REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

真核蛋白质合成的调控

• 批准号: 6290205
• 负责人: THOMAS E DEVER
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290205
• 关键词: Baculoviridae; eukaryote; gene induction /repression; gene mutation; genetic regulation; genetic strain; oxidative stress; phosphorylation; protein biosynthesis; protein kinase; protein tyrosine kinase; translation factor; vaccinia virus; yeasts

The binding of initiator methionyl-tRNA to ribosomes is catalyzed in eukaryotic organisms by the heterotrimeric factor eIF2, whereas in prokaryotes a single polypeptide factor IF2 performs the same function. We have identified and characterized IF2 homologs in archaea, the yeast Saccharomyces cerevisiae and humans. Previous studies demonstrated that the yeast IF2 homolog yIF2, encoded by the FUN12 gene, is a general translation initiation factor. We obtained a clone for a human IF2 homolog, and we found that human or archaeal IF2 proteins could functionally substitute in vivo for yIF2. In addition, the human and archaeal IF2 proteins could substitute for yIF2 and stimulate protein synthesis in extracts from fun12-deletion strains. These results demonstrate that IF2 is a universally conserved translation factor and that the mechanism of protein synthesis has been more highly conserved during evolution than previously anticipated. Biochemical assays demonstrated that the human IF2 protein promotes the ribosomal subunit joining step of protein synthesis. In recognition of this activity the eukaryotic IF2 homologs have been renamed eIF5B. Using an eIF5B mutant that utilizes XTP in place of GTP, we have demonstrated that at least two nucleotide hydrolysis events are required for subunit joining during eukaryotic translation initiation. A second research interest is phosphorylation of the translation initiation factor eIF2. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. We have demonstrated that the vaccinia virus K3L protein and the swine pox virus C8L protein are pseudosubstrate inhibitors of PKR, and can suppress PKR toxicity in yeast. A sequence motif located around 30 residues from the site of phosphorylation in eIF2alpha and conserved in K3L and C8L was critical for the inhibition of PKR in both yeast and mammalian cells. Mutations in this motif of eIF2alpha impaired phosphorylation by the GCN2 kinase in vivo, indicating that motif plays an important role in substrate recognition. Fourteen independent mutations in the carboxyl- terminal half of the PKR kinase domain rendered the kinase resistant to K3L inhibition, and these mutations are predicted to alter contacts between the kinase and substrate. Finally, experiments in yeast and mammalian cells demonstrated the importance of dimerization for PKR activation in vivo. Whereas an isolated PKR kinase domain was inactive in vivo, fusion of the kinase domain to heterologous dimerization domains was found to restore activity. - translation, factor, eIF, kinase, yeast, Saccharomyces cerevisiae

在真核生物中，异源三聚体因子eIF 2催化起始剂甲硫氨酰-tRNA与核糖体的结合，而在原核生物中，单一多肽因子IF 2执行相同的功能。我们已经确定和特点IF 2同源物在古细菌，酵母酿酒酵母和人类。先前的研究表明，酵母IF 2同源物yIF 2，由FUN 12基因编码，是一个通用的翻译起始因子。我们获得了人IF 2同源物的克隆，并且我们发现人或古细菌IF 2蛋白可以在体内功能上替代yIF 2。此外，人和古细菌IF 2蛋白可以替代yIF 2，并刺激fun 12缺失菌株提取物中的蛋白质合成。这些结果表明，IF 2是一个普遍保守的翻译因子，蛋白质合成的机制在进化过程中比以前预期的更高度保守。生物化学分析表明，人IF 2蛋白促进蛋白质合成的核糖体亚基连接步骤。为了确认这种活性，真核IF 2同源物已被重新命名为eIF 5 B。使用eIF 5 B突变体，利用XTP的GTP的地方，我们已经证明，至少有两个核苷酸水解事件所需的亚基连接在真核生物翻译起始。第二个研究兴趣是翻译起始因子eIF 2的磷酸化。哺乳动物激酶PKR和HRI以及酵母激酶GCN 2特异性磷酸化eIF 2 α亚基上的丝氨酸-51以调节应激条件下的翻译。我们已经证明牛痘病毒K3 L蛋白和猪痘病毒C8 L蛋白是PKR的假底物抑制剂，并且可以抑制酵母中的PKR毒性。在酵母和哺乳动物细胞中，位于eIF 2 α磷酸化位点约30个残基处并在K3 L和C8 L中保守的序列基序对于PKR的抑制至关重要。eIF 2 α基序中的突变损害了体内GCN 2激酶的磷酸化，表明该基序在底物识别中起重要作用。在PKR激酶结构域的羧基末端一半中的14个独立突变使得激酶对K3 L抑制具有抗性，并且预测这些突变改变激酶和底物之间的接触。最后，在酵母和哺乳动物细胞中的实验证明了二聚化对于PKR体内活化的重要性。而分离的PKR激酶结构域在体内是无活性的，发现激酶结构域与异源二聚化结构域的融合恢复活性。- 翻译，因子，eIF，激酶，酵母，酿酒酵母