A natively unfolded protein tau, which is encoded by the MAPT gene, regulates axonal transport and stabilizes microtubules in the human brain. Stabilization and binding to microtubules are enabled by the repeat domain and proline-rich domain of tau. On the other hand, pathologically modified tau causes tauopathies, which include Alzheimer´s disease (AD). The post-translation modifications – hyperphosphorylation and truncation lead to filament formation – paired helical and straight filaments are present in AD – that further build up tau neurofibrillary tangles. The presence of hexapeptides 275VQIINK280 and 306VQIVYK311 is known to cause protein aggregation [1]. However, the sequence 225KVAVVRT231 in the proline-rich domain, which contains hydrophobic residues, is homologous to the mentioned sequences and could represent another sequence that promotes the formation of aggregates [2].
In the present work, we investigated the mechanism and kinetics of in-vitro aggregation of different forms of tau – tau297-391, tau306-391, tau316-391, and tau321-391 induced by heparin but also without its addition. Dynamic Light Scattering and Thioflavin T fluorescence were used to confirm the presence of aggregates and to monitor the kinetics of this process. The results of the experiment illustrate the time dependence and the influence of different conditions like the addition of heparin and DTT on the formation of aggregates. We found that forms tau316-391 and tau321-391 are able to aggregate despite the fact that they are lacking the presence of hexapeptides 275VQIINK280 or 306VQIVYK311 in their sequence. Furthermore, this project should provide morphological data of the obtained filaments using microscopic methods such as Atomic Force Microscopy and Electron Microscopy. We hypothesize a possible formation of the steric zipper like interfaces that could be formed by sequences 337VEVKSE342, 375KLDF378 and 357LDNITH362. These sequences are forming interfaces in the structure of filaments isolated from patients with corticobasal degeneration [3, 4].
The project also focuses on the influence of the previously identified T-motif (220TRE222), which is located before the sequence 225KVAVVRT231, and its presence could affect the conformation of this sequence. It could have an impact on tau-microtubule binding as well [5]. To achieve our goals, we used the neuroblastoma cell line SH-SY5Y, into which we inserted a vector with mutation T220A that ensures the production of tau bound to a green fluorescent protein. Fluorescence Recovery After Photobleaching will be used to monitor the behavior of GFP-tau. The results of our work will further clarify the physiological function of the tau protein.
1. Y. Wang, E. Mandelkow. Nat Rev Neurosci, 17, (2016), 5-21.
2. A. Savastano, G. Jaipuria, L. Andreas, E. Mandelkow, M. Zweckstetter. Sci Rep, 10, (2020), 1-14.
3. O. Cehlar, O. Bagarova, L. Hornakova, R. Skrabana. Gen Physiol. Biophys, 40, (2021)., 479-493.
4. Y. Shi, W. Zhang, Y. Yang et al. Nature 598, (2021), 359-363.
5. M.D. Mukrasch, M. von Bergen, J. Biernat, D. Fischer, Ch. Griesinger, E. Mandelkow, M. Zweckstetter. J Biol Chem, 16, (2007), 12230-9.
This work was supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic (grant no. VEGA 2/0145/19).