REGULATION OF EUKARYOTE PROTEIN SYNTHESIS

真核生物蛋白质合成的调控

• 批准号: 6162466
• 负责人: T 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/6162466
• 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.

在应激下哺乳动物细胞中蛋白质合成的调节 条件以及对GCN4特异性翻译的调节 在氨基酸饥饿条件下酿酒酵母的酵母糖疗法是 通过真核翻译起始的磷酸化介导 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GCN2专门识别和磷酸化 EIF2亚基上的丝氨酸51调节翻译。 我们有 建立了一个抑制PKR表达在酵母中的毒性的系统 通过共表达PKR的病毒抑制剂。 离甲酸病毒K3L蛋白 是PKR的假基底抑制剂，可以抑制PKR毒性 酵母。 在附近发现的保守氨基酸序列基序 K3L的羧基末端以及在EIF2alpha中的残基73-83之间 对于K3L抑制PKR至关重要。 生化分析已揭示 这个图案位于位置的30多个残留物 EIF2alpha中的磷酸化对于K3L与PKR的结合很重要。 这 eIF2a中的相应残基是正确调节的 在EIF2Alpha中，该基序中的GCN4表达和突变受损 Ser-51体内的磷酸化。 这些结果表明接触 超过30多个磷酸化位点的残基对于激酶很重要 识别EIF2Alpha。 在研究激酶 - 底物的另一种方法中 识别我们已经确定了14个对K3L具有抗性的PKR等位基因 抑制。 羧基末端的PKR突变簇的一半 激酶结构域，并基于CAMP依赖性的X射线结构 蛋白激酶被预测会改变激酶和激酶之间的接触 基材。 来自杆状病毒Autographa加利福尼亚的PK2蛋白 类似于截短的EIF2Alpha激酶结构域，我们发现PK2 是EIF2Alpha激酶抑制剂。 PK2蛋白直接相互作用 使用PKR，发现PK2可保护杆状病毒免受抗病毒的侵害 PKR的影响。 EIF2alpha的底物特异性分析 激酶表明PKR是一种双特异性蛋白激酶 在残基51上，在Ser，Thr或Tyr上磷酸化EIF2Alpha。 第二 研究的主要领域涉及酵母蛋白FUN12 类似于细菌IF2，像EIF2这样的翻译因素 向核糖体大都会。 在蛋白质合成的模型测定中 Fun12蛋白可以代替EIF2，而多核糖体曲线 Fun12删除的菌株在翻译起始中显示出缺陷。 这些 结果，结合了Fun12-Deotion中的许多遗传观察 菌株，表明Fun12一般或替代机制的功能 翻译启动。 最后，Fun12蛋白也是 在古细菌中鉴定出来，我们已经确定了人类的fun12同源 暗示该因素在 细胞蛋白合成。