Structural memory of natively unfolded tau protein detected by small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) is a universal low-resolution method to study size and shape of globular proteins in solution but recent developments facilitate the quantitative characterization of the structure and structural transitions of metastable systems like partially or completely unfolded proteins. We present here a study of temperature induced transitions in tau, a natively unfolded protein involved in Alzheimer's disease. Previous studies on full length tau and several disease-related mutants provided information about the residual structure in different domains revealing a specific role and extended conformations of the so-called repeat domains, which are considered to be responsible for the formation of amyloid-like fibrils ("paired helical filaments"). Here, we employ SAXS to investigate the temperature dependent properties of tau. Slow heating/cooling of the full length protein from 10°C to 50°C did not lead to detectable changes in the overall size. Surprisingly, quick heating/cooling caused tau to adopt a significantly more compact conformation, which was stable over up to 3 h and represents a structural "memory" effect. This compaction is not observed for the shorter tau constructs containing largely the repeat domains. The structural and functional implications of the observed unusual behavior of tau under nonequilibrium conditions are discussed.     

Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.     

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.     

Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain

Tau protein, a neuronal microtubule-associated protein, forms insoluble fibers ("paired helical filaments") in Alzheimer's disease and other tauopathies. Conflicting views on the structure of the fibers have been proposed recently, ranging from mainly alpha-helical structure to mainly beta-sheet, or a mixture of mostly random coil and beta-sheet. We have addressed this issue by studying tau fibers immunopurified from Alzheimer brain tissue by a conformation-specific antibody and comparing them with fibers reassembled from recombinant tau or tau constructs in vitro, using a combination of electron microscopy and spectroscopic methods. Brain-derived fibers and reassembled fibers both exhibit a typical twisted appearance when examined by electron microscopy. The soluble tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs and reassembled fibers show a shift toward an increase in the level of beta-structure. The results support a model in which the repeat domain of tau (which lies within the core of PHFs) adopts an increasing level of beta-structure during aggregation, whereas the N- and C-terminal domains projecting away from the PHF core are mostly random coil.     

The effect of a DeltaK280 mutation on the unfolded state of a microtubule-binding repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer's disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a DeltaK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and DeltaK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of beta-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.     

The Effect of a ΔK280 Mutation on the Unfolded State of a Microtubule-Binding Repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer''s disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a ΔK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and ΔK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of β-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.     

Structure,stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis

By using tryptophan scanning mutagenesis, we observed the kinetics and structure of the polymerization of tau into paired helical filaments (PHFs) independently of exogenous reporter dyes. The fluorescence exhibits pronounced blue shifts due to burial of the residue inside PHFs, depending on Trp position. The effect is greatest near the center of the repeat domain, showing that the packing is tightest near the beta-structure inducing hexapeptide motifs. The tryptophan response allows measurement of PHF stability made by different tau isoforms and mutants. Unexpectedly, the stability of PHFs is quite low (denaturation half-points approximately 1.0 m GdnHCl), implying that incipient aggregation should be reversible and that the observed high stability of Alzheimer PHFs is due to other factors. The stability increases with the number of repeats and with tau mutants promoting beta-structure, arguing for a gain of toxic function in frontotemporal dementias. Fluorescence resonance energy transfer (FRET) was used to analyze the distances of Tyr(310) to tryptophans in different positions. The degree of FRET in the soluble protein was position-dependent, with highest signals within the second and third repeats but low or no signals further away. In PHFs most mutants showed FRET, indicating that tight packing results from assembly of tau into PHFs.     

Structural evaluation of conformational transition state responsible for self-assembly of tau microtubule-binding domain

In the brains of Alzheimer's disease patients, the tau protein abnormally aggregates to form an insoluble paired helical filament (PHF). Since the third repeat structure (R3) of the tau microtubule-binding domain plays an essential role in PHF formation and self-aggregates most significantly in an aqueous solution of 20-40% trifluoroethanol (TFE), its possible conformation was estimated from the combination of (i) the TFE-dependent deviations of NH and CalphaH proton chemical shifts from those of the random structure in water and (ii) the TFE-dependent NOE effect connectivity diagrams between the neighboring protons. Consequently, it was indicated that the extended structure of the N-terminal VQIVYK moiety and the alpha-helical-like structure of the LSKVTSKC region provide a structural scaffold for initiating the self-assembled filament formation of the R3 structure. To the best of our knowledge, this is the first study that demonstrated the initial structural moiety and its structural feature necessary for starting the tau PHF formation.     

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31-32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively (13)C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged (1)H-(13)C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain (2)H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.     

