Importance of local structures of second and third repeat fragments of microtubule-binding domain for tau filament formation.

To investigate the importance of the seventh residue of the second and third repeat fragments (R2 and R3 peptides) of the microtubule-binding domain (MBD) for tau filamentous assembly, the residues Lys and Pro were substituted (R2-K7P and R3-P7K). The filament formations of the R2 and R3 peptides were almost lost due to their substitutions despite their overall conformational similarities. The NOE analyses showed the importance of the conformational flexibility for the R2 peptide and the coupled extended and helical conformations for the R3 peptide in their limited N-terminal regions around their seventh residues. The result shows that the filament formation of MBD is initiated from a short fragment region containing the minimal conformational or functional motif.

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation.

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimer's disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C-terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau-induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.

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.

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.

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.