National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Mechanism and Regulation of Eukaryotic Protein Synthesis

Tom Dever
  • Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
  • Byung-Sik Shin, PhD, Staff Scientist
  • Chune Cao, Biological Laboratory Technician
  • Ivaylo P. Ivanov, PhD, Research Fellow
  • Arya Vindu, PhD, Visiting Fellow
  • Sara Young-Baird, PhD, Postdoctoral Research Associate Program (PRAT) Fellow
  • Leda Lotspeich-Cole, BS, Graduate Student
  • Joo-Ran Kim, BS, Special Volunteer

We study the mechanism and regulation of protein synthesis, focusing on GTPases and protein kinases that control this fundamental cellular process. We use molecular-genetic and biochemical studies in yeast and human cells to dissect the structure-function properties of translation factors, elucidate mechanisms that control protein synthesis, and characterize how mutations in the protein synthesis apparatus cause human disease. Of special interest are the translation initiation factors eIF2, a GTPase that binds methionyl-tRNA to the ribosome, and eIF5B, a second GTPase that catalyzes ribosomal subunit joining in the final step of translation initiation. We also investigate stress-responsive protein kinases that phosphorylate eIF2alpha, viral regulators of these kinases, and how cellular phosphatases are targeted to dephosphorylate eIF2alpha. We are characterizing eIF2gamma mutations that are associated with the MEHMO syndrome, a novel X-linked intellectual disability syndrome, and we are investigating the function of the translation factor eIF5A with a focus on its ability to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates such as proline. We are also examining the role of the hypusine modification on eIF5A and the role of this factor in gene-specific translational control mechanisms.

Analysis of eIF2gamma mutations that link intellectual disability with impaired translation initiation

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 conducted in collaboration with Lina Basel-Vanagaite and Guntram Borck, we described a human XLID syndrome characterized by intellectual disability and microcephaly. The patients carry a mutation in the EIF2S3 gene, which encodes eIF2gamma, and genetic and biochemical studies revealed that the mutation disrupts eIF2 complex integrity and translation start-codon selection. Over the past year, working in collaboration with Vera Kalscheuer, Daniela Gasperikova, and Clesson Turner, we characterized two additional mutations in eIF2gamma found in patients exhibiting intellectual (mental) disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. Based on this constellation of phenotypes, the disease has been termed the MEHMO syndrome, and we now conclude that the syndrome is caused by mutations in EIF2S3. Our studies on a yeast model of these newly described MEHMO syndrome mutations in eIF2gamma reveal impaired eIF2 function, altered translational control of specific mRNAs, and reduced stringency of translation start-site selection (Reference 1). Consistent with these properties, the Integrated Stress Response, a translational regulatory response typically associated with eIF2alpha phosphorylation, is induced in patient cells. The findings directly link intellectual disability with impaired translation initiation and provide a mechanistic basis for the MEHMO syndrome resulting from partial loss of eIF2 function (Reference 1). Based on our studies, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of symptoms.

Molecular analysis of eIF2alpha phosphorylation, dephosphorylation, and viral regulation

Phosphorylation of the eIF2alpha subunit is a common mechanism for down-regulating protein synthesis under stress conditions. Four distinct kinases phosphorylate eIF2alpha on Ser51 under different cellular stress conditions: GCN2 responds to amino-acid limitation, HRI to heme deprivation, PERK to ER (endoplasmic reticulum) stress, and PKR to viral infection. Consistent with their common activity to phosphorylate eIF2alpha on Ser51, the kinases show strong sequence similarity in their kinase domains. Phosphorylation of eIF2alpha converts eIF2 from a substrate to an inhibitor of its guanine-nucleotide exchange factor eIF2B. The inhibition of eIF2B impairs general translation, slowing the growth of yeast cells and, paradoxically, enhancing the translation of the GCN4 mRNA (GCN4 is a transcription factor) required for yeast cells to grow under amino-acid starvation conditions.

We previously used structural, molecular, and biochemical studies to define how the eIF2alpha kinases recognize their substrate. In collaboration with Frank Sicheri, we obtained the X-ray structure of eIF2alpha bound to the catalytic domain of PKR and elucidated an ordered mechanism of PKR activation by which catalytic domain dimerization triggers auto-phosphorylation, which in turn is required for specific eIF2alpha substrate recognition.

