Mechanism and Regulation of Eukaryotic Protein Synthesis

真核生物蛋白质合成机制及调控

• 批准号: 10001290
• 负责人: THOMAS E DEVER
• 金额: $ 180.83万
• 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/10001290
• 关键词: Amines; Amino Acids; Amino Acyl Transfer RNA; Anabolism; Antiviral Agents; Ascorbic Acid; Binding; Biochemical; Biological Assay; Biological Models; Bypass; Cell Line; Cells; Clinical; Code; Codon Nucleotides; Collaborations; Crystallization; Disease; Elements; Elongation Factor; Enzymes; Epilepsy; Eukaryotic Cell; Exhibits; Family; France; GTP Binding; GTP-Binding Proteins; Galactose; Gene Expression; Genes; Germany; Glucose; Goals; Growth and Development function; Guanosine Triphosphate; Human; Hydroxyl Radical; Hydroxylation; Hypogonadism; Hypopituitarism; Impairment; In Vitro; Initiator Codon; Israel; Learning; Link; London; MEHMO syndrome; Mammalian Cell; Medical center; Memory; Messenger RNA; Metabolism; Microcephaly; Military Personnel; Mixed Function Oxygenases; Modeling; Modification; Molecular; Molecular Genetics; Mutation; Neurons; Obesity; Open Reading Frames; Ornithine Decarboxylase; Orthologous Gene; Pathway interactions; Patients; Peptide Initiation Factors; Peptides; Phase; Phenotype; Phosphorylase a; Phosphorylases; Phosphorylation; Phosphotransferases; Plants; Play; Polyamines; Positioning Attribute; Post-Translational Protein Processing; Proline; Property; Protein Biosynthesis; Protein Kinase; Proteins; Regulation; Reporter; Reporting; Research; Research Personnel; Ribosomes; Roentgen Rays; Role; Running; Scanning; Serine; Side; Site; Slovakia; Spermidine; Stress; Structure; Symptoms; Syndrome; System; Trans-Activators; Transfer RNA; Translating; Translation Initiation; Translations; Universities; Viral; X-linked intellectual disability; Yeast Model System; Yeasts; arm; base; biological adaptation to stress; cell growth; cell growth regulation; disease-causing mutation; factor EF-P; human disease; hypusine; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; interest; mutant; peptidyl-tRNA; polypeptide; polyproline; prevent; prolyl-proline; reconstitution; release factor; sensor; stem; targeted treatment; tool; translation assay; translation factor; tryptophyl-proline

We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation. In addition, we are studying unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is composed of three subunits including the G protein eIF2gamma. During translation initiation, the GTP bound to eIF2gamma is hydrolyzed to GDP, and the factor eIF2B recycles eIF2-GDP to eIF2-GTP. Phosphorylation of eIF2alpha on serine 51, by a family of stress-responsive protein kinases, coverts eIF2 into an inhibitor of eIF2B. Our structure-function studies on eIF2 have provided insights into human disease. Protein synthesis plays a critical role in learning and memory in model systems, and our studies have linked a human X-linked intellectual disability (XLID) syndrome to altered function of eIF2. In previous studies, with collaborators in Israel, Germany, Slovakia, and at Walter Reed National Military Medical Center, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2. Over the past year we have generated yeast models of two additional EIF2S3 mutations linked to MEHMO syndrome. One of the mutations impaired methionyl-tRNA binding to eIF2, and both mutations impaired eIF2 function, altered translational control of specific mRNAs, and reduced stringency of translation start site selection. Our collaborators in London linked EIF2S3 mutations with hypopituitarism and glucose dysregulation, potentially expanding the clinical symptoms of MEHMO syndrome. More recently, we studied induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome. We observed a general reduction in protein synthesis, constitutive induction of the integrated stress response, and heightened expression of ATF4, CHOP and GADD34 under stress