PubMed · 3527493
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G H Rabbani. 1986. Cholera.. https://pubmed.ncbi.nlm.nih.gov/3527493/
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Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.
The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.
Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9A and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3'-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.