Dysregulation of ERAD and Stress Response Proteins by V493F FTO Over-expression: A Proteomic Perspective

Demethylase fat mass and obesity-related protein (FTO), which belongs to the AlkB homologous (ABH) family, is associated with various neurological diseases, cancer, and obesity. This protein, which contains many structurally and functionally different regions, contains a COOH-terminal domain whose function, unlike other ABH members, is not fully understood. This study aimed to investigate the effects of the exonic V493F mutation in this region of FTO on the soluble proteome.

SH-SY5Y cells stably over-expressing wild-type (WT-FTO) or mutant FTO (V493F-FTO) proteins under the control of the Tet promoter were created and used. Comparative proteomic analysis using two-dimensional gel electrophoresis (2DE) identified over 500 protein spots, with 10 showing significant (≥2-fold) differential expression. These proteins were identified by MALDI-TOF/TOF mass spectrometry and validated by western blotting.

WT-FTO over-expression primarily affected proteins related to DNA replication and repair, including PCNA, whereas V493F-FTO over-expression altered the expression of stress response and endoplasmic reticulum-associated degradation (ERAD) pathway proteins, such as HSPA4, ARHGDIA, and VCP. Although the mutation did not alter the nuclear localization or predicted 3D structure of FTO, it distinctly modulated pathways associated with protein homeostasis and cellular stress.

FTO participates in the regulation of the cellular stress response and the ubiquitin-dependent ERAD pathway, functions potentially independent of its demethylase activity. Importantly, dysregulation of these pathways has been implicated in cancer initiation, progression, and therapeutic resistance. Therefore, our findings provide new insights into how FTO mutations might influence oncogenic processes, highlighting FTO as a potential biomarker and therapeutic target in cancer biology.