conditions in the cells. Moreover, upon differentiation into neurons, the mutant cells exhibited reduced dendritic arborization. Based on our studies we propose that the mutations in eIF2gamma impair the efficiency and fidelity of protein synthesis, and that this altered control of protein synthesis underlies MEHMO syndrome. A second major research focus involves the translation factor eIF5A, the sole cellular protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. We also showed that eIF5A from yeast, like its bacterial ortholog EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we showed that eIF5A was critical for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays. In collaboration with researchers at Johns Hopkins University, we reported that, in addition to its critical requirement for polyproline synthesis, eIF5A functions globally to promote both translation elongation and termination. Working with x-ray crystallographers in France, we obtained the crystal structure of eIF5A bound to the yeast 80S ribosome. eIF5A occupies the E site of the ribosome with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies support a model in which eIF5A and its hypusine residue function to reposition the acceptor arm of the P site to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination. Over the past year, we have further investigated the hypusine modification on eIF5A. The modification is formed in two steps: first, transfer of an n-butyl amine moiety from spermidine to a specific Lys side chain on eIF5A, and then second, hydroxylation of the modified residue. Whereas the LIA1 gene encoding the hydroxylase is non-essential in yeast, we identified mutations in eIF5A that caused synthetic phenotypes in the absence of the hydroxylation. Our results are consistent with the notion that the hydroxyl modification helps to bind and position eIF5A and its hypusine residue to effectively promote the reactivity of the peptidyl-tRNA. Recently, we linked eIF5A to the regulation of polyamine metabolism in mammalian cells. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is regulated by a protein called antizyme, which, in turn, is regulated by another protein called antizyme inhibitor (AZIN1). The synthesis of AZIN1 is inhibited by polyamines and this regulation is dependent on a conserved upstream open reading frame-line (uORF-like) element in the leader of the AZIN1 mRNA. We refer to element as a uCC - for upstream conserved coding region because it lacks at AUG start codon and initiates at a near cognate codon instead. We found that high polyamines enhance translation initiation from the near-cognate start site of the uCC and that this regulation is dependent on the sequence of encoded polypeptide including a highly conserved Pro-Pro-Trp (PPW) motif. We proposed that scanning ribosomes typically bypass the near-cognate start codon of the uCC without initiating and then translate AZIN1. However, occasionally a ribosome will initiate translation at the uCC start codon. Under conditions of high polyamines, these elongating ribosomes pause on the PPW motif. The paused ribosome serves as a roadblock to subsequent scanning ribosomes that bypass the near-cognate start codon. The resultant queue of scanning ribosomes behind the paused elongating ribosome positions a ribosome near the start site of the uCC providing greater opportunity for initiation at the weak start site. Consistent with this queuing model, we found that impairing ribosome loading and thus queue formation reduced uCC translation and derepressed AZIN1 synthesis. In further studies on the AZIN1 regulatory mechanism, we identified eIF5A as a sensor and effector for polyamine control of uCC translation. Using reconstituted in vitro translation assays, we found that synthesis of a PPW peptide, like translation of polyproline sequences, requires eIF5A. Moreover, the ability