Mechanism and Regulation Of Eukaryotic Protein Synthesis

真核蛋白质合成机制及调控

• 批准号: 7594159
• 负责人: THOMAS E DEVER
• 金额: $ 128.11万
• 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/7594159
• 关键词: Active Sites; Address; Adopted; Alleles; Amino Acids; Back; Behavior; Binding; Biochemical; Biological Assay; Bypass; C-terminal; Catalytic Domain; Cells; Charge; Collaborations; Complex; Condition; Coupling; Data; Defect; Dimerization; Double-Stranded RNA; Elements; Eukaryotic Cell; Family; GTP Binding; GTP-Binding Proteins; Gene Expression; Goals; Growth; Growth and Development function; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Helix (Snails); Hydrolysis; In Vitro; Iodine; Lobe; Maps; Mediating; Molecular Conformation; Molecular Genetics; Movement; Mutate; Mutation; N-terminal; Nucleotides; Orthologous Gene; Peptide Initiation Factors; Phase; Phosphorylation; Phosphotransferases; Prokaryotic Initiation Factors; Property; Protein Biosynthesis; Protein Kinase; Protein Overexpression; Proteins; RNA, Transfer, Met; Reaction; Recruitment Activity; Regulation; Ribosomes; Role; SWI1; Serine; Shapes; Starvation; Stress; Structure; Suppressor Mutations; System; Tail; Testing; Thinking; Translation Initiation; Yeasts; alpha helix; analog; base; cell growth; design; dimer; ear helix; eukaryotic initiation factor-5B; eukaryotic peptide initiation factor-1A; in vitro Assay; in vivo; inhibitor/antagonist; insight; mouse Gdi2 protein; mutant; stem; tool; translation factor

We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding 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 stress-responsive protein 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, which is activated under conditions of amino acid starvation, PKR, which is activated by double-stranded RNA and downregulates protein synthesis in virally infected cells, and PERK, activated under conditions of ER stress. Previously, in collaboration with Frank Sicheri, we determined the structure of the PKR kinase domain in complex with eIF2alpha. This structural analysis revealed that eIF2alpha binds to the C-terminal lobe making intimate contact with helix alphaG, while catalytic domain dimerization is mediated by a back-to-back orientation of the kinase N-terminal lobes. Consistent with the structural data, charge-reversal mutations that disrupt a conserved salt-bridge between Arg262 and Asp266 in the dimer interface block PKR autophosphorylation and eIF2alpha phosphorylation. Importantly, combining the two charge-reversal mutations in the same PKR allele, designed to restore the salt-bridge interaction with opposite polarity, rescued PKR activity. We proposed an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation and specific substrate recognition. Interestingly, the residues forming the salt-bridge interaction in the PKR dimer interface are conserved among the eIF2alpha kinases. However, the crystal structure of the GCN2 kinase domain revealed a different dimerization interface than observed in the PKR structure. Whereas the PKR structure was of an active kinase, the GCN2 structure utilized a non-phosphorylated kinase domain and thus may represent an inactive kinase domain. The Arg262 and Asp266 