Site-specific nitration differentially influences tau assembly in vitro

Previously, we reported that the microtubule-associated tau protein, the major constituent of neurofibrillary tangles (NFTs) in Alzheimer's brain, undergoes site-selective nitration by peroxynitrite (ONOO-) and that this event inhibits tau polymerization in vitro [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we extend our analysis of tau nitration to include mutant tau proteins singly nitrated at each residue targeted by ONOO- in vitro (Tyr18, Tyr29, Tyr197, and Tyr394). Using our polymerization paradigm, we demonstrate that site-specific Tyr nitration differentially alters the rate and/or extent of tau assembly and generates robust changes in filament morphology. As determined by quantitative electron microscopy, select nitration of residues Tyr29 and Tyr197 increases the average length of synthetic tau filaments but does not alter the steady-state polymer mass. In contrast, site-specific nitration of residues Tyr18 and Tyr394 decreases the average length and/or number of synthetic filaments, resulting in a significant reduction in filamentous mass and an increase in tau critical concentration. Intriguingly, affinity measurements demonstrate that nitrative modifications do not preclude formation of the Alz-50 epitope, a pathological tau conformation detectable in authentic paired helical filaments (PHFtau). In fact, the Alz-50 antibody binds filaments assembled from nitrated mutant tau with higher avidity than wild-type filaments, even in instances where the overall filamentous mass is reduced. Taken together, our results suggest that site-specific nitration modulates the nucleation and/or elongation capacity of assembly-competent tau and that assumption of the Alz-50 conformation may be necessary, but not sufficient, to induce filament formation.     

Evidence for independent mechanisms and a multiple-hit model of tau assembly

Formation of inclusions containing polymerized tau protein is a hallmark of Alzheimer's disease and related disorders. In vitro studies have demonstrated the ability of polyglycosaminoglycans and fatty acids to promote tau polymerization. In this report, we examined their impact on tau polymerization separately and together. Tau assembly with only arachidonic acid was faster than that with only heparin. The presence of dithiothreitol reduced heparin-promoted tau assembly while enhancing arachidonic acid reactions. However, simultaneous use of these molecules increased the rate of filament assembly substantially, negated the effects of the reducing agent, and has very little effect on the morphology of filaments. The increases in polymerization resulted from accelerated nucleation. Finally, a FTDP-17 mutation was identified that could complement heparin to generate assembly kinetics similar to that of wild-type tau with both inducers. Our results support a multiple-hit model where several induced changes in tau can function independently to promote tau assembly.     

Differential assembly of human tau isoforms in the presence of arachidonic acid

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.     

Microtubule-associated protein tau. A component of Alzheimer paired helical filaments

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phosphocellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with perchloric acid. All of these tau preparations reacted specifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypeptides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results suggest that tau is a major component of Alzheimer paired helical filaments.     

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.     

The formation of straight and twisted filaments from short tau peptides

We studied fibril formation in a family of peptides based on PHF6 (VQIVYK), a short peptide segment found in the microtubule binding region of tau protein. N-Acetylated peptides AcVYK-amide (AcVYK), AcIVYK-amide (AcPHF4), AcQIVYK-amide (AcPHF5), and AcV-QIVYK-amide (AcPHF6) rapidly formed straight filaments in the presence of 0.15 m NaCl, each composed of two laterally aligned protofilaments approximately 5 nm in width. X-ray fiber diffraction showed the omnipresent sharp 4.7-A reflection indicating that the scattering objects are likely elongated along the hydrogen-bonding direction in a cross-beta conformation, and Fourier transform IR suggested the peptide chains were in a parallel (AcVYK, AcPHF6) or antiparallel (AcPHF4, AcPHF5) beta-sheet configuration. The dipeptide N-acetyl-YK-amide (AcYK) formed globular structures approximately 200 nm to 1 microm in diameter. The polymerization rate, as measured by thioflavin S binding, increased with the length of the peptide going from AcYK --> AcPHF6, and peptides that aggregated most rapidly displayed CD spectra consistent with beta-sheet structure. There was a 3-fold decrease in rate when Val was substituted for Ile or Gln, nearly a 10-fold decrease when Ala was substituted for Tyr, and an increase in polymerization rate when Glu was substituted for Lys. Twisted filaments, composed of four laterally aligned protofilaments (9-19 nm width, approximately 90 nm half-periodicity), were formed by mixing AcPHF6 with AcVYK. Taken together these results suggest that the core of PHF6 is localized at VYK, and the interaction between small amphiphilic segments of tau may initiate nucleation and lead to filaments displaying paired helical filament morphology.     

