REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

真核蛋白质合成的调控

• 批准号: 6432545
• 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/6432545
• 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, encoded by the FUN12 gene, is a general translation initiation factor. Biochemical assays demonstrated that the human IF2 protein possesses ribosome-dependent GTPase activity and promotes the ribosomal subunit joining step of protein synthesis. In recognition of this activity the eukaryotic IF2 homologs have been renamed eIF5B. Mutation of conserved residues in the eIF5B GTP-binding domain revealed a critical role for GTP-binding and hydrolysis by eIF5B for translation initiation. Using an eIF5B mutant that utilizes XTP in place of GTP, we have demonstrated that at least two nucleotide (GTP) hydrolysis events are required for eukaryotic translation initiation. Consistent with a role in subunit joining, yeast strains lacking eIF5B show increased levels of leaking scanning. Finally, yeast two-hybrid, in vitro protein binding assays and co-immunoprecipitation experiments revealed that yeast eIF5B directly interacts with the translation factor eIF1A (a homolog of the prokaryotic factor IF1). In addition, overexpression of eIF1A specifically exacerbated the growth defect of strains lacking, or expressing truncated forms of, eIF5B. This physical and functional interaction between the two evolutionarily conserved translation initiation factors may facilitate methionyl-tRNA binding to the ribosomal P site.A second research interest is phosphorylation of the translation initiation factor eIF2. The mammalian kinases PKR, HRI, and PERK and the yeast kinase GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. Mutational analysis of yeast eIF2alpha identified amino acid substitutions at residues 49 and 50 as well as in a conserved sequence motif around 30 residues C-terminal of the Ser-51 phosphorylation site that impair translational regulation. Biochemical studies revealed that a subset of the mutations in eIF2alpha blocked phosphorylation by the GCN2 and PKR kinases both in vivo and in vitro. These results demonstrate that kinase recognition of eIF2alpha utilizes residues both nearby and, surprisingly, remote from the phosphorylation site. We 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. This inhibition of PKR by K3L and C8L was dependent on residues conserved among eIF2alpha, K3L and C8L. 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.

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，由FUN 12基因编码的酵母IF 2同源物是一种通用的翻译起始因子。 生物化学分析表明，人IF 2蛋白具有核糖体依赖性GT3活性，并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。 eIF 5 B GTP结合结构域中保守残基的突变揭示了eIF 5 B对GTP结合和水解翻译起始的关键作用。 使用eIF 5 B突变体，利用XTP在GTP的地方，我们已经证明，至少有两个核苷酸（GTP）水解事件所需的真核生物翻译起始。 与亚基连接中的作用一致，缺乏eIF 5 B的酵母菌株显示出增加的泄漏扫描水平。 最后，酵母双杂交，体外蛋白结合试验和免疫共沉淀实验表明，酵母eIF 5 B直接与翻译因子eIF 1A（原核因子IF 1的同源物）相互作用。 此外，eIF 1A的过表达特别加剧了缺乏或表达截短形式的eIF 5 B的菌株的生长缺陷。 这两个进化上保守的翻译起始因子之间的物理和功能相互作用可能促进甲硫氨酰-tRNA与核糖体P位点的结合。哺乳动物激酶PKR、HRI和PERK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。 酵母eIF 2 alpha的突变分析发现，第49和50位残基以及Ser-51磷酸化位点C末端30个残基周围的保守序列基序中存在氨基酸取代，从而损害翻译调节。生物化学研究表明，eIF 2 α突变的一个子集在体内和体外都阻断了GCN 2和PKR激酶的磷酸化。 这些结果表明，eIF 2 α的激酶识别利用了磷酸化位点附近的残基，令人惊讶的是，远离磷酸化位点的残基。 我们证明了牛痘病毒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PKR在酵母中的毒性。 K3 L和C8 L对PKR的这种抑制依赖于eIF 2 α、K3 L和C8 L之间保守的残基。 在PKR激酶结构域的羧基末端的一半中的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与底物之间的接触。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。