A Conformation- and Phosphorylation-Dependent Antibody Recognizing the Paired Helical Filaments of Alzheimer's Disease

Abstract: Hyperphosphorylated tau (PHF-tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation-dependent epitopes on tau, and (c) those recognizing conformation-dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation-dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF-tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation-dependent phosphoepitopes on tau, typified by TG-3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site-directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG-3. Additional studies demonstrate that TG-3 reacts with neonatal mouse tau and PHF-tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG-3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.     

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.     

The natively unfolded character of tau and its aggregation to Alzheimer-like paired helical filaments

The abnormal aggregation of the microtubule-associated protein Tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer disease (AD). Tau in solution behaves as a natively unfolded or intrinsically disordered protein while its aggregation is based on the partial structural transition from random coil to beta-structure. Our aim is to understand in more detail the unfolded nature of Tau, to investigate the aggregation of Tau under different conditions and the molecular interactions of Tau in filaments. We show that soluble Tau remains natively unfolded even when its net charge is minimized, in contrast to other unfolded proteins. The CD signature of the random-coil character of Tau shows no major change over wide variations in charge (pH), ionic strength, solvent polarity, and denaturation. Thus there is no indication of a hydrophobicity-driven collapse, neither in the microtubule-binding repeat domain constructs nor in full-length Tau. This argues that the lack of hydrophobic residues but not the net charge accounts for unfolded nature of soluble Tau. The aggregation of the Tau repeat domain (that forms the core of PHFs) in the presence of nucleating polyanionic cofactors (heparin) is efficient in a range of buffers and pH values between approximately 5 and 10 but breaks down beyond that range, presumably because the pattern of charged interactions disappears. Similarly, elevated ionic strength attenuates aggregation, and the temperature dependence is bell-shaped with an optimum around 50 degrees C. Reporter dyes ThS and ANS record the aggregation process but sense different states (cross-beta-structure vs hydrophobic pockets) with different kinetics. Preformed PHFs are surprisingly labile and can be disrupted by denaturants at rather low concentration ( approximately 1.0 M GdnHCl), much less than required to denature globular proteins. Partial disaggregation of Tau filaments at extreme pH values monitored by CD and EM indicate the importance of salt bridges in filament formation. In contrast, Tau filaments are remarkably resistant to high temperature and high ionic strength. Overall, the stability of PHFs appears to depend mainly on directed salt bridges with contributions from hydrophobic interactions as well, consistent with a recent structural model of the PHF core derived from solid state NMR (Andronesi, O. C., von Bergen, M., Biernat, J., Seidel, K., Griesinger, C., Mandelkow, E., and Baldus, M. (2008) Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.     

Conformation of full-length Bruton tyrosine kinase (Btk) from synchrotron X-ray solution scattering

Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity.     

Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau(RD)) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation DeltaK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with DeltaK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau(RD) is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for beta-structure. (c) Soluble mutants of tau(RD) that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau(RD) in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau(RD) (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau(RD) disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau(RD) that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.     

Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods

The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the four-repeat microtubule binding domain (4RMBD) in tau protein and its three mutants, 4RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4RMBD mutation.     

Tau aggregation is driven by a transition from random coil to beta sheet structure

The abnormal aggregation of the microtubule associated protein tau into paired helical filaments (PHFs) is one the hallmarks of Alzheimer's disease. The soluble protein is one of the longest natively unfolded proteins, lacking significant amounts of secondary structure over a sequence of 441 amino acids in the longest isoform. Furthermore, the unfolded character is consistent with some notable features of the protein like stability towards heat and acid treatment. It is still unclear how these characteristics support the physiological function of binding to and stabilization of microtubules. We review here some recent studies on how an unfolded protein such as tau can adopt beta-structure, which then leads to the highly ordered morphology of the PHFs. The core sequence for both microtubule binding and PHF formation is the microtubule binding domain containing three or four repeats. This region alone is sufficient for PHF formation and mostly unfolded in the soluble state. A search for sequence motifs within this region crucial for PHF building revealed two hexapeptides in the second and the third repeat. Some of the genetically linked cases of FTDP-17 show missense mutations in or adjacent to these hexapeptide motifs. Proteins containing the P301L and the DeltaK280 mutations exhibit accelerated aggregation. The importance of the two hexapeptides stems from their capacity to undergo a conformational change from a random coil to a beta sheet structure. The increase of beta sheet structure is a typical feature of an amyloidogenic protein and is the basis of other characteristics like a decreased sensitivity towards proteolytic degradation and Congo red binding. PHFs aggregated in vitro and in vivo contain beta-sheet structure, as judged by circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction.     

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.     

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.     

A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.     

Small-angle X-ray Scattering of Apolipoprotein A-IV Reveals the Importance of Its Termini for Structural Stability

ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study''s low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.