Mechanism and Regulation of Eukaryotic Protein Synthesis

真核生物蛋白质合成机制及调控

• 批准号: 8553863
• 负责人: THOMAS E DEVER
• 金额: $ 138.65万
• 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/8553863
• 关键词: Affect; Amino Acids; Back; Bacterial Proteins; Binding; Biochemical; Biological Assay; Biological Models; Blood; C-terminal; Cells; Charge; Collaborations; Complex; Data; Defect; Dimerization; Disease; Double-Stranded RNA; Double-Stranded RNA Binding Domain; E3L protein; EEF1A1 gene; Elements; Elongation Factor; Eukaryota; Eukaryotic Cell; Family; GTP Binding; GTP-Binding Proteins; Gene Expression; Germany; Goals; Growth; Growth and Development function; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Hydroxyl Radical; Initiator Codon; Intellectual functioning disability; Israel; Learning; Link; Lysine; Malaria; Mammals; Maps; Mediating; Memory; Microcephaly; Missense Mutation; Modeling; Modification; Molecular Genetics; Mutation; N-terminal; New York; Nucleic Acid Binding; Peptide Initiation Factors; Peptides; Phase; Phosphorylation; Phosphotransferases; Plasmodium; Plasmodium falciparum; Play; Positioning Attribute; Poxviridae; Property; Protein Biosynthesis; Protein Kinase; Protein Synthesis Inhibition; Proteins; Puromycin; RNA, Transfer, Met; Reaction; Regulation; Relative (related person); Resistance; Ribosomal RNA; Ribosomes; Role; Scientist; Serine; Site; Smallpox; Smallpox Viruses; Staging; Starvation; Stress; Structure; Surface; Syndrome; System; Toxic effect; Transfer RNA; Translation Initiation; Translations; Vaccinia virus; Viral; Virus; Work; Yeasts; Z-Form DNA; base; cell growth; design; dimer; drug candidate; ds RNA-Binding Proteins; factor EF-P; genome sequencing; human disease; hypusine; in vivo; inhibitor/antagonist; insight; kinase inhibitor; meetings; member; mutant; nervous system disorder; overexpression; pathogen; phenylalanine-tRNA; polypeptide; research study; stem; tool; translation factor; transmission process

We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). The factor eIF2 is composed of three polypeptide chains. The gamma subunit of eIF2 is a GTPase that, based on sequence and the structure of the archaeal homolog aIF2gamma, resembles elongation factor EF-Tu. However, in contrast to EF-Tu, which binds tRNAs to the A-site of the 70S ribosome, eIF2 binds Met-tRNAi to the P-site of the 40S subunit. To gain insights into how eIF2 binds Met-tRNAi and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40SeIF1eIF1AeIF2GTPMet-tRNAimRNA (48S) complex. Based on the structure of the EF-Tu ternary complex, we predicted that linkage of Fe(II)-BABE, a hydroxyl radical generator, to domain III of eIF2gamma would result in cleavage of Met-tRNAi in the T-stem. However, this instead resulted in cleavage of the D-stem of Met-tRNAi and of 18S rRNA at the top of helix h44, a prominent landmark on the intersubunit surface of the 40S subunit. Based on the results of these and other cleavage experiments, and the fact that Met-tRNAi is bound to the P-site of the 40S subunit, we generated a model of the 48S complex in which domain III of eIF2gamma binds near 18S rRNA helix h44 and eIF2gammaMet-tRNAi contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAi is rotated nearly 180 degrees relative to the position of the tRNA in the EF-Tu ternary complex. Consistent with the alternate models of the eIF2 and EF-Tu ternary complexes, we found that a domain III mutation in EF-Tu severely impaired Phe-tRNA binding; whereas, the corresponding eIF2gamma mutation did not impair Met-tRNAi binding to eIF2. In addition, mutation of conserved positively charged residues on the surface of domain III of eIF2gamma impaired binding of the eIF2GTPMet-tRNAi ternary complex to the 40S ribosome. