| Popis |
Hyperphosphorylated and abnormally post-translationally modified Tau protein forms aggregates called neurofibrillary tangles (NFTs), which are typical hallmark of Alzheimer's disease (AD) in the human brain [1]. As the pattern of post-translational modifications (PTMs) within Tau is complex [2], AD-relevant Tau protein is not easily obtainable by recombinant production in E. coli and subsequent modification by individual enzymes in vitro. To prepare Tau protein with naturally occurring PTMs, we employed mammalian cell culture (HEK293). We aimed to identify the PTMs incorporated in the HEK293 cells, to compare them with published data from AD-patients and afterwards to study the impact of the PTMs on Tau properties in comparison to Tau from E. coli. We optimized the expression and purification protocols yielding sufficient amount of HEK-Tau for its characterization. Using LC-MS/MS, we identified around 20 phosphorylation sites with diverse extent of phosphorylation. The detected phosphorylation patterns were similar to those found in the brains of AD-patients. Afterwards, we performed interaction study between HEK-Tau and 14-3-3? protein, which provided surprising result compared to Tau expressed in E. coli and phosphorylated in vitro [3]. Finally, we used Thioflavin T assay and negative stain EM to obtain insight into aggregation propensities of HEK-Tau. The protein production in HEK293 cells was performed within an Industrial PhD programme in the biotech company BioVendor. 1. M. Goedert, R. A. Crowther, S. H. W. Scheres, M. G. Spillantini, Cytoskeleton, 81, (2024), 1. 2. H. Wesseling, W. Mair, M. Kumar, …, J. A. Steen, Cell, 183, (2020), 6. 3. R. Crha, A. Kozeleková, A. Hofrová, L. Iľkovičová, N. Gašparik, P. Kadeřávek, J. Hritz, Int. J. Biol. Macromol., 266, (2024), 130802. This project has received funding from the European Union’s Horizon Europe program under the grant agreement No. 101087124. We acknowledge CEITEC Proteomics Core Facility of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042, CZ.02.01.01/00/23_015/0008175, e-INFRA CZ (ID:90254)). We acknowledge Cryo-electron microscopy and tomography core facility CEITEC MU of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and European Regional Development Fund-Project „Innovation of Czech Infrastructure for Integrative Structural Biology“(No. CZ.02.01.01/00/23_015/0008175).
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