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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：
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
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/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

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. 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.