Mechanism and Regulation of Eukaryotic Protein Synthesis

真核生物蛋白质合成机制及调控

基本信息

批准号：
10684573
负责人：
THOMAS E DEVER
金额：
$ 203.26万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10684573
关键词：
2019-nCoV Adenosylmethionine Decarboxylase Affinity Amino Acid Motifs Amino Acids Amino Acyl Transfer RNA Anabolism Animals Binding Biochemical Biological Models Candida albicans Cell Line Cells Codon Nucleotides Collaborations Complement Country Data Defect Dependence Development Disease Elements Elongation Factor Enzymes Epilepsy Eukaryota Eukaryotic Cell Family France GTP Binding GTP-Binding Proteins Gene Expression Genes Goals Growth and Development function Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Homeobox Homeobox Genes Homeostasis Human Hypoglycemia Hypogonadism Hypopituitarism Impairment In Vitro Initiator Codon Learning Link MEHMO syndrome Maintenance Mammalian Cell Maps Mediating Memory Messenger RNA Metabolism Microcephaly Modification Molecular Molecular Genetics Mutation National Institute of Child Health and Human Development Neuronal Differentiation Nucleotides Obesity Open Reading Frames Ornithine Decarboxylase Orthologous Gene Pathogenicity Patients Peptide Initiation Factors Peptides Pharmaceutical Preparations Phase Phosphorylation Phosphotransferases Play Polyamines Post-Translational Protein Processing Proline Property Protein Biosynthesis Protein Kinase Protein Subunits Proteins Puromycin Reading Frames Regulation Reporter Reporting Research Personnel Ribosomal Frameshifting Ribosomes Roentgen Rays Role Running S-Adenosylmethionine Serine Site Spermidine Stress Structure Symptoms Syndrome System Therapeutic Intervention Trans-Activators Transfer RNA Translation Initiation Translations Universities Viral Work X-linked intellectual disability Yeasts antagonist arm base biological adaptation to stress cell growth cell growth regulation disease-causing mutation eukaryotic initiation factor-5B human disease hypusine induced pluripotent stem cell inhibitor insight interest mouse model mutant novel overexpression paralogous gene programs release factor sensor targeted treatment tool transcriptome translation factor

项目摘要

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 as well as the mRNA features and translational components that mediate the fidelity of translation start site selection. 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 and triggers the integrated stress response (ISR). Our recent studies on eIF2 have provided insights into the human disease MEHMO syndrome. 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 from multiple countries, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity as well as hypopituitarism and hypoglycemia is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2. Over the past year we have been characterizing additional novel EIF2S3 mutations identified in patients with MEHMO syndrome. This work complements our previous studies characterizing other EIF2S3 mutations linked to MEHMO syndrome and will further our understanding of the molecular defects in eIF2 that cause the varied symptoms of the disease. In previous studies we used induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome. Our studies revealed defects in general protein synthesis, constitutive induction of the ISR, and hyper-induction of the ISR under stress conditions The EIF2S3 mutation also impaired neuronal differentiation by the iPS cells. Interestingly, 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. Our current efforts are aimed at generating a mouse model of MEHMO syndrome. A second major focus is the translation factors eIF5B, a GTPase required for the last step of translation initiation joining of the large ribosomal subunit to small subunit poised on the start codon of an mRNA and eEF2, a GTPase that promotes translation elongation. In collaboration with researchers at Stanford, we helped show that eIF5B reorients the initiator Met-tRNAi to enable joining of the large ribosomal subunit. Our studies on the diphthamide modification of eEF2 revealed a critical role in reading frame maintenance including on the programmed frameshift site in SARS-CoV-2. Selection of the translation start site (typically an AUG codon) in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the factors eIF1 and eIF5. In our third major focus, we conducted a search of mammalian genes and identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. Our collaborators at the Johns Hopkins University mapped the 5 end of the Hox mRNAs, revealing that the mRNAs are much shorter than previously reported and lack proposed alternative translation elements. We found that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin lowers the fidelity of start codon selection. As the Hox genes encode developmental regulators of animal body plans, our findings reveal that alteration in start codon selection stringency has the potential to regulate global gene expression programs, including Hox gene-directed body plan formation in animals. A fourth major focus is the translation factor eIF5A and polyamines. The eIF5A is 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 for their translation both in cells and in vitro. Based, in part, on our studies with x-ray crystallographers in France, we propose eIF5A and its hypusine residue function to reposition the acceptor arm of the P site tRNA on the ribosome to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination. Interestingly, the hypusine modification on eIF5A is derived the polyamine spermidine, and we have discovered several connections between eIF5A and polyamines. We continue to study the link between eIF5A and 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. The regulation of AZIN synthesis is dependent on a conserved uORF in its mRNA. Our data indicate that polyamines interfere with eIF5A binding on the ribosome, triggering a ribosome pause that governs translation of the inhibitory uORF on the AZIN mRNA and thereby represses AZIN synthesis. We have also linked polyamine inhibition of eIF5A to stimulation of ribosomal frameshifting on the OAZ mRNA and to uORF-mediated inhibition of S-adenosylmethionine decarboxylase (AMD1) synthesis. Our studies thus identify eIF5A as a general sensor and effector for autoregulation of polyamine biosynthesis. Finally, we previously showed that Hol1 is the yeast high-affinity polyamine transporter. Together with Anirban Banerjees lab in the NICHD, we showed that purified Hol1 transports polyamines, and we found that polyamine inhibition of the translation factor eIF5A controls translation of the HOL1 mRNA. Thus, polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels. In ongoing studies, we are characterizing the HOL1 orthologs in the pathogenic yeast Candida albicans, and we are performing mutagenic structure-function studies on HOL1 to understand how the protein specifically recognizes and transports polyamines.