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNA in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. Whereas protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Moreover, the consequences of partial loss of eIF2gamma function or eIF2 integrity are unknown in mammals, including humans. Our collaborators in Israel and Germany recently identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change in resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Biochemical studies of human cells overexpressing the eIF2γ mutant and of yeast eIF2γ with the analogous mutation revealed a defect in binding the eIF2β subunit to eIF2γ. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2γ impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2β. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. Previously, we demonstrated that, when expressed in yeast, human PKR could phosphorylate the alpha subunit of eIF2 on Ser51 resulting in an inhibition of protein synthesis and yeast cell growth. We also identified the mechanism of activation of PKR that requires back-to-back dimerization of two PKR kinase domains. Further demonstrating the importance of kinase domain dimerization, we showed that appending heterologous dimerization domains to the PKR kinase domain activated the kinase. Kinases resembling the eIF2alpha kinases have been identified in the genome sequences of a variety of eukaryotes including pathogens such as Plasmodium falciparum, the protozoan that causes malaria. Plasmodium expresses three kinases related by sequence to the eIF2alpha kinases. Working in collaboration with scientists in New York, we demonstrated that the Plasmodium kinase PfPK4 is an eIF2alpha kinase. We first fused the PfPK4 kinase domain to the constitutive dimer GST, forming GST-PfPK4-KD. GST-PfPK4-KD phosphorylated yeast eIF2alpha on Ser51 in vivo leading to inhibition of yeast cell growth. This toxicity was suppressed in cells expressing a non-phosphorylatable form of eIF2alpha in which Ser51 was replaced by Ala. In addition, our collaborators showed that PfPK4 and eIF2alpha phosphorylation are essential for the blood stage growth of Plasmodium. Thus, PfPK4 is an attractive candidate for drugs to alleviate disease and inhibit malaria transmission. In order to subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively restored yeast cell growth. We are currently characterizing mutations in both the PKR kinase domain and its regulatory domain that confer resistance to E3L inhibition. As the kinase domain mutations enhance PKR dimerization, these results suggest that E3L may inhibit PKR by blocking kinase dimerization. We are also conducting structure-function studies on the E3L protein. Our preliminary data indicate that both the N-terminal Z-DNA binding domain (ZBD) and the C-terminal double-stranded RNA binding domain are required for PKR inhibition. However, mutations designed to disrupt nucleic acid binding to the ZBD do not affect PKR inhibition, suggesting that the ZBD has additional functions beyond nucleic acid binding. We are also studying the translation factor eIF5A. eIF5A is the sole protein containing the unusual amino acid hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and eIF5A was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A is unknown. Using molecular genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor. We are currently identifying and characterizing mutants of eIF5A.

