O-Phospho-L-tyrosine

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

O-phospho-L-tyrosine (P-Tyr) has been reported previously to inhibit growth of several cancer cell lines at mM concentrations. In the present study, we investigated the effect of this compound on tumor cells and normal cells in combination with radiation exposure. It could be demonstrated for the first time that P-Tyr at microM concentrations protects TP53 wild-type cells against ionizing radiation (SF4 minus BBI = 0.28, SF4 plus BBI = 0.45). On the contrary, human transformed or tumor cell lines characterized by mutated or functional inactivated TP53 were not altered or increased in their radiation sensitivity (SF4 minus BBI = 0.32, SF4 plus BBI = 0.22). Treatment of wild-type TP53 cells with P-Tyr induced stabilization of TP53 within 3 and 16 hours and a subsequent increase in CDKN1A expression after treatment. Consequently, a 16-hours pretreatment of cells with P-Tyr led to a significant radioprotective effect. This was not observed in cell lines with mutated TP53, which shows no radioprotection by P-Tyr. Thus, the present data suggest that P-Tyr-mediated radioprotection is dependent on preirradiation stabilization of TP53. The results indicate that P-Tyr is a radioprotective agent that can potentially be very useful and easy to deliver for radiation protection in general and especially in radiation therapy of TP53-mutated tumors.

Phosphorylation is one of the most important protein post-translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non-specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β-casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non-fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)-immobilized O-phospho-L-tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.