engleski

In vivo and in vitro analysis of plant seryl-tRNA synthetase interactome

Aminoacyl-tRNA synthetases (aaRS) play significant role in translation process by binding amino acids to their cognate tRNAs. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code. Beyond translation, these enzymes can be involved in diverse cellular functions. Characterization of these non-canonical functions broadens our knowledge in functional proteomics. The studies of aaRS assemblies in plants are scarce, therefore our main scientific goals were to determine and characterize potential protein- protein interacting partners of cytosolic seryl- tRNA synthetase (SerRS) from plant Arabidopsis thaliana. We conducted yeast-two hybrid (Y2H) screen on cDNA libraries followed by DNA sequencing, as well as tandem affinity purification combined with mass spectrometry analysis (TAP-MS). Among several SerRS potential interacting partners revealed by Y2H screen, BEN1, protein potentially involved in metabolism of brassinosteroid hormones was the most promising interacting partner. Interaction of BEN1 and SerRS was also analyzed in vitro using isothermal calorimetry titration (ITC), pull-down and surface plasmon resonance method (SPR). Probably due to the nature of interaction we were not able to retrieve positive results using pull-down assay and ITC, but SPR gave us positive confirmation and information about dissociation constant. To pinpoint regions responsible for protein- protein interaction we prepared shortened variants of both SerRS and BEN1 proteins which will be further subjected to biophysical analysis. Biophysical determination of possible interactions between SerRS and BEN1 variants will give us insight in additional cell functions and physiology of both SerRS and BEN1 proteins. TAP-MS analysis of transgenic plant overexpressing SerRS-TAP construct identified several SerRS potential interacting partners yet to be confirmed in vitro. Revealing plant SerRS interactome has great importance in shedding light on novel functions of SerRS beyond translation.

Seryl-tRNA synthetase; protein-protein interaction; surface plasmon resonance

nije evidentirano