engleski

BiFC as tool to investigate protein-protein interactions in methylation disorder AHCY deficiency

S-adenosylhomocysteine (AHCY) hydrolase deficiency is a multisystem methylation disorder caused by point mutations in exons of ahcy gene. Although many advances have been made since the discovery of the disease in 2004, molecular mechanisms of disease pathology are still elusive. Using AHCY deficiency as model system may allow new insights in understanding related methylation disorders thereby deciphering players of the complex epigenomic network. Therefore, we aim to develop versatile as well as reliable tools in order to study broad spectrum of AHCY cellular functions. For this purpose we have assembled a Gateway compatible vector system using split-GFP (Venus) as fluorescent tag to enable Bimolecular Fluorescence Complementation (BiFC). After transfection of HEK293T with various AHCY-BiFC constructs, cells were screened to evaluate protein interactions in vivo using fluorescence microscopy. Self-association of AHCY-BiFC constructs leads to strong fluorescence, indicating protein interactions, whereas lack of fluorescence is observed when protein interactions are absent. By using AHCY- BIFC constructs as bait, yet unknown protein interactions of AHCY may be investigated, as well as effects of point mutations found in patients on AHCY stability and macromolecular organization in vivo using live cell imaging. Additionally, we established HEK293 cell lines stably expressing WT and mutant AHCY tagged with regular EGFP. The amount of EGFP-AHCY was determined on mRNA level by qRT PCR and on protein level by Western blotting. Fluorescence microscopy was used to evaluate nucleocytoplasmic distribution of AHCY. Agreeable with findings observed in patient cells, expression of mutants is found to be lowered both on mRNA and protein level. Image analysis revealed changes of Icn factor meaning that significantly more mutant protein is shifted to cell nucleus when compared to WT. In summary, we show that our model system is able to mimic the changes observed in patient cells and thus serve as a starting point for future investigations of AHCY mRNA and protein processing or its cellular dynamics and trafficking. BiFC in combination with Gateway technology offers a fast and reliable approach to study the AHCY interactome, providing new insights in disease pathology and overall roles of AHCY at molecular level.

BiFC, methylation, AHCY

Best Poster Award