National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 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
  • Ivaylo P. Ivanov, PhD, Research Fellow
  • Margarito Rojas, PhD, Visiting Fellow
  • Chune Cao, Biological Laboratory Technician
  • Erik Gutierrez, BS, Graduate Student
  • Jason A. Murray, 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 to dissect the structure-function properties of 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. Our recent studies elucidated how an eIF2gamma mutation that is associated with intellectual disability impairs eIF2 function and revealed how eIF2 binds to methionyl-tRNA and the ribosome. Our studies also demonstrated that the hypusine-containing protein eIF5A promotes translation elongation by stimulating the peptidyl transferase activity of the ribosome and facilitating the reactivity of poor substrates such as proline.

Molecular analysis of eIF2alpha phosphorylation, dephosphorylation, and viral regulation

The translation factor eIF2 is composed of three distinct subunits. 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 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 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. Back-to-back dimerization enables each PKR protomer to engage a molecule of eIF2alpha in the crystal structure. Using site-directed mutagenesis studies, we demonstrated that a common mode of back-to-back dimerization is required for activation of PKR, GCN2, and PERK, and we proposed an ordered mechanism of PKR activation by which catalytic domain dimerization triggers autophosphorylation, which in turn is required for specific eIF2alpha substrate recognition. Our mutagenesis studies revealed that the position of the Ser51 residue in free eIF2alpha is restricted and that docking of eIF2alpha onto PKR helix alphaG disrupts a hydrophobic network and induces a conformational change that enables Ser51 to move by about 20 Å to engage the phospho-acceptor binding site of the kinase. We propose that the protected state of Ser51 in free eIF2alpha prevents promiscuous phosphorylation and attendant translational regulation by heterologous kinases, yet enables Ser51 phosphorylation upon binding of eIF2alpha to one of the canonical eIF2alpha kinase.

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; however, as there are no homologs of these targeting subunits in yeast, it was unclear how GLC7, the functional homolog of PP1 in yeast, is recruited to dephosphorylate eIF2alpha. We recently showed that a novel N-terminal extension on yeast eIF2gamma binds to GLC7 and targets it to dephosphorylate eIF2alpha. Truncation or point mutations designed to eliminate the PP1–binding motif in eIF2gamma impaired eIF2alpha dephosphorylation both in vivo and in vitro. Moreover, replacement of the N-terminus of eIF2gamma with the GLC7–binding domain from GAC1 or fusion of heterologous dimerization domains to eIF2gamma and GLC7 maintained eIF2alpha phosphorylation at basal levels. Taken together, our results indicate that, in contrast to the paradigm of distinct PP1 targeting or regulatory subunits, the unique N-terminus of yeast eIF2gamma functions in cis to target GLC7 to dephosphorylate eIF2alpha (Reference 1).

Over the last year, we continued our studies on eIF2alpha dephosphorylation and reconstituted human GADD34 function in yeast cells. We mapped a novel eIF2α-binding motif to the C-terminus of GADD34 in a region distinct from where PP1 binds to GADD34. Point mutations altering the 19–residue eIF2α–binding motif impaired the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, the eIF2α–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 eIF2α dephosphorylation. Taken together, our data demonstrate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs (Reference 2).

When expressed in yeast, human PKR phosphorylates the alpha subunit of eIF2 on Ser51, causing inhibition of protein synthesis and yeast cell growth. To subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. The poxviral protein E3L binds to double-stranded RNA and inhibits PKR by sequestering activators and forming heterodimers with the kinase. We previously showed that a Z-DNA–binding domain near the N-terminus of E3L, but not its Z-DNA–binding activity, is critical for E3L inhibition of PKR. We are currently characterizing mutations in PKR that confer resistance to E3L inhibition. Similarly, we previously discovered that the insect baculovirus PK2 protein is an eIF2alpha kinase inhibitor. PK2 structurally mimics the C-terminal lobe of a protein kinase domain. Using a genetic screen in yeast, and together with collaborators in Canada and Japan, we characterized mutations that enhance the ability of PK2 to inhibit eIF2 kinases. The mutations cluster to a surface of PK2 that, in bona fide protein kinases, forms the catalytic cleft through interactions with a kinase N-lobe. Yeast two-hybrid and protein-interaction assays revealed that PK2 associates with the N-lobe of PKR. Using yeast-based assays, we showed that PK2 was most effective in inhibiting an insect HRI–like kinase, and our collaborators showed that knockdown of the HRI–like kinase in insects rescued viral defects associated with loss of PK2. We propose an inhibitory mechanism whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism (Reference 3).

