12 research outputs found
Potent sensitisation of cancer cells to anticancer drugs by a quadruple mutant of the human deoxycytidine kinase
Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches
Yeast mitochondrial Gln-tRNAGln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS
It is impossible to predict which pathway, direct glutaminylation of tRNAGln or tRNA-dependent transamidation of glutamyl-tRNAGln, generates mitochondrial glutaminyl-tRNAGln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNAGln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNAGln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNAGln substrate for the AdT. We show that dual localization of cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNAGln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments
Kinetic parameters of the M36 mutant with respect to dC, gemcitabine and AraC.
<p>Kinetic parameters of the M36 mutant with respect to dC, gemcitabine and AraC.</p
Contribution of the individual mutations of G12 to the observed phenotype.
<p>Panel A. Structure of the genomic RNA generated by transcription after transfection of cells with pSDY-SIN plasmids. R, repeated sequence from HIV-1 genome; U5, 5' unique sequence of HIV-1 LTR; Cis-acting, sequences required for packaging and reverse transcription of the genomic RNA; EF1-alpha, human elongation factor 1-alpha promoter; hPGK, human phosphoglycerate kinase promoter; Puro, puromycin N-acetyl-transferase gene; ∆U3, partially deleted version of the 3' unique sequence of HIV-1 LTR. Panels B and C. The proportion of alive cells over the total number of cells as function of the concentration in gemcitabine is given as a ratio with respect to the alive cells observed in the absence of drug. White symbols represent reference populations in both panels and. grey symbols represent single and double mutants (as described in the main text). Since single and double mutants were tested in parallel in the same experiment. The data are the average of 3 independent experiments. Error bars are not shown for the sake of clarity.</p
The M36 mutant.
<p>Panel A. Sensitivity to gemcitabine of Messa10K cells induced by M36. The ratio of living cells over the total number of cells is plotted as a function of the concentration of gemcitabine. Data are the average of five independent experiments. Panel B. Localisation of the mutations in the human dCK that characterise M36. The nucleotide sequence (double stranded) is given above, with the aminoacid sequence below. The full-length protein is given by a pale blue bar with the major functional domains, as defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref028" target="_blank">28</a>], indicated in colour.</p
Mutation panel found in the dCK coding-sequence in F27-RL and in F10-DL.
<p>Frequencies of mutation are calculated as described in Materials and Methods; %G>A mutations: % of transitions from G to A among the number of mutated positions found; mutations/clone: range of mutations found in single clones (for the F10-DL library the three mutations present in G12 are not considered).</p><p>Mutation panel found in the dCK coding-sequence in F27-RL and in F10-DL.</p
Biochemical comparative characterisation of phosphorylation of AraC by wt dCK and M36.
<p>Panels A and C. Phosphorylation kinetics of purified wt dCK (black squares) and M36 (red circles). Steady state kinetic data were fitted according to the Michaelis-Menten equation. Panel A, phosphorylation of dC (average of four independent experiments). Panel B, phosphorylation of gemcitabine (average of four independent experiments). Panel C, phosphorylation of AraC (average of three independent experiments). Panel D, ratio of Km (black) and of Kcat (grey) for M36 vs G12, with respect to dC and to gemcitabine. The dotted line gives the reference of a ratio of 1.</p
M36 and sensitisation to anticancer compounds other than gemcitabine.
<p>Sensitivity to anticancer compounds of Messa10K cells induced by M36. The percentage of living cells over the total number of cells is plotted as a function of the concentration of the anticancer compound (the x axis is given in log scale). Panel A, sensitivity to fludarabine (average of three independent experiments, error bars are not shown for the sake of clarity). Panel B, sensitivity to AraC. Data in panel B give the average of five independent experiments.</p
Scheme of the procedure followed to generate F27-RL and F11-DL.
<p>The general structure of the genomic RNAs used for retrovolution is given at the top, on the left for the F16 population and on the right for the population containing only the G12 variant. The procedure has been previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref034" target="_blank">34</a>] and consists in the repeated transduction of HEK 293T cells with the lentiviral vectors followed by the selection, with puromycin, of cell populations that have stably integrated the proviral DNA that has been produced by reverse transcription. Transfection of this population with the transcomplementation plasmids pHCMV-G and pCMV∆8.91 (see main text) allows the production of the next vector generation that is then used to transduce fresh HEK 293T cells during repeated cycles of selection, transfection and transduction. For screening purposes, target cells are transduced by less than one lentivector particle per cell, and individual clones were isolated in the presence of puromycin. Screening for sensitisation to various compounds is carried out as described in the main text.</p
Position of the mutations found in the individual clones.
<p>In each panel, the dCK protein is represented (top of the drawing) as a pale blue box with the main structural motifs given in color (the first and last aminoacid of each motif is given below the box): purple, P-loop; yellow, insert; green, ERS; blue, lid; pink, base sensing loop; as defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140741#pone.0140741.ref028" target="_blank">28</a>]. For each panel, a cumulative map of the positions that were mutated is provided at the top of the box with aminoacid substitutions given in red, while aminoacids that did not change but with associated codons that carried synonymous mutations given in black. Panels A and B respectively show the patterns observed for the random and directed libraries, respectively. Two deletion were also found: one single nucleotide deletion in clone 6 indicated as "193 out of frame", and a two-nucleotide deletion (positions 141–142) in clone 36 indicated by a red horizontal line. In Panel B, clone 36 (corresponding to M36 in the main text, for Mutant 36) is given in bold.</p