Structure of tau protein and assembly into paired helical filaments

Over the past few years the systematic investigation of paired helical filament assembly from tau protein in vitro has become feasible. We review our current understanding of the structure and conformations of tau protein and how this affects tau's assembly into the pathological paired helical filaments in Alzheimer's disease.     

Sites of tau important for aggregation populate {beta}-structure and bind to microtubules and polyanions

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.     

Structural and functional implications of tau hyperphosphorylation: information from phosphorylation-mimicking mutated tau proteins

Abnormal tau-immunoreactive filaments are a hallmark of tauopathies, including Alzheimer's disease (AD). A higher phosphorylation ("hyperphosphorylation") state of tau protein may represent a critical event. To determine the potential role of tau hyperphosphorylation in these disorders, mutated tau proteins were produced where serine/threonine residues known to be highly phosphorylated in tau filaments isolated from AD patients were substituted for glutamate to simulate a paired helical filament (PHF)-like tau hyperphosphorylation. We demonstrate that, like hyperphosphorylation, glutamate substitutions induce compact structure elements and SDS-resistant conformational domains in tau protein. Hyperphosphorylation-mimicking glutamate-mutated tau proteins display a complete functional loss in its ability to promote microtubule nucleation which can partially be overcome by addition of the osmolyte trimethylamine N-oxide (TMAO), which is similar to phosphorylated tau. In addition, glutamate-mutated tau proteins fail to interact with the dominant brain protein phosphatase 2A isoform ABalphaC, and exhibit a reduced ability to assemble into filaments. Interestingly, wild-type tau and phosphorylation-mimicking tau similarly bind to microtubules when added alone, but the mutated tau is almost completely displaced from the microtubule surface by equimolar concentrations of wild-type tau. The data indicate that glutamate-mutated tau proteins provide a useful model for analyzing the functional consequences of tau hyperphosphorylation. They suggest that several mechanisms contribute to the abnormal tau accumulation observed during tauopathies, in particular a selective displacement of hyperphosphorylated tau from microtubules, a functional loss in promoting microtubule nucleation, and a failure to interact with phosphatases.     

Self assembly of microtubule associated protein tau into filaments resembling those found in Alzheimer disease

Microtubule associated protein tau factor self-assembles into filamentous structures resembling the paired helical filaments found in Alzheimer disease. Tau polymerization requires of a previous modification; conversion of glutamine into glutamic acid by deamination.     

Ligand-dependent inhibition and reversal of tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.     

Prediction of nucleating sequences from amyloidogenic propensities of tau-related peptides

Physical properties, including amyloid morphology, FTIR and CD spectra, enhancement of Congo red absorbance, polymerization rate, critical monomer concentration, free energy of stabilization, hydrophobicity, and the partition coefficient between soluble and amyloid states, were measured for the tau-related peptide Ac-VQIVYK amide (AcPHF6) and its single site mutants Ac-VQIVXK amide (X not equal Cys). Transmission electron microscopy showed that 15 out of the 19 peptides formed amyloid in buffer, with morphologies ranging from straight and twisted filaments to sheets and rolled sheets. Using principal component analysis (PCA), measured properties were treated in a comprehensive manner, and scores along the most significant principal components were used to define individual amino acid amyloidogenic propensities. Quantitative structure-activity modeling (QSAM) showed that residues with greater size and hydrophobicity made the largest contributions to the propensity of peptides to form amyloid. Using individual amino acid propensities, sequences within tau with high amyloid-forming potential were estimated and found to include 226VAVVR230 in the proline-rich region, 275VQIINK280 (PHF6) and 306VQIVYK311 (PHF6) within the microtubule binding region, and 392IVYK395 in the C-tail region of the protein. The results suggest that regions outside the microtubule-binding region may play important roles in tau aggregation kinetics or paired helical filament structure.     

