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

Abstract

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.

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase

“…Here, size exclusion chromatography (SEC) was performed for the purified recombinant FTO obtained from the baculovirus expression system ( BES FTO), as well as for samples from the HNSCC tissues and specific cell lines, both cancerous (HeLa and U87) and non-cancerous (HEK293). In the absence of other proteins, FTO (58 kDa) showed the ability to form dimers [ 28 ]. This form predominates in vitro outside the cell.…”

“…The FTO demethylase is a member of the ALKBH family of proteins connected with both adipogenesis, osteogenesis, maintaining bone mass and heart regeneration, as well as civilization diseases such as obesity, type 2 diabetes, cancer and others [ 28 ]. Many studies have demonstrated that FTO is involved in crucial cell processes; however, not much is known regarding the regulation of its activity.…”

“…Moreover, the interplay between FTO and CaM was only slightly affected by the type of the expression system used and only in the part of the experiments. Thus, the presence of posttranslational modifications, especially serine phosphorylation [ 28 ], occurring mostly in the FTO N-terminal domain did not significantly affect FTO–CaM binding. The obtained results showing nanomolar K D are likely to be biologically relevant, because FTO in tissues occurs at nanomolar—or even lower—concentrations that can be detected by sensitive methods, such as Western blot [ 30 ].…”

“…Here, our structural analysis showed that the interaction with FTO destabilized the CaM, especially the calcium-binding regions; nevertheless, the reciprocal effect was negligible. This may be explained by FTO homodimerization, when interfaces of the FTO–FTO homodimer and the FTO–CaM heterodimer are similar [ 28 ]. Possibly, both interactions require FTO to maintain the same particular structure.…”

“…For investigation of the molecular state of endogenous human FTO, fast protein liquid chromatography (FPLC) was performed. Recombinant FTO from baculovirus expression system ( BES FTO) was expressed and purified as described previously [ 28 ]. Protein extracts from selected cancer tissues and HeLa, HEK293, and U87 cell lines were prepared as described previously [ 25 ].…”