Structure of core domain of fibril-forming PHF/Tau fragments

Short peptide sequences within the microtubule binding domain of the protein Tau are proposed to be core nucleation sites for formation of amyloid fibrils displaying the paired helical filament (PHF) morphology characteristic of neurofibrillary tangles. To study the structure of these proposed nucleation sites, we analyzed the x-ray diffraction patterns from the assemblies formed by a variety of PHF/tau-related peptide constructs containing the motifs VQIINK (PHF6*) in the second repeat and VQIVYK (PHF6) in the third repeat of tau. Peptides included: tripeptide acetyl-VYK-amide (AcVYK), tetrapeptide acetyl-IVYK-amide (AcPHF4), hexapeptide acetyl-VQIVYK-amide (AcPHF6), and acetyl-GKVQIINKLDLSNVQKDNIKHGSVQIVYKPVDLSKVT-amide (AcTR4). All diffraction patterns showed reflections at spacings of 4.7 A, 3.8 A, and approximately 8-10 A, which are characteristic of an orthogonal unit cell of beta-sheets having dimensions a=9.4 A, b=6.6 A, and c=approximately 8-10 A (where a, b, and c are the lattice constants in the H-bonding, chain, and intersheet directions). The sharp 4.7 A reflections indicate that the beta-crystallites are likely to be elongated along the H-bonding direction and in a cross-beta conformation. The assembly of the AcTR4 peptide, which contains both the PHF6 and PHF6* motifs, consisted of twisted sheets, as indicated by a unique fanning of the diffuse equatorial scattering and meridional accentuation of the (210) reflection at 3.8 A spacing. The diffraction data for AcVYK, AcPHF4, and AcPHF6 all were consistent with approximately 50 A-wide tubular assemblies having double-walls, where beta-strands constitute the walls. In this structure, the peptides are H-bonded together in the fiber direction, and the intersheet direction is radial. The positive-charged lysine residues face the aqueous medium, and tyrosine-tyrosine aromatic interactions stabilize the intersheet (double-wall) layers. This particular contact, which may be involved in PHF fibril formation, is proposed here as a possible aromatic target for anti-tauopathy drugs.     

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.     

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.     

Chemical dissection and reassembly of amyloid fibrils formed by a peptide fragment of transthyretin

We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

蛋白磷酸酯酶对Alzheimer神经原纤维缠结的松解作用

神经原纤维缠结是Ａｌｚｈｅｉｍｅｒ患者的特征性脑病理损伤,其形成机制至今不明．根据神经原纤维缠结的基本组分是异常磷酸化ｔａｕ蛋白的聚集形式双螺旋丝（ｐａｉｒｅｄｈｅｌｉｃａｌｆｉｌａｍｅｎｔｓ,ＰＨＦ）的研究结果,推测蛋白磷酸酯酶与蛋白激酶的失衡可能与ＰＨＦ的形成有关．将蛋白磷酸酯酶ＰＰ－２Ａ和ＰＰ－２Ｂ与ＰＨＦ一起在３７℃保温３０ｍｉｎ可使ＰＨＦ缠结结构松解,成为单个ＰＨＦ原纤维,延长去磷酸化反应时间至３ｈ可使ＰＨＦ结构进一步松解,释放一些游离ＰＨＦ原纤维片段．放免印迹定量分析结果表明：ＰＰ－２Ａ处理的ＰＨＦ样品比对照者释放游离ｔａｕ蛋白的量增加２５％．此外,ＰＰ－２Ａ和ＰＰ－２Ｂ去磷酸化的ＰＨＦ对脑中钙激活的中性蛋白水解酶的抗性降低．这些研究资料从结构上显示了Ａｌｚｈｅｉｍｅｒ病脑病理损伤的可逆性,为Ａｌｚｈｅｉｍｅｒ病治疗的可能性提供了实验依据     

beta-Amyloid induces paired helical filament-like tau filaments in tissue culture

Paired helical filaments (PHF) are the principal pathologic components of neurofibrillary tangles in Alzheimer's disease (AD). To reproduce the formation of PHF in tissue culture, we stably expressed human tau with and without pathogenic mutations in human SH-SY5Y cells and exposed them for 5 days to aggregated synthetic beta-amyloid peptide (A beta 42). This caused a decreased solubility of tau along with the generation of PHF-like tau-containing filaments. These were 20 nm wide and had periodicities of 130-140 nm in the presence of P301L mutant tau or 150-160 nm in the presence of wild-type tau. Mutagenesis of the phosphoepitope serine 422 of tau prevented both the A beta 42-mediated decrease in solubility and the generation of PHF-like filaments, suggesting a role of serine 422 or its phosphorylation in tau filament formation. Together, our data underscore a role of A beta 42 in the formation of PHF-like filaments. Our culture system will be useful to map phosphoepitopes of tau involved in PHF formation and to identify and characterize modifiers of the tau pathology. Further adaptation of the system may allow the screening and validation of compounds designed to prevent PHF formation.     

