Atomic Force Microscopy ◽  
Conformational Changes ◽  
Force Microscopy ◽  
Atomic Force

High-resolution transmission electron microscopic investigations of molybdenum thin films on faceted α-Al2O3

2005 ◽  
Vol 38 (2) ◽  
pp. 260-265 ◽  
Author(s):  
Leonore Wiehl ◽  
Jens Oster ◽  
Michael Huth
Keyword(s):  
High Resolution ◽  
Three Dimensional ◽  
Epitaxial Relationship ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
High Resolution Images ◽  
Α Al2o3 ◽  
Relationship Of

Epitaxially grown Mo films on a faceted corundum (α-Al2O3)mplane were investigated by transmission electron microscopy. Low- and high-resolution images were taken from a cross-section specimen cut perpendicular to the facets. It was possible to identify unambiguously the crystallographic orientation of these facets and explain the considerable deviation (∼10°) of the experimental interfacet angle, as measured with atomic force microscopy (AFM), from the expected value. For the first time, proof is given for a smooth \{10\bar{1}1\} facet and a curvy facet with orientation near to \{10\bar{1}\bar{2}\}. Moreover, the three-dimensional epitaxial relationship of an Mo film on a faceted corundummsurface was determined.

scholarly journals FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

2020 ◽  
Author(s):  
Senthilvelrajan Kaniyappan ◽  
Katharina Tepper ◽  
Jacek Biernat ◽  
RamReddy Chandupatla ◽  
Sabrina Hübschmann ◽  
...  
Keyword(s):  
Recipient Cell ◽  
Pathological State ◽  
Tau Pathology ◽  
Amyloid Fibers ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
Repeat Domain ◽  
Scanning Transmission ◽  
Tau Aggregation

Abstract Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~3nm x 4nm) is ~7 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused a process distinct from assembly of TauRD filaments.

scholarly journals Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

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