residues that form an intermolecular salt-bridge in PKR are conserved in GCN2 and PERK; however, these two residues are too remote to interact in the GCN2 structure. To test the importance of this potential salt-bridge interaction in PKR, GCN2 and PERK, the residues constituting the salt-bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu in PERK) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases the double mutation designed to restore the salt-bridge interaction with opposite polarity, resulted in a functional kinase (reference 1). Thus, the salt-bridge interaction and back-to-back dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. Our results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain while the GCN2 structure may represent an inactive state of the kinase. The GTP-binding (G) protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. The eIF5B is an ortholog of prokaryotic translation initiation factor IF2. Previous studies revealed that eIF5B consists of four domains that structurally assemble to form a chalice-shaped molecule. The G domain plus domains II and III form the cup of the chalice, a long alpha helix forms the stem, and domain IV is the base of the chalice. In addition, we previously showed that the domain IV of eIF5B binds to the C-terminal tail of the factor eIF1A (an ortholog of prokaryotic factor IF1). We have shown that the eIF5B-eIF1A interaction promotes ribosomal subunit joining and possibly provides a checkpoint for correct ribosome formation, with full activation of eIF5B GTP hydrolytic activity dependent on formation of a properly organized initiation complex. Whereas in vitro assays established that eIF5B catalyzes ribosomal subunit joining, supporting in vivo data has been lacking. To address the essential function of eIF5B in vivo, rapidly depleted eIF5B in yeast cells. Analysis of translation initiation complexes from these cells revealed a defect in subunit joining. Mutation of the eIF1A C-terminal tail impaired eIF5B binding to eIF1A, and overexpression of eIF5B suppressed the growth and translation initiation defects in yeast expressing the eIF1A mutant, indicating that eIF1A helps recruit eIF5B to the ribosome prior to subunit joining. Blocking GTP hydrolysis by eIF5B led to the accumulation of both eIF1A and eIF5B on the 80S products of subunit joining both in vivo and in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of a non-hydrolyzable GTP analog, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining, and that subsequent release of eIF1A is dependent on GTP hydrolysis and release of eIF5B from the 80S ribosome (reference 2). Our previous studies on an eIF5B Switch I mutant revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. To gain further insights into the eIF5B GTP-binding properties and regulatory switch, and by extension the switch and guanine-nucleotide binding behavior of other GTP-binding proteins, we conducted a mutational and suppressor analysis of the conserved Switch II Gly479 residue of yeast eIF5B. Based on studies of other G proteins, it has generally been thought that movement of this Gly residue is critical for the structural transition of Switch II during GTP