REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

真核蛋白质合成的调控

• 批准号: 6108045
• 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/6108045
• 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 regulation of protein synthesis in mammalian cells under stress conditions as well as the regulation of GCN4-specific translation in the yeast Saccharomyces cerevisiae under amino acid starvation conditions is mediated by phosphorylation of the eukaryotic translation initiation factor (eIF)-2. We have been studying how the mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically recognize and phosphorylate serine-51 on the a subunit of eIF2 to regulate translation. We have established a system to suppress the toxicity of PKR expression in yeast by co-expressing viral inhibitors of PKR. The vaccinia virus K3L protein is a pseudosubstrate inhibitor of PKR, and can suppress PKR toxicity in yeast. A conserved amino acid sequence motif found near the carboxyl-terminus of K3L and between residues 73-83 in eIF2alpha is critical for K3L inhibition of PKR. Biochemical analyses have revealed that this motif, located over 30 residues from the site of phosphorylation in eIF2alpha is important for K3L binding to PKR. The corresponding residues in eIF2a are required for proper regulation of GCN4 expression and mutations in this motif in eIF2alpha impair phosphorylation of Ser-51 in vivo. These results suggest that contacts over 30 residues from site of phosphorylation are important for kinase recognition of eIF2alpha. In another approach to study kinase-substrate recognition we have identified 14 PKR alleles that are resistant to K3L inhibition. The PKR mutations cluster in the carboxyl-terminal half of the kinase domain and, based on the x-ray structure of the cAMP-dependent protein kinase, are predicted to alter contacts between the kinase and substrate. The pk2 protein from the baculovirus Autographa californica resembles a truncated eIF2alpha kinase domain and we have found that pk2 is an eIF2alpha kinase inhibitor. The pk2 protein directly interacted with PKR, and pk2 was found to protect baculovirus from the anti-viral effects of PKR. Analysis of substrate specificity of the eIF2alpha kinases revealed that PKR is a dual-specificity protein kinase capable of phosphorylating eIF2alpha on Ser, Thr, or Tyr at residue-51. A second major area of investigation involves the yeast protein FUN12 which resembles bacterial IF2, a translation factor that like eIF2 delivers Met-tRNAiMet to the ribosome. In model assays of protein synthesis the FUN12 protein could substitute for eIF2, and polyribosome profiles from fun12-deleted strains showed defects in translation initiation. These results, combined with a number of genetic observations in fun12-deletion strains, suggest that FUN12 functions in general or alternate mechanisms of translation initiation. Finally, the FUN12 protein has also been identified in archaea and we have identified a human homolog of FUN12 suggesting that this factor plays an important, and conserved, role in cellular protein synthesis.

应激条件下哺乳动物细胞蛋白质合成的调控 条件以及GCN 4特异性翻译的调节， 在氨基酸饥饿条件下的酵母酿酒酵母， 由真核翻译起始磷酸化介导 因子（eIF）-2。 我们一直在研究哺乳动物激酶PKR和 HRI和酵母激酶GCN 2特异性识别并磷酸化 丝氨酸-51在eIF 2的α亚基上调节翻译。 我们有 建立了一个抑制PKR在酵母中表达的毒性的系统 通过共表达PKR的病毒抑制剂。 牛痘病毒K3 L蛋白 是PKR的假底物抑制剂，可抑制PKR毒性， 酵母 一个保守的氨基酸序列基序发现附近的 K3 L的羧基末端和eIF 2 α中的残基73-83之间是 对于K3 L抑制PKR至关重要。 生化分析显示 这个基序，位于30多个残基的网站， eIF 2 α的磷酸化对于K3 L与PKR的结合是重要的。 的 eIF 2a中的相应残基是适当调节 eIF 2 α损伤中GCN 4的表达和该基序的突变 Ser-51的磷酸化。 这些结果表明，接触 来自磷酸化位点的30多个残基对于激酶是重要的 识别eIF 2alpha。 在另一种研究激酶底物的方法中， 我们已经鉴定了14个PKR等位基因， 抑制作用 PKR突变集中在羧基末端的一半， 激酶结构域，并基于cAMP依赖性的 蛋白激酶，预测改变激酶和 衬底 苜蓿银纹夜蛾杆状病毒PK 2蛋白的研究 类似于截短的eIF 2 α激酶结构域，我们发现pk 2 是eIF 2 α激酶抑制剂。 pk 2蛋白直接作用于 与PKR一起使用，发现pk 2可以保护杆状病毒免受抗病毒药物的侵害 PKR的影响。 eIF 2 α的底物特异性分析 PKR是一种双特异性蛋白激酶， 使eIF 2 α在Ser、Thr或Tyr残基-51处磷酸化。 第二 研究的主要领域涉及酵母蛋白FUN 12， 类似于细菌IF 2，一种翻译因子，像eIF 2一样传递 Met-tRNAiMet与核糖体的结合。 在蛋白质合成的模型测定中， FUN 12蛋白可以替代eIF 2，并且来自 fun 12缺失的菌株显示翻译起始缺陷。 这些 结果，结合fun 12缺失中的一些遗传观察， 菌株，表明FUN 12在一般或替代机制中起作用 翻译的开始。 最后，FUN 12蛋白也被 在古生菌中发现了FUN 12，我们也发现了FUN 12的人类同源物 这表明该因子在以下方面起着重要且保守的作用： 细胞蛋白质合成