Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
NanoBioMedical Centre, Adam Mickiewicz University, Wszechnicy Piastowskiej 3, 61-614 Poznan, Poland.
Int J Mol Sci. 2021 Apr 26;22(9):4512. doi: 10.3390/ijms22094512.
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 -methyladenosine (-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/Mn and 2-OG), Ca that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either (FTO) or baculovirus (FTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that FTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in FTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca, 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.
FTO 蛋白参与广泛的生理过程，包括脂肪生成和骨生成。这种双结构域蛋白属于 AlkB 家族的 2-氧戊二酸（2-OG）和 Fe(II)依赖性双加氧酶，具有 -甲基腺苷（-meA）去甲基酶活性。本研究旨在研究 FTO 的结构与活性之间的关系。使用生物物理方法（DSF、MST、SAXS）和生化技术（分子筛层析、酶活性测定）测定了辅因子（Fe/Mn 和 2-OG）、不结合在催化部位的 Ca2+以及蛋白浓度对 FTO 在 （FTO）或杆状病毒（FTO）系统中表达的性质的影响。我们发现 FTO 带有三个磷酸丝氨酸（S184、S256、S260），而 FTO 中没有这些修饰。模拟 S256 磷酸化的 S256D 突变适度降低了 FTO 的催化活性。在 Ca2+存在下，观察到 FTO 结构略有稳定，同时催化活性降低。分子筛层析和 MST 数据证实了来自两种表达系统的 FTO 形成同源二聚体的能力。MST 测定的 FTO 同源二聚体的解离常数与它们在人细胞中的体内形成一致。最后，根据 SAXS 数据构建了 FTO 同源二聚体的低分辨率结构。
