Mechanism and Regulation of Eukaryotic Protein Synthesis

真核生物蛋白质合成机制及调控

• 批准号: 10266469
• 负责人: THOMAS E DEVER
• 金额: $ 201.27万
• 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/10266469
• 关键词: Amines; Amino Acid Motifs; Amino Acids; Amino Acyl Transfer RNA; Anabolism; Antiviral Agents; Ascorbic Acid; Binding; Biochemical; Biological Models; Bypass; Cell Line; Cells; Central obesity; Clinical; Code; Codon Nucleotides; Collaborations; Defect; Dependence; Diabetes Mellitus; Disease; Elements; Elongation Factor; Enzymes; Epilepsy; Eukaryotic Cell; Exhibits; Family; France; GTP Binding; GTP-Binding Proteins; Galactose; Gene Expression; Genes; Genetic; Germany; Goals; Growth and Development function; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Homeostasis; Human; Hydroxyl Radical; Hydroxylation; Hypogonadism; Impairment; In Vitro; Initiator Codon; Israel; Learning; Link; MEHMO syndrome; Mammalian Cell; Medical center; Memory; Messenger RNA; Metabolism; Microcephaly; Military Personnel; Mixed Function Oxygenases; Modeling; Modification; Molecular; Molecular Genetics; Motor; Mutation; Neuronal Differentiation; Neurons; Obesity; Open Reading Frames; Ornithine Decarboxylase; Pathway interactions; Patients; Peptide Initiation Factors; Peptides; Pharmaceutical Preparations; Phase; Phenotype; Phosphorylase a; Phosphorylases; Phosphorylation; Phosphotransferases; Plants; Play; Polyamines; Positioning Attribute; Post-Translational Protein Processing; Proline; Property; Protein Biosynthesis; Protein Kinase; Proteins; Regulation; Reporting; Research Personnel; Ribosomes; Roentgen Rays; Role; Running; Scanning; Serine; Side; Site; Slovakia; Spermidine; Stress; Structure; Symptoms; Syndrome; System; Therapeutic Intervention; Trans-Activators; Transfer RNA; Translating; Translation Initiation; Translations; United Kingdom; Universities; Viral; X-linked intellectual disability; Yeast Model System; Yeasts; arm; base; biological adaptation to stress; cell growth; cell growth regulation; disease-causing mutation; experimental study; human disease; hypusine; induced pluripotent stem cell; inhibitor/antagonist; insight; interest; mutant; novel; peptidyl-tRNA; 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, the United Kingdom 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 (2). These new mutations, which lie in the G domain of eIF2gamma, impaired yeast cell growth, altered translation and reduced stringency of translation start site selection. Our collaborators in Germany linked the EIF2S3 mutations with variable levels of motor delay, microcephaly, ID, epilepsy, central obesity and diabetes, thus broadening the genetic spectrum and clinical expressivity of MEHMO syndrome. More recently, we studied induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome (1). 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. Addition of the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescued the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for MEHMO syndrome (1). A second major focus is the translation factor eIF5A, the sole cellular protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we previously showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. Moreover, certain amino acid motifs like runs of consecutive proline residues showed a heightened dependency on eIF5A both in cells and in vitro. 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 found that 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. In ongoing studies, we are further investigating 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 indicate 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 (OAZ), which, in turn, is regulated by another protein called antizyme inhibitor (AZIN). The synthesis of OAZ is stimulated by polyamines while AZIN synthesis is inhibited by polyamines. The regulation of AZIN synthesis is dependent on a conserved upstream open reading frame-like (uORF-like) element in the leader of the AZIN mRNA. We refer to this 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 indirectly enhance translation initiation from the near-cognate start site of the uCC by pausing translation elongation within the uCC at a highly conserved Pro-Pro-Trp (PPW) motif. We proposed that scanning ribosomes typically bypass the near-cognate start codon of the uCC and then translate AZIN. However, occasionally a ribosome initiates 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 AZIN 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. In ongoing studies, we have also linked polyamine inhibition of eIF5A to translational control of OAZ synthesis, suggesting that eIF5A might be a general sensor for autoregulation of polyamine biosynthesis. In ongoing studies we have identified a novel uCC in the mRNA encoding plant GDP-L-galactose phosphorylase (GGP), a control enzyme in the vitamin C biosynthetic pathway. In vitro and mammalian cell experiments revealed that this uCC senses vitamin C. 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 triggers increased initiation on the uCC and prevents ribosome access to the GGP ORF. We hypothesize that this uCC mechanism, whereby a paused elongating ribosome promotes initiation at an upstream weak start site via ribosome queuing, may underlie the uORF control of translation of other mRNAs.

