Aylin KANLI, Murat KASAP, Gürler AKPINAR, Sevinç YANAR

Fat Mass and Obesity Associated (FTO) Protein Ekspresyonunun Neden Olduğu SH-SY5Y Hücrelerinin Proteomunda Meydana Gelen Değişiklikler, FTO Proteininin Çok Yönlü Özellikleri Ortaya Çıkarır

Amaç: Fat mass and obesity associated (FTO) proteini postranskripsiyonel modifikasyonlar, DNA tamiri ve yağ asidi metabolizması gibi çeşitli hücresel işlevlerde rol oynayan bir RNA dematilazdır. Başlangıçta obesite ile yakından ilişkilendirilen FTO proteininin daha sonraki çalışmalarla nörolojik hastalıklar ve çeşitli kanser türleri ile ilişkili olduğu gösterilmiştir. Bu çalışmanın amacı SH-SY5Y hücrelerinde FTO geninin ekzonik R316Q mutasyonunun çözünür proteom üzerindeki etkilerini araştırmaktır.Yöntem: İki boyutlu Difference Gel Electrophoresis (2D-DIGE) deneylerinde, Tet promotörün kontrolü altında yabanıl (WT) veya mutant FTO proteinlerini stabil şekilde eksprese eden SH-SY5Y hücreleri kullanılmıştır. 2-kat regülasyon kriterine göre WT ve mutant FTO proteinini fazla eksprese eden örneklerde 500’den fazla protein spotu karşılaştırılmış ve ekspresyonlarında değişiklik görülen protein-spotları jellerden kesilerek MALDI-TOF/TOF ile tanımlanmıştır.Bulgular: Genel olarak, WT ve mutant FTO ifadesi çözünür proteomda büyük değişikliklere neden olmamıştır. Ancak, altı protein noktasında bazı küçük değişiklikler gözlemledik. Bu protein noktalarının üçü Hsp70'e aitti ve mutant FTO eksprese eden hücrelerde daha fazla eksprese idi. Bu, Hsp70'in yalnızca fazla eksprese olduğunu değil, aynı zamanda translasyon sonrası değişikliğe uğradığını da gösterir. İfadesi regüle edilen diğer proteinler fosfogliserat kinaz-1 (PGK1), kalmodulin ve keratindir.Sonuç: Bu sonuçlar FTO’nun enerji metabolizması ile ilişkisinin yanı sıra hücresel strese cevabı indükleyebileceğinin bir göstergesi olabilir. Ek olarak, FTO Wnt sinyal iletim yolağını etkiyelebilir. Genel olarak, çalışmamız FTO'nun çok yönlü özelliklerini vurgulamış ve nöroblastom hücrelerinin proteomunda meydana gelen değişiklikleri yansıtmıştır.

Anahtar Kelimeler:

FTO (Fat mass and obesity associated) proteini, R316Q mutasyonu, FTO-ilişkili hastalıklar, SH-SY5Y, Proteomiks, 2D-DIGE

Changes Occurring in the Proteome of SH-SY5Y Cells Caused by Fat Mass and Obesity Associated (FTO) Protein Expression Reveals Multifaceted Properties of the FTO Protein

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:

FTO(Fat mass and obesity associated) protein, R316Q mutation, FTO-related-diseases, SH-SY5Y, Proteomics, 2D-DIGE,

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