Positional effects of phosphorylation on the stability and morphology of tau-related amyloid fibrils

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer's disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V(306)QIVYK(311) sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-β structure. In addition, the phosphoserine-containing peptides showed the characteristics of β-sandwiches that could interact with both faces of the β-sheet. On the basis of these observations, possible protofilament models with four β-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between β-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.     

The conformations of filamentous and soluble tau associated with Alzheimer paired helical filaments

Paired helical filaments (PHF) occur in Alzheimer's diseased brains and are known to be composed of the microtubule-associated protein, tau. In the present report, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM) were used to characterize PHF suspended in Tris-buffered saline (TBS), sodium acetate buffer, and water. In TBS the CD spectrum of PHF was observed to have a spectral pattern consistent with 31-37% alpha-helix, 15-20% beta-sheet, 20-23% turn, and 26-29% unordered structure. The TBS sample was found to undergo a cooperative thermal transition between 70 and 75 degrees C, consistent with the changes observed in filament morphology, and it suggests that filamentous tau in the PHF (PHF-tau) makes a substantial contribution to the overall CD. Observed changes in the CD spectrum following removal of PHF by centrifugation suggest that PHF-tau possesses a higher fraction of alpha-helical structure than soluble tau. In acetate buffer, where only straight filaments were observed, the CD was consistent with a marked decrease in the fraction of alpha-helix and an increase in the fraction of beta-sheet relative to the sample in TBS. In water, where only rudimentary filaments remain, the CD was consistent with a Type II or II' beta-turn conformation. Only noncooperative thermal transitions were observed for the PHF samples in acetate buffer and water, consistent with the presence of a heterogeneous population of folded structures. Taken cumulatively, the results are consistent with immunological data showing the presence of folded forms of tau and suggest that phosphorylation or nonproteinaceous components are able to induce conformations of tau other than the random coil conformation previously reported for cloned or purified human tau.     

The carboxyl third of tau is tightly bound to paired helical filaments

To obtain definitive evidence that tau is a component of paired helical filaments (PHF) in Alzheimer's disease, we fractionated and sequenced PHF-derived peptides according to a previously described procedure. In the PHF digest, we found four independent tau peptides that were located in the carboxyl third of tau. Subsequent extensive analysis of the PHF digest did not provide any other tau peptides. The conventional PHF antiserum and a new antiserum directed toward formic acid-denatured PHF reacted with the distinct CNBr fragments of tau localized on the carboxy-terminal portion of tau by protein sequencing. From these observations, we conclude that the carboxyl third of tau is tightly bound to PHF.     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

Deamidation and isoaspartate formation in smeared tau in paired helical filaments. Unusual properties of the microtubule-binding domain of tau

An extensive loss of a selected population of neurons in Alzheimer's disease is closely related to the formation of paired helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160), smeared tau was purified, and its peptide map was compared with that of soluble (normal) tau. A CNBr fragment from soluble tau (CN5; residues 251-419 according to the 441-residue isoform) containing the microtubule-binding domain migrated at 15 and 18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from smeared tau exhibited two larger, unusually broad bands at approximately 30 and approximately 45 kDa, presumably representing dimers and trimers of CN5. In the peptide map of smeared tau-derived CN5, distinct peaks eluting at unusual locations were noted. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other than aspartyl or isoaspartyl peptide were found in the digests of smeared tau-derived CN5, it is likely that site-specific deamidation and isoaspartate formation are involved in its dimerization and trimerization and thus in PHF formation in vivo.     

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments.

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross-linked with the lysine specific cross-linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross-links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15-20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino-terminal helices 1A, 1B and 2A. Both EGS cross-links identified in the rod were also isolated from cross-linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross-linked filaments yielded a third EGS cross-link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy-terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross-links on current filament models are discussed.     

N-terminal fragments of tau inhibit full-length tau polymerization in vitro

The polymerization of the microtubule-associated protein, tau, into insoluble filaments is a common thread in Alzheimer's disease and in a variety of frontotemporal dementias. The conformational change required for tau to transition from an extended monomeric state to a filamentous state with a high beta-sheet content involves the extreme N-terminus coming into contact with distal portions of the molecule; however, these exact interactions are incompletely understood. Here we report that a construct representing amino acids 1-196 (Tau196), which itself does not polymerize, inhibits polymerization of full-length tau (hTau40) in vitro. In addition, we trace the inhibitory effect of Tau196 to amino acids 18-42 of the construct. We also provide evidence that the N-terminal tau fragments require a specific C-terminal region of tau (residues 392-421) to exert their inhibitory effect. The fragments are most effective at inhibiting polymerization when present during the initial 5 min; they remain in the soluble fraction of the polymerization reaction, and they increase the amount of soluble hTau40. The fragments also reduce the number and average length of filaments that are formed. Taken together, these results suggest that the N-terminal tau fragments inhibit hTau40 polymerization by interacting with a specific C-terminal sequence, thereby stabilizing a soluble conformation of tau.     

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.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.