of eIF5A to stimulate PPW synthesis was inhibited by polyamines and could be rescued by increasing eIF5A levels. We propose that polyamines interfere with eIF5A binding on the ribosome and that inhibition of eIF5A serves as the trigger to cause the ribosome pause that governs uCC translation. We are now exploring the possibility that polyamine regulation of eIF5A underlies translational control of mRNAs encoding other enzymes and regulators of polyamine biosynthesis. In recent studies, we have searched for additional mRNAs containing potential uCCs. Reporter assays in mammalian cells and in vitro revealed that a uORF-like element in the mRNA encoding plant GDP-L-galactose phosphorylase (GGP), a control enzyme in the vitamin C biosynthetic pathway, is a UCC. We propose that interaction of vitamin C with the GGP uCC nascent peptide in the ribosome exit tunnel causes the ribosome to pause and that queuing of subsequent scanning ribosomes results in increased initiation on the uCC and prevents ribosome access to the GGP ORF. We believe that this mechanism of a paused elongating ribosome promoting initiation at an upstream weak start site via ribosome queuing may underlie the control of translation of other mRNAs, especially those whose translation is derepressed by conditions that impair ribosome loading.

我们研究真核细胞中蛋白质合成的机制和调节。特别感兴趣的是通过GTP结合（G）蛋白质和蛋白质磷酸化来调节蛋白质合成。此外，我们正在研究有助于核糖体合成蛋白质的因素的异常翻译后修饰。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由三个亚基组成，包括G蛋白EIF2GAMMA。在翻译启动过程中，与EIF2GAMMA结合的GTP被水解为GDP，而EIF2B因子回收EIF2-GDP到EIF2-GTP。由胁迫响应蛋白激酶家族的EIF2Alpha在丝氨酸51上的磷酸化，将EIF2秘密地覆盖为EIF2B的抑制剂。我们对EIF2的结构功能研究提供了对人类疾病的见解。 蛋白质合成在模型系统中的学习和记忆中起着至关重要的作用，我们的研究将人类X连锁的智力障碍（XLID）综合征与EIF2的功能改变了。 In previous studies, with collaborators in Israel, Germany, Slovakia, and at Walter Reed National Military Medical Center, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2.在过去的一年中，我们生成了与Mehmo综合征相关的两个EIF2S3突变的酵母模型。其中一个突变损害了与EIF2结合的蛋白质基-TRNA，并且两个突变都损害了EIF2功能，改变了对特定mRNA的翻译控制，并降低了翻译起始位点选择的严格性。我们在伦敦的合作者将EIF2S3突变与垂体性和葡萄糖失调联系起来，可能会扩大MEHMO综合征的临床症状。最近，我们研究了源自MEHMO综合征患者的诱导多能茎（IPS）细胞。我们观察到蛋白质合成，综合应力反应的组成型诱导以及在细胞中应力条件下的ATF4，CHOP和GADD34的表达增强。此外，在分化为神经元后，突变细胞表现出降低的树突状树博化。基于我们的研究，我们提出EIF2GAMMA中的突变损害了蛋白质合成的效率和保真度，并且这种改变了对蛋白质合成的控制是MEHMO综合征的基础。 第二个主要的研究重点涉及翻译因子EIF5A，含有异常氨基酸胰岛素的独一细胞蛋白。使用分子遗传学和生化研究，我们表明EIF5A促进了翻译的伸长率，并且该活性取决于次量修饰。我们还表明，来自酵母的EIF5A（与其细菌直源性EF-P一样）刺激了含有连续脯氨酸残基的蛋白质的合成。与这些体内发现一致，我们表明EIF5A对于在体外翻译测定中重构的酵母菌中多生肽的合成至关重要。我们与约翰·霍普金斯大学（Johns Hopkins University）的研究人员合作，我们报告说，除了对多产合成的关键要求外，EIF5A在全球范围内发挥作用，以促进翻译伸长和终止。与法国的X射线晶体学家一起工作，我们获得了与酵母80S核糖体结合的EIF5A的晶体结构。 EIF5A占据了核糖体的E位点，而无uSine残留物向P位置tRNA的受体茎突出。我们的研究支持了一个模型，其中EIF5A及其无usine残基功能重新定位P位点的受体组，以增强对氨基酰基TRNA的反应性，以形成肽键或释放因子，以终止翻译。在过去的一年中，我们进一步研究了EIF5A的无偶联修饰。修饰分为两个步骤：首先，将N-丁基胺部分从精子定为EIF5A上的特定LYS侧链，然后是修饰残基的羟基化。尽管编码羟化酶的LIA1基因在酵母中是非必需的，但我们在EIF5a中鉴定了在没有羟基化的情况下引起合成表型的EIF5A突变。我们的结果与羟基修饰有助于结合和定位EIF5A及其无uSINE残基以有效促进肽基-TRNA的反应性的观念是一致的。 最近，我们将EIF5A与哺乳动物细胞中多胺代谢的调节联系起来。酶鸟氨酸脱羧酶（ODC）催化多胺合成的第一步。 ODC受一种称为抗酶的蛋白质调节，该蛋白质反过来由另一种称为抗酶抑制剂的蛋白质调节（Azin1）。多胺抑制了Azin1的合成，并且该调节取决于Azin1 mRNA领导者中保守的上游上游的开放式阅读框架（UORF样）元素。我们将元素称为UCC-对于上游保守的编码区域，因为它在Aug Start Codon上缺乏，并在接近同源密码子上启动。我们发现，高多胺从UCC的近认知起始位点增强了翻译起始，并且该调节取决于编码多肽的序列，包括高度保守的Pro-Pro-TRP（PPW）基序。我们提出扫描核糖体通常绕过UCC的近同名起始密码子而无需启动，然后翻译Azin1。但是，有时核糖体会在UCC开始密码子上启动翻译。在高聚胺的条件下，这些伸长的核糖体在PPW基序上暂停。暂停的核糖体是随后绕过接近同名起始密码子的扫描核糖体的障碍。扫描核糖体在暂停伸长的核糖体位置后面的扫描核糖体的队列在UCC的起始位置附近一个核糖体，为弱起始地点提供了更大的启动机会。与这个排队模型一致，我们发现核糖体负荷会损害核糖体负载，因此队列的形成减少了UCC的翻译和过度压制的Azin1合成。 在有关Azin1调节机制的进一步研究中，我们将EIF5A确定为对UCC翻译的多胺控制的传感器和效应器。使用重构的体外翻译测定法，我们发现PPW肽的合成（如聚生序序列的翻译）需要EIF5A。此外，多胺抑制了EIF5A刺激PPW合成的能力，可以通过增加EIF5A水平来挽救。我们建议多胺干扰核糖体上的EIF5A结合，并且抑制EIF5A是引起控制UCC翻译的核糖体暂停的触发因素。现在，我们正在探讨eIF5A的多胺调节基于编码其他酶和多胺生物合成调节剂的mRNA转化控制的可能性。 在最近的研究中，我们搜索了含有潜在UCC的其他mRNA。在哺乳动物细胞和体外的记者分析表明，编码植物GDP-L-乳糖磷酸化酶（GGP）中的UORF样元素是维生素C生物合成途径中的对照酶，是一个UCC。我们提出，在核糖体出口隧道中，维生素C与GGP UCC新生肽的相互作用会导致核糖体暂停，并排队随后的扫描核糖体导致UCC的起始增加并预防核糖体访问GGP ORF。我们认为，这种暂停的拉长核糖体通过核糖体排队在上游弱开始部位促进启动的机制可能是其他mRNA翻译的控制的基础，尤其是那些受损核糖体负载条件的翻译压力。