A complex mechanism for inducer mediated tau polymerization

The accumulation of polymers of the microtubule associated protein tau is correlative with increased neurodegeneration in Alzheimer's disease and other related tauopathies. In vitro models have been developed in order to investigate molecular mechanisms that regulate the polymerization of tau. Arachidonic acid and heparin have been proposed to induce tau polymerization via a ligand dependent nucleation-elongation mechanism. However, certain aspects of these in vitro results are inconsistent with a classic nucleation-elongation mechanism. Using steady state and kinetic analyses of tau polymerization at a variety of protein and inducer concentrations, we have found that the thermodynamic barrier for nucleation in the presence of inducers is negligible, which was manifested by increases in protein polymerization at low tau concentrations and very rapid kinetics of polymerization. However, the mechanism of polymerization is complicated by the observation that high concentrations of inducer molecules result in the inhibition of tau fibril formation through different mechanisms for arachidonic acid and heparin. These observations indicate that the molar ratio of inducer to protein is a greater determinant of the rate and extent of tau polymerization than the concentration of tau itself. Our results are therefore not consistent with a canonical nucleation-elongation reaction but rather are more consistent with an allosteric regulation model in which the presence of small molecules induce a conformational change in the protein that decreases the thermodynamic barrier for polymerization essentially to zero.     

Quinones facilitate the self-assembly of the phosphorylated tubulin binding region of tau into fibrillar polymers

The fragment of tau containing the first and third tubulin-binding motifs, involved in self-assembly of tau, was phosphorylated by protein kinase A (PKA). In the presence of hydroxynonenal (HNE) or in the presence of quinones such as juglone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q(0) or DMM), or menadione, the polymerization of this phosphorylated tau fragment is catalyzed, whereas polymerization of the unmodified fragment takes place in a lesser extent. The quinones coenzyme Q(0) and menadione are found in every cell, including neural cells, and may interact with tau protein to facilitate its assembly into filamentous structures. These tau filaments, assembled in the presence of quinones, have a fibrillar morphology very similar to that of paired helical filaments present in the brains of patients with Alzheimer's disease.     

Effect of capping protein on the kinetics of actin polymerization

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.     

Residual structure in the repeat domain of tau: echoes of microtubule binding and paired helical filament formation

The microtubule-associated protein tau is found aggregated into paired helical filaments in the intraneuronal neurofibrillary tangle deposits of victims of Alzheimer's disease (AD) and other related dementias. Tau contains a repeat domain consisting of three or four 31-32-residue imperfect repeats that forms the core of tau filaments and is capable of self-assembling into filaments in vitro. We have used high-resolution NMR spectroscopy to characterize the structural properties of the three-repeat domain of tau at the level of individual residues. We find that three distinct regions of the polypeptide corresponding to previously mapped microtubule interaction sites exhibit a preference for helical conformations, suggesting that these sites adopt a helical structure when bound to microtubules. In addition, we directly observe a marked preference for extended or beta-strand-like conformations in a stretch of residues between two of the helical regions, which corresponds closely to a region previously implicated as an early site of beta-strand structure formation and intermolecular interactions leading to paired helical filament (PHF) formation. This observation supports the idea that this region of the protein plays a crucial role in the formation of tau aggregates. We further show that disulfide-bond-mediated dimer formation does not affect and is not responsible for the observed structural preferences of the protein. Our results provide the first high-resolution view of the structural properties of the protein tau, are consistent with an important role for beta structure in PHF formation, and may also help explain recent reports that tau filaments contain helical structure.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

In vitro assembly of tau protein: mapping the regions involved in filament formation

Unraveling the mechanism of self-assembly of the protein tau into paired helical filaments (PHFs) is a crucial step toward the understanding of Alzheimer's and other neuropathological diseases at the molecular level. In an effort to map the role of different regions of tau in the mechanism of self-assembly, we have studied the polymerization ability of different tau fragments using an in vitro assay. Our results indicate that the N-terminal domain interferes with tau's ability to polymerize in vitro. The effect seems to be size dependent. Particularly, an isoform of tau from the peripheral nervous system, which has a much larger N-terminal domain, was found unable to form filaments in our in vitro assay. This finding can explain why in Alzheimer's patients PHFs only accumulate in the neurons from the central nervous system. We also report that a short segment of tau located in the third microtubule binding repeat (residues 317 to 335, peptide 1/2R) is probably the minimal segment of that region able to grow into filaments in vitro and in the presence of heparin. In contrast with whole peptide 1/2R, peptides corresponding to either the N-terminal or C-terminal halves of this segment were unable to form filaments. Finally, our polymerization studies of peptides from the C-terminal domain reveal a short sequence spanning residues 391 to 407 that grows into filaments in vitro. This tau segment forms filaments regardless of whether is incubated with heparin. Moreover, such filaments differ in diameter and morphology, suggesting a different mechanism of self-assembly.     

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.