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，我们还研究了有助于核糖体合成蛋白质的因素的异常翻译后修饰，以及介导翻译起始位点选择的忠诚度的mRNA特征和翻译成分。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。 EIF2Alpha在丝氨酸51上的磷酸化，由胁迫响应蛋白激酶家族掩盖EIF2，将EIF2掩盖到EIF2B的抑制剂中，并触发综合应力反应（ISR）。我们最近对EIF2的研究提供了对人类疾病MEHMO综合征的见解。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 from multiple countries, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity as well as hypopituitarism and hypoglycemia is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2.在过去的一年中，我们一直在表征MEHMO综合征患者发现的其他新型EIF2S3突变。这项工作补充了我们以前的研究表征了与Mehmo综合征有关的其他EIF2S3突变，并将进一步了解EIF2中引起疾病症状各异的分子缺陷。在先前的研究中，我们使用了源自MEHMO综合征患者的诱导多能茎（IPS）细胞。我们的研究表明，在应力条件下，IIF2S3突变还损害了IPS细胞的神经元分化，ISR的一般蛋白质合成，ISR的本构诱导和ISR的过度诱导的缺陷。有趣的是，EIF2鸟嘌呤核苷酸交换因子的激活剂的药物挽救了与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。我们目前的努力旨在产生Mehmo综合征的鼠标模型。第二个主要重点是翻译因子eIF5b，这是翻译启动的最后一步所需的GTPase，将大型核糖体亚基与小亚基与mRNA和EEF2的起始密码子保持一致，这是一种促进翻译延长的GTPase。在与斯坦福大学的研究人员合作的情况下，我们帮助表明EIF5B重新启动器Met-trnai可以使大型核糖体亚基的加入。我们对EEF2的双乙酰胺修饰的研究表明，在阅读框架维护中至关重要的作用，包括在SARS-COV-2中的编程装置位点上。在真核生物中选择翻译起始位点（通常是AUG密码子）受环境核苷酸侧面的核苷酸以及EIF1和EIF5因子的水平的影响。在我们的第三个主要重点中，我们对哺乳动物基因进行了搜索，并确定了Aug密码子在保守的次优上下文中发起的五个同源基因旁系同源物以及13个hox基因，其中包含在较差的序列上下的Aug Codons启动的进化上保守的上游开放式阅读框（UORF）。我们在约翰霍普金斯大学（Johns Hopkins University）的合作者绘制了HOX mRNA的5端，表明mRNA比以前报道的短得多，并且缺乏拟议的替代翻译元素。我们发现，保守的UORF抑制HOX报告基因的表达，并且通过过表达EIF1或EIF5来改变起始密码子选择的严格度可调节HOX报告者的表达。我们还表明，通过耗尽大型核糖体亚基蛋白或用蛋白酶浓度降低细胞来改变核糖体稳态，从而降低了起始密码子选择的忠诚度。当HOX基因编码动物体计划的发育调节剂时，我们的发现表明，开始密码子选择的变化有可能调节全球基因表达程序，包括HOX基因指导的身体计划形成动物。第四个主要重点是翻译因子EIF5A和多胺。 EIF5a是含有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）对EIF5A在细胞和体外的翻译都具有增强的依赖性。在某种程度上，基于我们在法国的X射线晶体学家的研究中，我们提出EIF5A及其无uSine残留功能，以重新定位核糖体对P位点tRNA的受体臂的重新定位，以增强对氨基酰基-TRNA的反应性，以提高对肽键的形成或释放因子的反应性。有趣的是，EIF5A上的次肌修饰是多胺精子的，我们发现了EIF5A和多胺之间的几个连接。我们继续研究EIF5A与哺乳动物细胞中多胺代谢的调节之间的联系。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。在抑制氮杂合成的同时，多胺刺激了OAZ的合成。阿唑合成的调节取决于其mRNA中保守的UORF。我们的数据表明，多胺干扰了核糖体上的EIF5A结合，引发了核糖体暂停，该核糖体暂停，该核糖体在azin mRNA上控制抑制性uorf的翻译，从而抑制了阿津蛋白的合成。我们还将对eIF5A的多胺抑制与刺激OAZ mRNA上的核糖体框架以及UORF介导的抑制S-腺苷甲硫代氨基氨酸脱羧酶（AMD1）合成。因此，我们的研究将EIF5A鉴定为多胺生物合成自动调节的一般传感器和效应子。最后，我们先前表明Hol1是酵母高亲和力多胺转运蛋白。我们与NICHD中的Anirban Banerjees实验室一起表明，纯化的HOL1传输了多胺，并且发现多胺抑制翻译因子EIF5A控制了HOL1 mRNA的翻译。因此，多胺类似多胺的生物合成，在酵母中被多胺的转化自动调节，强调了广泛的控制细胞对多胺水平施加。在正在进行的研究中，我们正在表征致病性酵母白色念珠菌中的Hol1直系同源物，并且我们正在对HOL1进行诱变结构 - 功能研究，以了解蛋白质如何特异性地识别和运输多胺。