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.
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