While the protein kinases GCN2, HRI, PKR, and PERK specifically phosphorylate eIF2alpha on Ser51 to regulate global and gene-specific mRNA translation, eIF2alpha is dephosphorylated by the broadly acting serine/threonine protein phosphatase 1 (PP1). In mammalian cells, the regulatory subunits GADD34 and CReP target PP1 to dephosphorylate eIF2alpha. We showed that a novel N-terminal extension on yeast eIF2gamma binds to yeast PP1 (GLC7) and targets GLC7 to dephosphorylate eIF2alpha. Moreover, we reconstituted human GADD34 function in yeast cells and mapped a novel eIF2alpha–binding motif to the C-terminus of GADD34 (Reference 2). Interestingly, the eIF2alpha–docking motif is conserved among several viral orthologs of GADD34, and we showed that it is necessary for the proteins produced by African swine fever virus, canarypox virus, and herpes simplex virus to promote eIF2alpha dephosphorylation. Taken together, our data demonstrate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2alpha by interacting with both PP1 and eIF2alpha through independent binding motifs (Reference 2).

The eIF2alpha kinase PKR is part of the cellular antiviral defense mechanism. When expressed in yeast, human PKR phosphorylates eIF2alpha, resulting in inhibition of protein synthesis and yeast cell growth. To subvert the antiviral defense mediated by PKR, viruses produce inhibitors of the kinase. We are studying the inhibition of PKR by the poxviral double-stranded RNA–binding protein E3L, and we are currently characterizing mutations in PKR that confer resistance to E3L inhibition. In a related project, we characterized the insect baculovirus PK2 protein, an eIF2alpha kinase inhibitor that structurally mimics the C-terminal lobe of a protein kinase domain. Together with collaborators Frank Sicheri and Susuma Katsuma, we revealed that PK2 targets an insect HRI–like kinase through an unusual lobe-swapping mechanism to generate a nonfunctional pseudo-kinase complex.

Molecular analysis of the hypusine-containing protein eIF5A

The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine], was originally identified based on its ability to stimulate a model assay for first peptide-bond synthesis. However, the precise cellular role of eIF5A was unknown. Using molecular-genetic and biochemical studies, we previously showed that eIF5A promotes translation elongation and that this activity depends on the hypusine modification. Given that eIF5A is a structural homolog of the bacterial protein EF-P, we proposed that eIF5A/EF-P is a universally conserved translation elongation factor.

Using in vivo reporter assays, we showed that eIF5A in yeast, like bacterial EF-P, stimulates the synthesis of proteins containing runs of three or more 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, and, using directed hydroxyl radical probing experiments, we mapped eIF5A binding to near the E site of the ribosome. Thus, we proposed that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates such as proline.

Over the past year, in collaboration with Rachel Green, we reported that eIF5A functions globally to promote both translation elongation and termination. Moreover, exploiting our in vitro reconstituted assay system, we used mis-acylated tRNAs to show that the imino acid proline but not tRNA(Pro) imposes the requirement for eIF5A. In addition, we found that the more flexible proline analog azetidine-2-carboxylic acid relaxes the eIF5A requirement for peptide synthesis. We also found that eIF5A could functionally substitute for polyamines to stimulate general protein synthesis (Reference 3).

Over the last year, and working with the X-ray crystallographer Marat Yusupov, we obtained a 3.25 Å–resolution crystal structure of eIF5A bound to the yeast 80S ribosome (Reference 4). The structure reveals interactions between eIF5A and conserved ribosomal proteins and rRNA bases. Moreover, eIF5A occupies the E site of the ribosome, with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies reveal a previously unseen conformation of an eIF5A–ribosome complex, suggest a function for eIF5A and its hypusine residue in repositioning the peptidyl-tRNA in the P site to alleviate stalling, and highlight a possible functional link between conformational changes of the ribosome during protein synthesis and eIF5A–ribosome association (Reference 4). In related studies, we reported the structure of a diproline–tRNA analog bound to the ribosome, revealing that proline affects nascent peptide positioning in the ribosome exit tunnel.

Taken together, our studies support a model in which eIF5A and its hypusine residue function to reposition the acceptor arm of polyprolyl–tRNA in the P site to alleviate stalling and that the body of eIF5A functions like polyamines to enhance general protein synthesis. In ongoing studies, we have linked eIF5A to the regulation of polyamine metabolism in mammalian cells. Synthesis of the antizyme inhibitor AZIN1, a positive regulator of polyamine synthesis, is inhibited by polyamines. We found that translational control of AZIN1 synthesis relies on polyamine inhibition of eIF5A function. Thus, eIF5A functions generally in protein synthesis, and modulation of eIF5A function by polyamines can be exploited to regulate specific mRNA translation.

