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Article

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
2
NanoBioMedical Centre, Adam Mickiewicz University, Wszechnicy Piastowskiej 3, 61-614 Poznan, Poland
3
Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, Poland
4
National Synchrotron Radiation Centre SOLARIS, Jagiellonian University, Czerwone Maki 98, 30-392 Kraków, Poland
5
Veterinary Research Centre, Department of Large Animal Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 100, 02-797 Warsaw, Poland
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Christina Piperi
Int. J. Mol. Sci. 2021, 22(9), 4512; https://doi.org/10.3390/ijms22094512
Received: 12 March 2021 / Revised: 19 April 2021 / Accepted: 22 April 2021 / Published: 26 April 2021
(This article belongs to the Section Molecular Toxicology)
The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data. View Full-Text
Keywords: ECFTO; BESFTO; phosphorylation; calcium; dimerization; nanoDSF; MST; HDX; SAXS ECFTO; BESFTO; phosphorylation; calcium; dimerization; nanoDSF; MST; HDX; SAXS
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MDPI and ACS Style

Marcinkowski, M.; Pilžys, T.; Garbicz, D.; Piwowarski, J.; Mielecki, D.; Nowaczyk, G.; Taube, M.; Gielnik, M.; Kozak, M.; Winiewska-Szajewska, M.; Szołajska, E.; Dębski, J.; Maciejewska, A.M.; Przygońska, K.; Ferenc, K.; Grzesiuk, E.; Poznański, J. Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase. Int. J. Mol. Sci. 2021, 22, 4512. https://doi.org/10.3390/ijms22094512

AMA Style

Marcinkowski M, Pilžys T, Garbicz D, Piwowarski J, Mielecki D, Nowaczyk G, Taube M, Gielnik M, Kozak M, Winiewska-Szajewska M, Szołajska E, Dębski J, Maciejewska AM, Przygońska K, Ferenc K, Grzesiuk E, Poznański J. Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase. International Journal of Molecular Sciences. 2021; 22(9):4512. https://doi.org/10.3390/ijms22094512

Chicago/Turabian Style

Marcinkowski, Michał; Pilžys, Tomaš; Garbicz, Damian; Piwowarski, Jan; Mielecki, Damian; Nowaczyk, Grzegorz; Taube, Michał; Gielnik, Maciej; Kozak, Maciej; Winiewska-Szajewska, Maria; Szołajska, Ewa; Dębski, Janusz; Maciejewska, Agnieszka M.; Przygońska, Kaja; Ferenc, Karolina; Grzesiuk, Elżbieta; Poznański, Jarosław. 2021. "Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase" Int. J. Mol. Sci. 22, no. 9: 4512. https://doi.org/10.3390/ijms22094512

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