Mechanism and Regulation of Eukaryotic Protein Synthesis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the control of translation start site selection, the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation, and the function of 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 converts eIF2 into an inhibitor of eIF2B, thereby reducing eIF2 activity and triggering the Integrated Stress Response (ISR). Our recent studies on eIF2 have provided insights into the human disease MEHMO syndrome. We previously showed that the human X-linked intellectual disability (XLID) called MEHMO syndrome is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2. We have generated yeast models of different patient mutations to confirm that the mutations are pathogenic and to reveal how the mutations impair eIF2 function. Using induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome, we previously revealed that the mutation impaired general protein synthesis, induced the Integrated Stress Response (ISR), and impaired neuronal differentiation of 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. In addition to intellectual disability, patients with MEHMO syndrome exhibit epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. In addition, some patients exhibit hypopituitarism and hypoglycemia. Over the past year we have been characterized an additional novel EIF2S3 mutation identified in a patient with MEHMO syndrome that appears to alter eIF2 function by disrupting the assembly of the eIF2 complex. We have also generated and continued our characterization of a mouse model of MEHMO syndrome to further study this ultrarare disease and to test the efficacy of ISRIB and related therapeutics. Initial characterization of the mouse models has revealed pan-hypopituitarism, small size, muscle weakness, and a rudimentary reproductive tract in the mutants, consistent with phenotypes observed in patients with MEHMO syndrome. Interestingly, several of the MEHMO mouse phenotypes are shared in a mouse model carrying mutations in an eIF2 phosphatase subunit (Reineke et al), suggesting that both diseases are caused by persistent ISR activation. A second major research focus has been the translational GTPases eIF5B, required for joining the large ribosomal subunit to the small subunit poised on the mRNA start codon, and eEF2, required for translation elongation. Whereas we previously helped show that eIF5B reorients the initiator Met-tRNAi to enable joining of the large ribosomal subunit, we are currently characterizing mutations in eIF5B that cause ribosomes to scan past the normal start codon and to initiate at downstream AUG codons instead. Our prior studies on eEF2 revealed the role of the novel diphthamide post-translational modification on the factor. The disease diphtheria is caused by a bacterial toxin that ADP-ribosylates the diphthamide residue to inactivate eEF2. We found that diphthamide helps maintain translational fidelity by preventing spurious frameshifting by ribosomes during elongation, and we propose that the modification has been conserved through evolution to maintain translational fidelity despite conferring sensitivity to bacterial toxins. A third major research focus involves the translation factor eIF5A and polyamines. The eIF5A is the sole cellular protein containing the unusual amino acid hypusine. We previously showed that eIF5A promotes translation elongation and termination, with certain amino acid motifs like polyproline sequences showing a heightened requirement for eIF5A. In ongoing studies, we are characterizing yeast eIF5A mutants that show heightened dependency on the second (hydroxylation) step of hypusine biosynthesis catalyzed by the enzyme DOHH. Our studies indicate that hypusine contributes to functional binding of eIF5A to the ribosome, and we are exploiting these yeast mutants to characterize human mutations in DOHH associated with a novel neurodevelopmental disorder. In additional ongoing studies, we have identified eIF5A as a sensor and effector for polyamine control of translation of mRNAs encoding enzymes or regulators of polyamine biosynthesis. The first step in polyamine synthesis is catalyzed by ornithine decarboxylase (ODC). ODC is inhibited by the protein antizyme (OAZ), which, in turn, is regulated by the protein antizyme inhibitor (AZIN). We previously showed that polyamines interfere with eIF5A function on the ribosome, causing ribosomes to pause on a uORF in the AZIN mRNA and thereby repress AZIN synthesis. Recently, we 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. We also showed that polyamine inhibition of eIF5A controls translation of the yeast HOL1 mRNA, which, together with Anirban Banerjee in NICHD, we showed encodes the high-affinity polyamine transporter in fungi. More recently, in collaboration with Michail Lionakis in NIAID, we found that combined inhibition of polyamine synthesis and transport (Hol1) prevents hyphal growth by the pathogenic fungi C. albicans and suppresses C. albicans virulence in a mouse infection model. Selection of the start codon is a crucial step for synthesis of the proper proteins. The small ribosomal subunit with bound Met-tRNAi associates with an mRNA at the 5’ cap and then scans down the mRNA in search of a start codon. While translation typically initiates at an AUG codon, near-cognate codons that differ from AUG by a single nucleotide can sometimes be used, albeit with lower efficiency. Selection of the translation start sites is influenced by context nucleotides flanking the start codon and by levels of the factors eIF1 and eIF5. In a recent collaborative study (Grosely et al), we showed that overexpression of eIF1 or knockdown of eIF5 reduced initiation at near-cognate start codons whereas knockdown of eIF1 and overexpression of eIF5 had the opposite effects. Consistent with these findings, eIF1 and eIF5 were found to rapidly and transiently sample initiation complexes providing a rationale for how start-site selection is tuned to the levels of these two factors. Finally, in a previous study, we identified a collection of mammalian genes including several homeobox (Hox) gene paralogs in which the main ORF or a regulatory upstream open reading frame (uORF) is initiated by an AUG codon in conserved suboptimal context. As these features sensitize translation to the levels of eIF1 and eIF5, we hypothesize that alterations in start codon selection stringency could contribute to developmental or tissue-specific regulation of Hox gene-directed body plan formation in animals as well as other global gene expression programs.