Conserved mechanism of action of a phosphate analogue in cultured cells

Abstract

The present study was undertaken to further examine a model for the mechanism of action of thio-phosphate in cells using the cultured human cell line HEK 293 and the bacterium E. coli. Previous work has shown that thio-phosphate is incorporated into the nucleic acids of all types of cells and that this modification leads to the stabilization and accumulation of mRNA in vivo. Evidently, competition for translation occurs between cellular mRNAs, due to the limited number of ribosomes in the cell, which impacts the proteins synthesized. In both E. coli and HEK 293 cells, a global shift in the distribution of proteins on 2D gels was observed when cells were grown in the presence of thio-phosphate. In both cases, a significant fraction of the proteins were impacted, namely 17 % in E. coli and 28 % in HEK 293 cells. In general, those proteins originally expressed at lower levels were enhanced while those at higher levels were reduced. In addition, a correlation was observed between the % initial mass and the fold change observed, supporting the idea of a global shift. Furthermore, the potential identity of some proteins spots was obtained by comparisons with computed MW and pI values for known genes. The candidates were cross-referenced with known mRNA half-lives when available. The analysis supports the idea that a preferential accumulation of proteins derived from mRNAs with half-lives less than the cellular mRNA average occurred, at the expense of those proteins derived from mRNAs with half-lives greater than the cellular average. In summary, thio-phosphate is shown to be a useful tool for predictably shifting the distribution of proteins in a variety of cell types. The fold enhancement is typically 5- to 10-fold. Interestingly, there were some proteins elevated more than 20-fold, identifying potential regulatory responses. Some of these were further characterized using LC–MS/MS.