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.     

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.     

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.     

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.     

Carbamylation promotes amyloidogenesis and induces structural changes in Tau-core hexapeptide fibrils

Background

Carbamylation is a non-enzymatic post-translational modification (PTM), which involves the covalent modification of N-terminus of protein or ε-amino group of Lys. The role of carbamylation in several age-related disorders is well documented, however, the relationship between carbamylation and neurodegenerative disorders including Alzheimer's disease remains uncharted.

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.     

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.     

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule‐binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule‐binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule‐binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N‐terminus of repeat 2 has a lower propensity to form strand‐like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle‐bound state. Interestingly, the behavior of the Module‐B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module‐B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand‐like structure and the resistance of these propensities to changes in tau's environment.     

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.     

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.     

Formation and Growth of Oligomers: A Monte Carlo Study of an Amyloid Tau Fragment

Small oligomers formed early in the process of amyloid fibril formation may be the major toxic species in Alzheimer's disease. We investigate the early stages of amyloid aggregation for the tau fragment AcPHF6 (Ac-VQIVYK-NH2) using an implicit solvent all-atom model and extensive Monte Carlo simulations of 12, 24, and 36 chains. A variety of small metastable aggregates form and dissolve until an aggregate of a critical size and conformation arises. However, the stable oligomers, which are β-sheet-rich and feature many hydrophobic contacts, are not always growth-ready. The simulations indicate instead that these supercritical oligomers spend a lengthy period in equilibrium in which considerable reorganization takes place accompanied by exchange of chains with the solution. Growth competence of the stable oligomers correlates with the alignment of the strands in the β-sheets. The larger aggregates seen in our simulations are all composed of two twisted β-sheets, packed against each other with hydrophobic side chains at the sheet–sheet interface. These β-sandwiches show similarities with the proposed steric zipper structure for PHF6 fibrils but have a mixed parallel/antiparallel β-strand organization as opposed to the parallel organization found in experiments on fibrils. Interestingly, we find that the fraction of parallel β-sheet structure increases with aggregate size. We speculate that the reorganization of the β-sheets into parallel ones is an important rate-limiting step in the formation of PHF6 fibrils.     

The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.     

Amyloidogenic sequences in native protein structures

Numerous short peptides have been shown to form β‐sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 proteins containing 45 sequences known to form amyloid, to see how the proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen‐bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form β structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a β structure. Secondly, amyloidogenic sequences that are found in β structure are usually buried within the protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to protein aggregation via assembly of the amyloidogenic regions. The use of α‐helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for β structure, is thus a widespread mechanism to avoid protein aggregation.     

Therapeutic Strategies for Alzheimer’s Disease

Therapeutic approaches for Alzheimer's disease (AD) are guided by four disease characteristics: amyloid plaques, neurofibrillar tangles (NFT), neurodegeneration, and dementia. Amyloid plaques are composed largely of 4 kDa beta-amyloid (Abeta) peptides, with the more amyloidogenic, 42 amino acid form (Abeta42) as the primary species. Because multiple, rare mutations that cause early-onset, familial AD lead to increased production or aggregation of Abeta42, amyloid therapeutics aim to reduce the amount of toxic Abeta42 aggregates. Amyloid-based therapies include gamma-secretase inhibitors and modulators, BACE inhibitors, aggregation blockers, catabolism inducers, and anti-Abeta biologics. Tangles are composed of paired helical filaments of hyperphosphorylated tau protein. Tau-based therapeutics include kinase inhibitors, microtubule stabilizers, and catabolism inducers. Therapeutic strategies for neurodegeneration target multiple mechanisms, including excitotoxicity, mitochondrial dysfunction, oxidative damage, and inflammation or stimulation of neuronal viability. Although not disease modifying, cognition enhancers are important to treat the symptom of dementia. Strategies for cognition enhancement include cholinesterase inhibitors, and other approaches to enhance the signaling of cholinergic and glutamatergic neurons. In summary, plaques, tangles, neurodegeneration and dementia guide the development of multiple therapeutic approaches for AD and are the subject of this review.     

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.     

Integrating in vitro and in silico approaches to evaluate the “dual functionality” of palmatine chloride in inhibiting and disassembling Tau-derived VQIVYK peptide fibrils

Background

Alzheimer's disease (AD) is the most common neurodegenerative disorder which is characterized by the deposits of intra-cellular tau protein and extra-cellular amyloid-β (Aβ) peptides in the human brain. Understanding the mechanism of protein aggregation and finding compounds that are capable of inhibiting its aggregation is considered to be highly important for disease therapy.

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.     

Expression of tau reduces secretion of Abeta without altering the amyloid precursor protein content in CHOsw cells

Insoluble deposits of tau and amyloid precursor protein (APP) peptides Abeta characterize Alzheimer's disease. We studied the role of tau in the metabolism of APP in cells stably expressing APP Swedish mutation (CHOsw). Transient expression of tau in CHOsw cells caused morphological changes, bundling of microtubules and perinuclear aggregation of Golgi-derived vesicles. It also reduced the secretion of Abeta(1-40) and Abeta(1-42) without altering the APP steady state levels. This was accompanied by a reduction in the gamma-secretase and an increase in the insulin degrading enzyme activities. Our results suggest that tau may play an inhibitory role in the amyloidogenic activity of APP.     

Exploring amyloid oligomers with peptide model systems

The assembly of amyloidogenic peptides and proteins, such as the β-amyloid peptide, α-synuclein, huntingtin, tau, and islet amyloid polypeptide, into amyloid fibrils and oligomers is directly linked to amyloid diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, frontotemporal dementias, and type II diabetes. Although amyloid oligomers have emerged as especially important in amyloid diseases, high-resolution structures of the oligomers formed by full-length amyloidogenic peptides and proteins have remained elusive. Investigations of oligomers assembled from fragments or stabilized β-hairpin segments of amyloidogenic peptides and proteins have allowed investigators to illuminate some of the structural, biophysical, and biological properties of amyloid oligomers. Here, we summarize recent advances in the application of these peptide model systems to investigate and understand the structures, biological properties, and biophysical properties of amyloid oligomers.     

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.