RESUMO
The promyelocytic leukemia (PML) tumor suppressor protein accumulates in PML nuclear bodies (PML-NBs), and can induce growth arrest, cellular senescence and apoptosis. PML has also been localized in the cytoplasm, although its function in this localization remains elusive. A general property of primary cancers is their high glycolytic rate which results from increased glucose consumption. However, the mechanism by which cancer cells up-regulate glycolysis is not well understood. Here, we have shown that cytoplasmic PML (cPML) directly interacts with M2-type pyruvate kinase (PKM2), a key regulator of carbon fate. PKM2 determines the proportion of carbons derived from glucose that are used for glycolytic energy production. Over-expression of PML-2KA mutant in the cytoplasm, which was generated by mutagenesis of the nuclear localization signals of PML, in MCF-7 breast cancer cells suppressed PKM2 activity and the accumulation of lactate. PKM2 exists in either an active tetrameric form which has high affinity for its substrate phosphoenolpyruvate (PEP) or a less active dimeric form which has low affinity for its substrate. Over-expression of PML-2KA suppressed the activity of the tetrameric form of PKM2, but not the dimeric form. Our findings suggest that cPML plays a role in tumor metabolism through its interaction with PKM2.
Assuntos
Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Piruvato Quinase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Metabolismo Energético/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Camundongos , Camundongos SCID , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Piruvato Quinase/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The mitochondrial seryl-tRNA synthetase (mt SerRS) from Bos taurus was overexpressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion method. Crystals grew in a very narrow range of conditions using PEG 8000 as precipitant at room temperature. An appropriate concentration of lithium sulfate was critical for crystal nucleation. Crystals diffracted well beyond a resolution of 1.6 A and were found to belong to the orthorhombic space group C222(1), with unit-cell parameters a = 79.89, b = 230.42, c = 135.60 A. There is one dimer (M(r) approximately 113 kDa) in the asymmetric unit, with a solvent content of 55%. Efforts to solve the phase problem by molecular replacement are under way.
Assuntos
Mitocôndrias/enzimologia , Serina-tRNA Ligase/química , Animais , Sequência de Bases , Bovinos , Cristalização , Cristalografia por Raios X , Expressão Gênica , Mitocôndrias/genética , Conformação de Ácido Nucleico , Serina-tRNA Ligase/genética , Serina-tRNA Ligase/isolamento & purificaçãoRESUMO
It is known that each aminoacyl-tRNA synthetase (aaRS) specifically recognizes its cognate tRNAs to ensure the correct translation of the genetic information. However, we had previously demonstrated that mammalian mitochondrial seryl-tRNA synthetase (mt SerRS) can markedly misacylate mitochondrial (mt) tRNA(Gln). To investigate extensively misacylation reactions in mammalian mitochondrion, we purified overall twenty-two mt tRNAs from bovine liver, and determined their misacylation activities using mt SerRS. In addition to tRNA(Gln), tRNA(Ala) and tRNA(Asn) showed weak but significant serylation activities, which raises the possibility that each mammalian mt aaRS can misacylate several non-cognate mt tRNAs in varying degrees, but translational fidelity might be maintained by kinetic discrimination of tRNAs in the network of aaRSs.