Analysis of the role of eEF2 and its diphthamide modification in translation elongation and CrPV IRES translation

We are also studying the translation elongation factor eEF2. Like its bacterial ortholog EF-G, eEF2 promotes translocation of tRNAs and mRNA from the A site to the P site on the ribosome following peptide bond formation. In all eukaryotes and archaea, a conserved histidine residue at the tip of eEF2 is post-translationally modified to diphthamide through the action of seven non-essential proteins. The function of diphthamide and rationale for its evolutionary conservation are not well understood, and to date the only known function of diphthamide is to serve as a substrate for inactivation by diphtheria toxin. To gain insights into the role of eEF2 and diphthamide, we examined peptide synthesis in a reconstituted yeast in vitro translation system using unmodified eEF2 or eEF2 containing the diphthamide modification. Using the canonical initiation pathway to direct the synthesis of the peptide Met-Phe-Lys revealed no distinction between unmodified eEF2 and eEF2 with the diphthamide modification. In contrast, synthesis of the same peptide directed by the novel cricket paralysis virus (CrPV) internal ribosome entry site (IRES), which byasses canonical translation initiation and instead requires novel eEF2-directed pseudo-translocation reactions prior to peptide bond formation, was sensitive to the loss of diphthamide. As the pseudo-translocation steps are the main distinguishing feature of the CrPV IRES system, we propose that the precise phasing of pseudo-translocation, in which a tRNA–mimicking RNA element from the virus is translocated through the ribosome, is dependent on the diphthamide modification on eEF2.

Consistent with this interpretation, using electron cryomicroscopy, our collaborators in Venki Ramakrishnan’s lab in Cambridge revealed that eEF2 interacts with the CrPV–IRES on the ribosome and stabilizes the IRES in a conformation reminiscent of a hybrid tRNA state. Interestingly, diphthamide appears to interact directly with the tRNA–mimicking element of the CrPV IRES, perhaps to facilitate its precise translocation in the ribosome and to break decoding interactions between conserved rRNA bases and the IRES. Thus, our studies provide the first evidence that diphthamide plays a role in protein synthesis, and we propose that diphthamide functions to disrupt the decoding interactions of rRNA in the A site and to maintain codon-anticodon interactions as the A-site tRNA is translocated to the P site (Reference 5).

Publications

  1. Skopkova M, Hennig F, Shin BS, Turner CE, Stanikova D, Brennerova K, Stanik J, Fischer U, Henden L, Müller U, Steinberger D, Leshinsky-Silver E, Bottani A, Kurdiova T, Ukropec J, Nyitrayova O, Kolnikova M, Klimes I, Borck G, Bahlo M, Haas SA, Kim JR, Lotspeich-Cole LE, Gasperikova D, Dever TE, Kalscheuer VM. EIF2S3 mutations associated with severe X-linked intellectual disability syndrome MEHMO. Hum Mutat 2017 38:409-425.
  2. Rojas M, Vasconcelos G, Dever TE. An eIF2alpha-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2alpha. Proc Natl Acad Sci USA 2015 112:E3466-3475.
  3. Shin BS, Katoh T, Gutierrez E, Kim JR, Suga H, Dever TE. Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis. Nucleic Acids Res 2017 45:8392-8402.
  4. Melnikov S, Mailliot J, Shin BS, Rigger L, Yusupova G, Micura R, Dever TE, Yusupov M. Crystal structure of hypusine-containing translation factor eIF5A bound to a rotated eukaryotic ribosome. J Mol Biol 2016 428:3570-3576.
  5. Murray J, Savva CG, Shin BS, Dever TE, Ramakrishnan V, Fernández IS. Structural characterization of ribosome recruitment and translocation by type IV IRES. Elife 2016 5:e13567.

Collaborators

  • Lina Basel-Vanagaite, MD, Tel Aviv University, Tel Aviv, Israel
  • Guntram Borck, MD, PhD, Universität Ulm, Ulm, Germany
  • Daniela Gasperikova, PhD, Slovak Academy of Sciences, Bratislava, Slovakia
  • Rachel Green, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
  • Vera Kalscheuer, PhD, Max Planck Institute for Molecular Genetics, Berlin, Germany
  • Susumu Katsuma, PhD, University of Tokyo, Tokyo, Japan
  • Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
  • Frank Sicheri, PhD, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
  • Hiroaki Suga, PhD, University of Tokyo, Tokyo, Japan
  • Clesson Turner, MD, Walter Reed National Military Medical Center, Bethesda, MD
  • Marat Yusupov, PhD, L'Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Strasbourg, France

Contact

For more information, email thomas.dever@nih.gov or visit http://deverlab.nichd.nih.gov.

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