我们研究真核细胞中蛋白质合成的机制和调控，重点关注 GTP 结合 (G) 蛋白和蛋白质磷酸化的调控。 蛋白质合成的第一步是通过因子 eIF2 将起始子 Met-tRNA 与核糖体小亚基结合。 eIF2 是一种 GTP 结合蛋白，在翻译起始过程中，GTP 被水解为 GDP。 eIF2 从核糖体中与 GDP 复合释放，需要鸟嘌呤核苷酸交换因子 eIF2B 将 eIF2-GDP 转化为 eIF2-GTP。 这种交换反应由一系列激酶调节，这些激酶特异性磷酸化 eIF2 丝氨酸残基 51 上的 α 亚基，从而将 eIF2 转化为 eIF2B 抑制剂。 eIF2α 激酶家族包括 GCN2（在氨基酸饥饿条件下激活）、PKR（由双链 RNA 激活并下调病毒感染细胞中的蛋白质合成）和 PERK（在内质网应激条件下激活）。 因子eIF2由三个多肽链组成。 eIF2 的 γ 亚基是一种 GTP 酶，基于古菌同源物 aIF2gamma 的序列和结构，它类似于延伸因子 EF-Tu。然而，与将 tRNA 结合到 70S 核糖体 A 位点的 EF-Tu 不同，eIF2 将 Met-tRNAi 结合到 40S 亚基的 P 位点。为了深入了解 eIF2 如何结合 Met-tRNAi，然后与 40S 核糖体结合，我们使用定向羟基自由基探测来识别 40SeIF1eIF1AeIF2GTPMet-tRNAimRNA (48S) 复合物内的 eIF2 接触。基于EF-Tu三元复合物的结构，我们预测羟基自由基产生剂Fe(II)-BABE与eIF2gamma的结构域III的连接将导致T-茎中Met-tRNAi的裂解。然而，这反而导致 Met-tRNAi 的 D 茎和螺旋 h44 顶部的 18S rRNA 裂解，螺旋 h44 是 40S 亚基亚基间表面上的显着标志。基于这些和其他切割实验的结果，以及 Met-tRNAi 与 40S 亚基的 P 位点结合的事实，我们生成了 48S 复合物的模型，其中 eIF2gamma 的结构域 III 结合在 18S rRNA 螺旋 h44 附近，并且 eIF2gammaMet-tRNAi 接触仅限于 tRNA 的受体茎。在这个 eIF2 三元复合物模型中，Met-tRNAi 相对于 EF-Tu 三元复合物中 tRNA 的位置旋转了近 180 度。与 eIF2 和 EF-Tu 三元复合物的替代模型一致，我们发现 EF-Tu 中的结构域 III 突变严重损害了 Phe-tRNA 结合；然而，相应的 eIF2gamma 突变不会损害 Met-tRNAi 与 eIF2 的结合。此外，eIF2gamma 结构域 III 表面保守带正电残基的突变会损害 eIF2GTPMet-tRNAi 三元复合物与 40S 核糖体的结合。因此，尽管它们结构相似，eIF2和EF-Tu以实质上不同的方式结合tRNA，我们提出EF-Tu的tRNA结合域III在eIF2gamma中获得了结合核糖体的新功能。 尽管已知蛋白质合成在不同模型系统的学习和记忆中发挥着关键作用，但人类智力障碍综合征尚未与蛋白质合成的改变直接相关。此外，在包括人类在内的哺乳动物中，eIF2gamma 功能或 eIF2 完整性部分丧失的后果尚不清楚。我们在以色列和德国的合作者最近发现了一种以智力障碍和小头畸形为特征的人类 X 染色体神经系统疾病。绘图研究将致病突变确定为单碱基变化，导致 eIF2gamma（由 EIF2S3 编码）发生错义突变。对过表达 eIF2γ 突变体的人类细胞和具有类似突变的酵母 eIF2γ 的生化研究揭示了 eIF2β 亚基与 eIF2γ 结合的​​缺陷。与 eIF2 完整性的丧失相一致，酵母 eIF2γ 的突变损害了翻译起始密码子选择和体内 eIF2 的功能，而 eIF2β 的过度表达会抑制这种功能。这些发现将智力障碍与翻译起始受损直接联系起来，并为由于 eIF2 功能部分丧失而导致的人类疾病提供了机制基础。 此前，我们证明，当在酵母中表达时，人 PKR 可以磷酸化 eIF2 Ser51 上的 α 亚基，从而抑制蛋白质合成和酵母细胞生长。我们还确定了 PKR 激活机制，需要两个 PKR 激酶结构域的背靠背二聚化。进一步证明激酶结构域二聚化的重要性，我们表明将异源二聚化结构域附加到 PKR 激酶结构域会激活激酶。类似于 eIF2α 激酶的激酶已在多种真核生物的基因组序列中被发现，包括恶性疟原虫（引起疟疾的原生动物）等病原体。疟原虫表达三种与 eIF2α 激酶序列相关的激酶。 我们与纽约的科学家合作，证明了疟原虫激酶 PfPK4 是一种 eIF2α 激酶。 我们首先将 PfPK4 激酶结构域与组成型二聚体 GST 融合，形成 GST-PfPK4-KD。 GST-PfPK4-KD 在体内磷酸化酵母 eIF2α Ser51，从而抑制酵母细胞生长。 这种毒性在表达不可磷酸化形式的 eIF2α 的细胞中受到抑制，其中 Ser51 被 Ala 取代。此外，我们的合作者表明，PfPK4 和 eIF2α 磷酸化对于疟原虫的血液阶段生长至关重要。因此，PfPK4 是减轻疾病和抑制疟疾传播的药物的有吸引力的候选者。 为了破坏 PKR 介导的抗病毒防御，病毒会产生激酶抑制剂。 痘病毒家族的一些成员表达两种不同类型的 PKR 抑制剂：一种假底物抑制剂（例如类似于 eIF2α N 末端三分之一的痘苗病毒 K3L 蛋白）和一种称为 E3L 的双链 RNA 结合蛋白。人PKR的高水平表达抑制了酵母的生长，而痘苗病毒K3L或E3L蛋白或相关天花病毒C3L或E3L蛋白的共表达分别恢复了酵母细胞的生长。我们目前正在鉴定 PKR 激酶结构域及其调节结构域中赋予 E3L 抑制抗性的突变。由于激酶结构域突变增强了 PKR 二聚化，这些结果表明 E3L 可能通过阻断激酶二聚化来抑制 PKR。我们还在对 E3L 蛋白进行结构功能研究。 我们的初步数据表明，N 端 Z-DNA 结合域 (ZBD) 和 C 端双链 RNA 结合域都是 PKR 抑制所必需的。 然而，旨在破坏核酸与 ZBD 结合的突变不会影响 PKR 抑制，这表明 ZBD 具有核酸结合之外的其他功能。 我们还在研究翻译因子 eIF5A。 eIF5A 是唯一含有不常见氨基酸海布辛 N-ε-(4-氨基-2-羟丁基)赖氨酸的蛋白质，eIF5A 最初是根据其刺激甲硫氨酰-嘌呤霉素合成（一种用于第一个肽键合成的模型测定）的产量（终点）的能力而被识别的。然而，eIF5A 的确切细胞作用尚不清楚。通过分子遗传学和生化研究，我们最近表明 eIF5A 促进翻译延伸，并且这种活性依赖于 hypusine 修饰。由于 eIF5A 是细菌蛋白 EF-P 的结构同源物，因此我们提出 eIF5A/EF-P 是普遍保守的翻译延伸因子。我们目前正在鉴定和表征 eIF5A 突变体。