Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, vol.6, no.2, pp.101-112, 2020 (National Refreed University Journal)
Objective: Fat mass and obesity associated (FTO) protein is an RNA-demethylase which is employed in various metabolic functions such as post-transcriptional modifications, DNA repair and fatty acid utilization. Fat mass and obesity associated protein was initially found to be closely associated with obesity and increased body-mass-index and later studies have established association of FTO with neurological diseases and cancer. The aim of this study was to investigate the effect of R316Q FTO mutation on soluble proteome in SH-SY5Y cells.
Methods: SH-SY5Y cells stably expressing the wild-type (WT) and the mutant FTO proteins under the control of Tet-promoter were used to study changes in overall proteome using two-dimentional Difference Gel Electrophoresis approach. More than 500 protein spots were compared in samples that overexpressed the WT-FTO or the mutant-FTO protein according to 2-fold-criteria. Spots displaying differences were cut from the gels and identified by MALDI-TOF/TOF.
Results: In overall, the expression of neither the WT nor the mutant FTO caused major changes in the soluble proteome. However, we observed some minor changes in six protein spots. Three of those protein spots belonged to Hsp70 and were up-regulated in the mutant-FTO-expressing cells. This indicated that Hsp70 was not only up-regulated but also post-translationally modified. The other proteins regulated were phosphoglycerate kinase-1, calmodulin and keratin.
Conclusion: These results indicated that FTO appear to be associated with energy metabolism and might induce the cellular stress. In addition, FTO might affect to the Wnt signalling pathway. In overall, our study highlighted the multifaceted properties of the FTO and reflected onto the changes occurring in the proteome of neuroblastoma cells.
Keywords: Fat mass and obesity associated (FTO) protein, R316Q-mutation, FTO-related-diseases, SH-SY5Y, Proteomics, 2D-DIGE