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, the United Kingdom 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综合征相关的另外两个EIF2S3突变的酵母模型（2）。这些新突变位于EIF2GAMMA的G结构域，损害了酵母细胞的生长，改变了翻译的翻译和降低的翻译开始位点选择的严格度。我们在德国的合作者将EIF2S3突变与运动延迟，小头畸形，ID，癫痫，中央肥胖和糖尿病联系起来，从而扩大了MEHMO综合征的遗传谱和临床表达性。 最近，我们研究了源自MEHMO综合征患者的诱导多能茎（IPS）细胞（1）。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。添加了ISRIB的添加是EIF2鸟嘌呤核苷酸交换因子的激活剂，挽救了与EIF2S3突变相关的细胞生长，翻译和神经元分化缺陷，为MEHMO综合征提供了治疗性干预的可能性（1）。 第二个主要重点是翻译因子EIF5A，即包含异常氨基酸次氨酸的唯一细胞蛋白。使用分子遗传和生化研究，我们先前表明EIF5A促进了翻译的伸长率，并且该活性取决于次次修饰。此外，某些氨基酸基序（例如连续的脯氨酸残基运行）在细胞和体外对EIF5A的依赖性增强。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射线晶体学家合作，我们发现EIF5A占据了核糖体的E位点，而无素残基向P位置tRNA的受体茎投射了。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。在正在进行的研究中，我们正在进一步研究EIF5A上的无偶联修饰。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。我们的结果表明，羟基修饰有助于结合和定位EIF5A及其无uSine残基，从而有效促进肽基-TRNA的反应性。 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（OAZ）的蛋白质调节，而蛋白质又受到另一种称为抗酶抑制剂（Azin）的蛋白质调节。多胺刺激了OAZ的合成，而多胺则抑制了Azin合成。阿赞合成的调节取决于azin mRNA领导者中的保守上游开放式阅读框架（UORF样元素）。我们将此元素称为UCC-对于上游保守的编码区域，因为它在Aug Start Codon中缺乏并在几乎同源密码子上启动。我们发现，高聚胺通过在高度保守的Pro-Pro-TRP（PPW）基序中暂停UCC的翻译伸长来间接增强UCC的近认知起始位点的翻译起始。我们提出扫描核糖体通常绕过UCC的近同名起始密码子，然后翻译Azin。但是，有时核糖体在UCC起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。 在有关阿赞调节机制的进一步研究中，我们将EIF5A确定为多胺控制UCC翻译的传感器和效应器。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。在正在进行的研究中，我们还将eIF5A的多胺抑制与OAZ合成的转化控制联系起来，这表明EIF5A可能是多胺生物合成自动调节的一般传感器。 在正在进行的研究中，我们在编码植物GDP-L-半乳糖磷酸化酶（GGP）的mRNA中确定了一种新颖的UCC，这是一种维生素C生物合成途径中的对照酶。 In vitro and mammalian cell experiments revealed that this uCC senses vitamin C. 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 triggers increased initiation on the uCC and prevents ribosome access to the GGP ORF.我们假设这种UCC机制通过核糖体排队在上游弱开始部位促进启动的UCC机制可能是其他mRNA翻译的UORF控制。