| Description |
Phosphorylation is a post-translational modification that affects structure, function, and interactions of proteins. 14-3-3? protein, an abundant human regulatory protein, in non-phosphorylated state exists as a dimer [1]. However, after phosphorylation at Ser58 (pS58), it monomerizes and changes its properties [2, 3]. Hyperphosphorylation of Tau protein, a microtubule-associated protein, causes detachment of Tau from microtubules in neurons and leads to neurodegeneration [4]. Hyperphosphorylated Tau aggregates into neurofibrillary tangles (NFTs) - one of the hallmarks of Alzheimer’s disease (AD). As 14-3-3? proteins were found colocalized in the NFTs [5], their interconnection with Tau in AD needs to be comprehended. In our study, we aimed to compare interaction properties of dimeric 14-3-3? WT and monomeric 14-3-3? pS58 with respect to Tau protein. The interaction with Tau protein phosphorylated by protein kinase A (PKA) was inspected from various points of view. The binding affinity, stoichiometry, and interacting residues were studied using native-PAGE, chemical cross-linking, tandem MS, and NMR spectroscopy. We revealed that phosphorylation of 14-3-3? at Ser58 decreases its affinity to Tau protein and changes binding stoichiometry. Both NMR and cross-linking results suggested that Tau is in contact with 14-3-3? proteins via the proline-rich domain and microtubule-binding domain. Moreover, cross-linking data showed that not only the binding channel of 14-3-3? protein is responsible for Tau binding, but also the outer 14-3-3? protein surface and exposed dimeric interface of monomeric 14-3-3? pS58 are involved. In summary, we provide novel insight into the 14-3-3?+Tau interaction and its regulation by phosphorylation of both partners. 1. V. Obsilova & T. Obsil, Front. Mol. Biosci., 9, (2022), 1-15. 2. A. Kozeleková, A. Náplavová, T. Brom, N. Gašparik, J. Šimek, J. Houser, J. Hritz, Front. Chem., 10, (2022), 1-17. 3. Z. Trošanová, P. Louša, A. Kozeleková, T. Brom, N. Gašparik, J. Tungli, V. Weisová, E. Župa, G. Žoldák, J. Hritz, J. Mol. Biol., 434, (2022), 167479. 4. T. Arendt, J. T. Stieler, M. Holzer, Brain. Res. Bull., 126, (2016), 238-292. 5. R. Layfield, J. Fergusson, A. Aitken, J. Lowe, M. Landon, R. J. Mayer, Neurosci. Lett., 209, (1996), 57-60. This project has received funding from the European Union’s Horizon Europe program under the grant agreement No. 101087124 and from Czech Science Foundation (GF20-05789L). We acknowledge CEITEC Proteomics Core Facility and Josef Dadok National NMR Centre of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and European Regional Development Fund-Project „UP CIISB“ (No. CZ.02.1.01/0.0/0.0/18_046/0015974). Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by MEYS CR.
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