engleski

AHCY interaction with galectin-3 identified by new Gateway vectors for bi-molecular fluorescence complementation and pooled ORFs screening strategy

AHCY is the single mammalian enzyme that removes S-adenosyl homocysteine (SAH) – a byproduct in cellular methylation reactions. AHCY controls the methylation potential of the cell, thereby ensuring the efficient methylation of DNA, mRNA, tRNA, lipid and protein (1). Methylation is the key epigenetic regulator of expression of many vital proteins, thus it is no surprise that AHCY deficiency causes developmental defects and is potentially lethal. Our aim was to study AHCY interaction network since it shapes intracellular dynamics and function of a protein. Interaction studies often use bi-molecular fluorescence complementation (BiFC) to reveal the formation and cellular localization of protein complexes, however large-scale approach is lacking. We developed the tool for fast human protein interactions screening that combines Gateway- ready BiFC vectors and ORF pooling. First we have validated the vectors, and in a high-throughput screen we identified galectin-3 as the new AHCY interaction partner. Using Cell Cognition, machine-learning free software, we analyzed the intracellular localization of this complex based on BiFC fluorescence pattern. Complex localized in vesicles, identified as compartments of the endosomal pathway, a known galectin-3 trafficking route. We report this interaction here for the first time in a live cell system, after it was first captured by the pull-down screen in 2014 (2). Galectin-3 is a ubiquitous protein involved in intracellular traffic and transport of nuclear proteins, a possible route for AHCY nuclear translocation. This approach offers a platform to rapidly identify and localize new protein interactions inside living human cells, a basis for understanding the function of an interaction.

AHCY, BiFC, galectin-3, Gateway, high-throughput

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