binding and hydrolysis. We found that the G479A mutation in eIF5B impaired yeast cell growth and the guanine-nucleotide binding, GTP hydrolytic, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement of this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element (A444V) and at a residue in domain 2 of eIF5B that interacts with Switch II (D740R). The intragenic suppressors restored yeast cell growth and eIF5B nucleotide binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain 2 suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis, and thereby re-establish coupling between GTP binding and eIF5B domain movements (reference 3). As the switch 2 mutation and the switch 1 suppressor mutation map to elements conserved in all GTP-binding proteins, we believe that this interaction may be of importance for all GTP-binding proteins.

我们研究了通过GTP结合蛋白和蛋白质磷酸化调节的真核细胞中蛋白质合成的机制和调节。 蛋白质合成的第一步是通过因子EIF2将引发剂Met-tRNA与小核糖体亚基结合。 EIF2是GTP结合蛋白，在翻译启动过程中，GTP被水解为GDP。 EIF2从与GDP复杂的核糖体中释放，并要求鸟嘌呤核苷酸交换因子EIF2B将EIF2-GDP转换为EIF2-GTP。 这种交换反应由一个应力反应性蛋白激酶家族调节，该蛋白激酶专门磷酸化在残基51处的丝氨酸上的EIF2的α亚基，从而将EIF2掩盖到EIF2B的抑制剂中。 在EIF2Alpha激酶的家族中，GCN2在氨基酸饥饿的条件下被激活，PKR被双链RNA激活，并下调病毒感染细胞中的蛋白质合成，并在ER应力条件下激活了PERK。 以前，我们与Frank Sicheri合作，确定了与EIF2alpha复杂的PKR激酶结构域的结构。 该结构分析表明，EIF2Alpha与C末端叶结合，与螺旋alphag紧密接触，而催化域二聚化是由激酶N末端裂片的背靠背方向介导的。 与结构数据一致，在二聚体界面阻止PKR自磷酸化和EIF2Alpha磷酸化中，在ARG262和ASP266之间破坏了ARG262和ASP266之间的保守盐桥的电荷反转突变。重要的是，将两个电荷反转突变组合在同一PKR等位基因中，旨在以相反的极性恢复盐桥相互作用，并挽救了PKR活性。 我们提出了一种有序的PKR激活机制，其中催化结构域二聚化触发自磷酸化和特定的底物识别。 有趣的是，在PKR二聚体界面中形成盐桥相互作用的残基在EIF2Alpha激酶之间是保守的。但是，GCN2激酶结构域的晶体结构显示出与PKR结构中观察到的二聚化界面不同。 尽管PKR结构是活性激酶的，但GCN2结构利用了非磷酸化激酶结构域，因此可能代表一个无活跃的激酶结构域。 在PKR中形成分子间盐桥的ARG262和ASP266残基在GCN2和PERK中保守。但是，这两个残基太遥远而无法在GCN2结构中相互作用。 为了测试这种潜在的盐桥相互作用在PKR，GCN2和PERK中的重要性，将构成盐桥的残留物独立或将相反电荷的残基突变为残基。 ASP的单个突变（或PERK中的GLU）和ARG残基阻断了酵母细胞和体外的激酶功能。 然而，对于所有三种激酶，双突变旨在恢复盐桥相互作用的极性相反，导致功能激酶（参考文献1）。 因此，在PKR结构中观察到的盐桥相互作用和背对背二聚体界面对于所有三种EIF2alpha激酶的活性至关重要。 我们的结果与PKR结构代表EIF2Alpha激酶结构域的活性状态的观念一致，而GCN2结构可能代表激酶的无活性状态。 GTP结合（G）蛋白EIF5B催化核糖体亚基在翻译起始的最后一步。 EIF5B是原核翻译起始因子IF2的直系同源。 先前的研究表明，EIF5B由四个结构组装以形成圣杯形分子的结构域组成。 G域和域II和III形成了圣杯的杯子，长α螺旋形成茎，而IV域则是圣杯的基础。 此外，我们先前证明了EIF5B的域IV与EIF1A因子的C末端尾巴（原核因子IF1的正源性）结合。我们已经表明，EIF5B-EIF1A相互作用促进了核糖体亚基连接，并可能提供了一个检查点以进行正确的核糖体形成，并完全激活EIF5B GTP GTP水解活性取决于形成适当组织的起始络合物。 尽管在体外测定中表明，EIF5B催化核糖体亚基连接，但缺乏支持体内数据。 为了解决EIF5B在体内的基本功能，在酵母细胞中迅速耗尽的EIF5B。 对这些细胞的翻译起始复合物的分析显示，亚基连接的缺陷。 EIF1A C末端尾部的突变受损EIF5B与EIF1A的结合，EIF5B的过表达抑制了表达EIF1A突变体的酵母的生长和翻译起始缺陷，这表明EIF1A在加入前核糖体的核糖体中有助于将EIF5B募集到核糖体上。 通过EIF5B阻断GTP水解，导致EIF1A和EIF5B在80年代的亚基产物上均积累了体内和体外。 同样，EIF5B和EIF1A仍然与在不可溶解的GTP类似物存在下形成的80S复合物相关联，而这些因素是在包含GTP的测定中从80年代复合物中释放出来的。 我们建议EIF1A促进EIF5B与40S亚基的结合以促进亚基连接，随后的EIF1A的释放取决于GTP水解和从80S核糖体中EIF5B释放（参考文献2）。 我们先前对EIF5B开关I突变体的研究表明，EIF5B的GTP水解激活了亚基连接后核糖体释放EIF5B所需的调节开关。 为了进一步了解EIF5B GTP结合特性和调节开关，并通过扩展其他GTP结合蛋白的开关和鸟嘌呤核苷酸结合行为，我们对保守的开关II GLY479 GLY479酵母EIF5B残基进行了突变和抑制分析。 基于对其他G蛋白的研究，通常认为该GLY残基的运动对于GTP结合和水解过程中开关II的结构过渡至关重要。 我们发现，EIF5B中的G479A突变受损酵母细胞的生长以及鸟嘌呤核苷酸结合，GTP水解和核糖体亚基联合活性。 在绕过EIF5B中此开关II GLY的临界要求的突变的屏幕中，在开关I元素（A444V）和EIF5B域2中与开关II相互作用的域2中鉴定了基因内抑制器。基因内抑制剂恢复了酵母细胞的生长和EIF5B核苷酸结合，GTP水解和亚基连接活性。我们提出，开关II突变会扭曲GTP结合活性位点的几何形状损害核苷酸结合和与GTP结合相关的EIF5B结构域运动。 因此，开关I和域2抑制突变诱导开关II采用有利于核苷酸结合和水解的构象，从而在GTP结合和EIF5B域运动之间重新建立耦合（参考文献3）。 当开关2突变和开关1抑制剂突变图与所有GTP结合蛋白中保守的元素时，我们认为这种相互作用对于所有GTP结合蛋白可能都很重要。