Preparation of Alzheimer's-disease-relevant Tau protein in HEK293 mammalian cells

KOZELEKOVÁ, Aneta, Marie CHOVANČÍKOVÁ, Petr KAŠPÁREK, Helena REUTOVÁ, Martina HLOŽÁNKOVÁ and Jozef HRITZ. Preparation of Alzheimer's-disease-relevant Tau protein in HEK293 mammalian cells. In CEITEC PhD School Conference 2024, Brno. 2024.

Basic information

• Original name: Preparation of Alzheimer's-disease-relevant Tau protein in HEK293 mammalian cells
• Name in Czech: Příprava proteinu Tau relevantního pro Alzheimerovu chorobu v savčích HEK293 buňkách
• Authors: KOZELEKOVÁ, Aneta, Marie CHOVANČÍKOVÁ, Petr KAŠPÁREK, Helena REUTOVÁ, Martina HLOŽÁNKOVÁ and Jozef HRITZ.
• Edition: CEITEC PhD School Conference 2024, Brno, 2024.

Other information

• Original language: English
• Type of outcome: Conference abstract
• Country of publisher: Czech Republic
• Confidentiality degree: is not subject to a state or trade secret
• Organization unit: Central European Institute of Technology

Abstract

Tau protein is a microtubule-binding protein that is usually post-translationally modified (PTM). Hyperphosphorylation and abnormal PTMs of Tau have been identified as key determinants connecting Tau and Alzheimer’s disease. After such abnormal modification, Tau detaches from microtubules and aggregates into neurotoxic neurofibrillary tangles. However, the molecular mechanism and exact factors causing Tau aggregation are so far not well understood. Here, we aimed to prepare Tau protein in mammalian HEK293 cells to obtain Tau bearing natural PTMs. Tau protein was successfully expressed, purified from the cell lysate, and its purity and identity were verified using SDS-PAGE, Western blot, and mass spectrometry (MS). Currently, the presence of PTMs within Tau protein is inspected by advanced MS analysis. Next step will be to study the effects of the PTMs on Tau structural, interaction, and aggregation properties in comparison to un-modified Tau expressed in E. coli or in vitro phosphorylated Tau (already published in article by Crha R, Kozeleková A, Hofrová A, Iľkovičová L, Gašparik N, Kadeřávek P, Hritz J. Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3ζ protein variants. Int J Biol Macromol. 2024, doi: 10.1016/j.ijbiomac.2024.130802). The majority of results were obtained within Industrial PhD programme in biotech company BioVendor. Project acknowledgement: 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, e-INFRA CZ (ID:90254)).

Links

• LM2023042, research and development project: Name: Česká infrastruktura pro integrativní strukturní biologii

Investor: Ministry of Education, Youth and Sports of the CR, CIISB - Czech Infrastructure for Integrative Structural Biology

• 101087124, interní kód MU: Name: Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe

Investor: European Union, Widening participation and strengthening the European Research Area

• 90254, large research infrastructures: Name: e-INFRA CZ II