Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase
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 <i>N</i><sup>6</sup>-methyladenosine (&l...
| Main Authors: | , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-04-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/9/4512 |
| _version_ | 1797536232993980416 |
|---|---|
| author | Michał Marcinkowski Tomaš Pilžys Damian Garbicz Jan Piwowarski Damian Mielecki Grzegorz Nowaczyk Michał Taube Maciej Gielnik Maciej Kozak Maria Winiewska-Szajewska Ewa Szołajska Janusz Dębski Agnieszka M. Maciejewska Kaja Przygońska Karolina Ferenc Elżbieta Grzesiuk Jarosław Poznański |
| author_facet | Michał Marcinkowski Tomaš Pilžys Damian Garbicz Jan Piwowarski Damian Mielecki Grzegorz Nowaczyk Michał Taube Maciej Gielnik Maciej Kozak Maria Winiewska-Szajewska Ewa Szołajska Janusz Dębski Agnieszka M. Maciejewska Kaja Przygońska Karolina Ferenc Elżbieta Grzesiuk Jarosław Poznański |
| author_sort | Michał Marcinkowski |
| collection | DOAJ |
| description | 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 <i>N</i><sup>6</sup>-methyladenosine (<i>N</i><sup>6</sup>-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe<sup>2+</sup>/Mn<sup>2+</sup> and 2-OG), Ca<sup>2+</sup> that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either <i>E. coli</i> (<sup>EC</sup>FTO) or baculovirus (<sup>BES</sup>FTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that <sup>BES</sup>FTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in <sup>EC</sup>FTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca<sup>2+</sup>, 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. |
| first_indexed | 2024-03-10T11:56:45Z |
| format | Article |
| id | doaj.art-2b261959dc6147a9b636ed04b983367f |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T11:56:45Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2b261959dc6147a9b636ed04b983367f2023-11-21T17:12:47ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-04-01229451210.3390/ijms22094512Effect of Posttranslational Modifications on the Structure and Activity of FTO DemethylaseMichał Marcinkowski0Tomaš Pilžys1Damian Garbicz2Jan Piwowarski3Damian Mielecki4Grzegorz Nowaczyk5Michał Taube6Maciej Gielnik7Maciej Kozak8Maria Winiewska-Szajewska9Ewa Szołajska10Janusz Dębski11Agnieszka M. Maciejewska12Kaja Przygońska13Karolina Ferenc14Elżbieta Grzesiuk15Jarosław Poznański16Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandNanoBioMedical Centre, Adam Mickiewicz University, Wszechnicy Piastowskiej 3, 61-614 Poznan, PolandDepartment of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, PolandDepartment of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, PolandDepartment of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandVeterinary Research Centre, Department of Large Animal Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 100, 02-797 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandThe 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 <i>N</i><sup>6</sup>-methyladenosine (<i>N</i><sup>6</sup>-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe<sup>2+</sup>/Mn<sup>2+</sup> and 2-OG), Ca<sup>2+</sup> that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either <i>E. coli</i> (<sup>EC</sup>FTO) or baculovirus (<sup>BES</sup>FTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that <sup>BES</sup>FTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in <sup>EC</sup>FTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca<sup>2+</sup>, 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.https://www.mdpi.com/1422-0067/22/9/4512<sup>EC</sup>FTO<sup>BES</sup>FTOphosphorylationcalciumdimerizationnanoDSF |
| spellingShingle | Michał Marcinkowski Tomaš Pilžys Damian Garbicz Jan Piwowarski Damian Mielecki Grzegorz Nowaczyk Michał Taube Maciej Gielnik Maciej Kozak Maria Winiewska-Szajewska Ewa Szołajska Janusz Dębski Agnieszka M. Maciejewska Kaja Przygońska Karolina Ferenc Elżbieta Grzesiuk Jarosław Poznański Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase International Journal of Molecular Sciences <sup>EC</sup>FTO <sup>BES</sup>FTO phosphorylation calcium dimerization nanoDSF |
| title | Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase |
| title_full | Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase |
| title_fullStr | Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase |
| title_full_unstemmed | Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase |
| title_short | Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase |
| title_sort | effect of posttranslational modifications on the structure and activity of fto demethylase |
| topic | <sup>EC</sup>FTO <sup>BES</sup>FTO phosphorylation calcium dimerization nanoDSF |
| url | https://www.mdpi.com/1422-0067/22/9/4512 |
| work_keys_str_mv | AT michałmarcinkowski effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT tomaspilzys effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT damiangarbicz effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT janpiwowarski effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT damianmielecki effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT grzegorznowaczyk effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT michałtaube effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT maciejgielnik effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT maciejkozak effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT mariawiniewskaszajewska effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT ewaszołajska effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT januszdebski effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT agnieszkammaciejewska effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT kajaprzygonska effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT karolinaferenc effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT elzbietagrzesiuk effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase AT jarosławpoznanski effectofposttranslationalmodificationsonthestructureandactivityofftodemethylase |