Unique properties of Tau protein expressed in mammalian cells
compared to E. coli

a 2024

Unique properties of Tau protein expressed in mammalian cells compared to E. coli

KOZELEKOVÁ, Aneta; Marie CHOVANČÍKOVÁ; Petr KAŠPÁREK; Martina HLOŽÁNKOVÁ; Jozef HRITZ et al.

Základní údaje

Originální název

Unique properties of Tau protein expressed in mammalian cells compared to E. coli

Autoři

KOZELEKOVÁ, Aneta ; Marie CHOVANČÍKOVÁ; Petr KAŠPÁREK; Martina HLOŽÁNKOVÁ a Jozef HRITZ

Vydání

XXIII. Meeting of Biochemists and Molecular Biologists, 2024

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Označené pro přenos do RIV

Organizační jednotka

Přírodovědecká fakulta

ISBN

978-80-280-0621-1

Změněno: 5. 3. 2025 09:58, Mgr. Aneta Kozeleková

Anotace

V originále

Neurofibrillary tangles composed of aggregated hyperphosphorylated Tau protein are one of the typical hallmarks of Alzheimer's disease (AD) in the brain [1]. As the patterns of phosphorylation and other post-translational modifications (PTMs) within Tau are complex [2], AD-relevant Tau protein is not easily obtainable by recombinant production in E. coli and then modification by individual enzymes in vitro. To overcome this problem, we employed mammalian cell culture (HEK293) to produce Tau protein with natural PTMs. The expression and purification conditions were optimized, and Tau protein purity and identity were verified by SDS-PAGE, Western blot, and mass spectrometry. Using LC-MS/MS, we identified around 20 phosphorylation sites with diverse extent of phosphorylation. The detected phosphorylation patterns were comparable to those found in the brains of AD-patients. Moreover, in contrast to Tau phosphorylated by protein kinase A (PKA) in vitro [3], Tau isolated from HEK293 and potentially AD-brains did not interact with 14-3-3ζ protein, a well-recognized Tau partner [4]. In the future, we plan to analyse also other properties of Tau purified from HEK293 cells – such as protein compactness, aggregation propensities and fibril folds – all in comparison to Tau produced in E. coli and phosphorylated by selected kinases in vitro [3]. The protein production in HEK293 cells was performed within an Industrial PhD programme in the 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)). References [1] M. Goedert, R. A. Crowther, S. H. W. Scheres, and M. G. Spillantini, “Tau and neurodegeneration,” Cytoskeleton, vol. 81, no. 1, pp. 95–102, 2024, doi: 10.1002/cm.21812. [2] H. Wesseling et al., “Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer’s Disease,” Cell, vol. 183, no. 6, pp. 1699-1713.e13, 2020, doi: 10.1016/j.cell.2020.10.029. [3] R. Crha et al., “Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3ζ protein variants,” Int. J. Biol. Macromol., vol. 266, no. 130802, pp. 1–14, 2024, doi: 10.1016/j.ijbiomac.2024.130802. [4] J. F. Neves et al., “Phosphorylated full-length Tau interacts with 14-3-3 proteins via two short phosphorylated sequences, each occupying a binding groove of 14-3-3 dimer,” FEBS J., vol. 288, no. 6, pp. 1918–1934, 2021, doi: 10.1111/febs.15574.

Návaznosti

LM2023042, projekt VaV

Název: Česká infrastruktura pro integrativní strukturní biologii

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, CIISB - Česká infrastruktura pro integrativní strukturní biologii

101087124, interní kód MU

Název: Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe

Investor: Evropská unie, Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe, Rozšiřování účasti a posílení ERA

90254, velká výzkumná infrastruktura

Název: e-INFRA CZ II

Citovat

KOZELEKOVÁ, Aneta; Marie CHOVANČÍKOVÁ; Petr KAŠPÁREK; Martina HLOŽÁNKOVÁ a Jozef HRITZ. Unique properties of Tau protein expressed in mammalian cells compared to E. coli. In XXIII. Meeting of Biochemists and Molecular Biologists. 2024. ISBN 978-80-280-0621-1.

@proceedings{2481787,
   author = {Kozeleková, Aneta and Chovančíková, Marie and Kašpárek, Petr and Hložánková, Martina and Hritz, Jozef},
   booktitle = {XXIII. Meeting of Biochemists and Molecular Biologists},
   language = {eng},
   isbn = {978-80-280-0621-1},
   title = {Unique properties of Tau protein expressed in mammalian cells compared to E. coli},
   year = {2024}
}

TY  - CONF
ID  - 2481787
AU  - Kozeleková, Aneta - Chovančíková, Marie - Kašpárek, Petr - Hložánková, Martina - Hritz, Jozef
PY  - 2024
TI  - Unique properties of Tau protein expressed in mammalian cells compared to E. coli
SN  - 9788028006211
N2  - Neurofibrillary tangles composed of aggregated hyperphosphorylated Tau protein are one of the typical hallmarks of Alzheimer's disease (AD) in the brain [1]. As the patterns of phosphorylation and other post-translational modifications (PTMs) within Tau are complex [2], AD-relevant Tau protein is not easily obtainable by recombinant production in E. coli and then modification by individual enzymes in vitro. To overcome this problem, we employed mammalian cell culture (HEK293) to produce Tau protein with natural PTMs. The expression and purification conditions were optimized, and Tau protein purity and identity were verified by SDS-PAGE, Western blot, and mass spectrometry. Using LC-MS/MS, we identified around 20 phosphorylation sites with diverse extent of phosphorylation. The detected phosphorylation patterns were comparable to those found in the brains of AD-patients. Moreover, in contrast to Tau phosphorylated by protein kinase A (PKA) in vitro [3], Tau isolated from HEK293 and potentially AD-brains did not interact with 14-3-3ζ protein, a well-recognized Tau partner [4]. In the future, we plan to analyse also other properties of Tau purified from HEK293 cells – such as protein compactness, aggregation propensities and fibril folds – all in comparison to Tau produced in E. coli and phosphorylated by selected kinases in vitro [3]. The protein production in HEK293 cells was performed within an Industrial PhD programme in the 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)). References [1] M. Goedert, R. A. Crowther, S. H. W. Scheres, and M. G. Spillantini, “Tau and neurodegeneration,” Cytoskeleton, vol. 81, no. 1, pp. 95–102, 2024, doi: 10.1002/cm.21812. [2] H. Wesseling et al., “Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer’s Disease,” Cell, vol. 183, no. 6, pp. 1699-1713.e13, 2020, doi: 10.1016/j.cell.2020.10.029. [3] R. Crha et al., “Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3ζ protein variants,” Int. J. Biol. Macromol., vol. 266, no. 130802, pp. 1–14, 2024, doi: 10.1016/j.ijbiomac.2024.130802. [4] J. F. Neves et al., “Phosphorylated full-length Tau interacts with 14-3-3 proteins via two short phosphorylated sequences, each occupying a binding groove of 14-3-3 dimer,” FEBS J., vol. 288, no. 6, pp. 1918–1934, 2021, doi: 10.1111/febs.15574.
ER  -

KOZELEKOVÁ, Aneta; Marie CHOVANČÍKOVÁ; Petr KAŠPÁREK; Martina HLOŽÁNKOVÁ a Jozef HRITZ. Unique properties of Tau protein expressed in mammalian cells compared to E. coli. In \textit{XXIII. Meeting of Biochemists and Molecular Biologists}. 2024. ISBN~978-80-280-0621-1.

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