RESUMO
To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA(Phe), indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3'-terminal adenosine, reduced Tyr-tRNA(Phe) deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.
Assuntos
Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/metabolismo , Pyrococcus horikoshii/enzimologia , Tirosina/química , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Aminoacil-RNA de Transferência/biossíntese , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
OBJECTIVE: To evaluate the efficacy of primary treatment for ovarian cancer from overall survival and progression-free survival. PATIENTS AND METHODS: A total of 28 patients with epithelial ovarian cancer in stages III and IV who were primarily treated in our ward from 1993 were examined retrospectively. The Kaplan-Meier method and Harrington-Fleming test were carried out for the cumulative survival rate and analysis. RESULTS: There were significant differences in the progression-free survival rate depending on whether or not optimal debulking was possible through primary treatment (p = 0.0128) and whether the histological diagnosis was serous adenocarcinoma (p = 0.038). In the serous adenocarcinoma group, the periods of both overall survival and progression-free survival were longer in treatment with taxanes and platinum than by other regimens, but in the endometrioid adenocarcinoma group, the period of progression-free survival was very short. CONCLUSIONS: Optimal debulking through primary treatment is critical in advanced ovarian cancer. Therapy with taxanes and platinum is efficacious for serous adenocarcinoma. The chemo-sensitivity of endometrioid adenocarcinoma is high, but the chemotherapeutic effect is only temporary.