Analysis of an eIF2gamma mutation that links intellectual disability with impaired translation initiation

We have a long-standing interest in the structure-function properties of eIF2, and recently our studies have enabled us to provide insights into human disease. While protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Moreover, the consequences of partial loss of eIF2gamma function or eIF2 integrity are unknown in mammals, including humans. Our collaborators Lina Basel-Vanigaite and Guntram Borck identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Biochemical studies of human cells overexpressing the eIF2gamma mutant and of yeast eIF2gamma with the analogous mutation revealed a defect in binding of the eIF2beta subunit to eIF2gamma. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2gamma impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2beta. The findings directly link intellectual disability with impaired translation initiation and provide a mechanistic basis for the human disease as a result of partial loss of eIF2 function (Borck et al., Mol Cell 2012;48:641-646). Over the past year, we have been characterizing new mutations in eIF2gamma that cause intellectual disability.

Molecular analysis of the hypusine-containing protein eIF5A

The translation factor eIF5A, the only 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 is dependent on the hypusine modification. Given that eIF5A is a structural homologue of the bacterial protein EF-P, we proposed that eIF5A/EF-P is a universally conserved translation elongation factor.

Recently, it was shown that EF-P promotes translation of polyproline sequences by bacterial ribosomes. Using in vivo reporter assays, we showed that eIF5A in yeast stimulates the synthesis of proteins containing runs of three or more consecutive proline residues. Consistently, the expression of native yeast proteins containing homopolyproline sequences was impaired in eIF5A mutant strains. To support the in vivo findings, we used reconstituted yeast in vitro translation assays to monitor the impact of eIF5A on protein synthesis. We found that the synthesis of polyproline peptides, but not of polyphenylalanine peptides, was critically dependent on addition of eIF5A. Toe-printing experiments revealed that addition of eIF5A relieved ribosomal stalling during translation of three consecutive proline residues in vitro. Consistent with the functions of eIF5A in promoting peptide bond synthesis, directed hydroxyl radical probing experiments localized eIF5A binding to near the E site of the ribosome, with the hypusine residue of eIF5A adjacent to the acceptor stem of the P-site tRNA. Thus, we propose that eIF5A, like its bacterial orthologue EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates such as proline (Reference 4).

Analysis of HAC1 mRNA translational control

In addition to studying translation factors and regulators, we also studied the impact of mRNA structure on translation. The HAC mRNA in yeast encodes a transcription factor that up-regulates genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the leader of the HAC1 mRNA represses Hac1 protein production under basal conditions. An unusual cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under conditions of ER stress, relieves the inhibition and enables Hac1 synthesis. Using random and site-directed mutations, we showed that disruption of the base-pairing interactions derepresses translation of the unspliced HAC1 mRNA. With our collaborator Madhusudan Dey, we also showed that insertion of an in-frame AUG start codon upstream of the base-pairing interaction releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of the translation initiation factor eIF4A, a helicase, enhanced production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. As the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes, we conclude that the 5′ UTR–intron interaction represses translation initiation on the unspliced HAC1 mRNA (Reference 5).

Publications

  1. Rojas M, Gingras AC, Dever TE. Protein phosphatase PP1/GLC7 interaction domain in yeast eIF2 bypasses targeting subunit requirement for eIF2 dephosphorylation. Proc Natl Acad Sci USA 2014; 111:E1344-1353.
  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. Li JJ, Cao C, Fixsen SM, Young JM, Ono C, Bando H, Elde NC, Katsuma S, Dever TE, Sicheri F. Baculovirus protein PK2 subverts eIF2alpha kinase function by mimicry of its kinase domain C-lobe. Proc Natl Acad Sci USA 2015; 112:E4364-E4373.
  4. Gutierrez E, Shin BS, Woolstenhulme CJ, Kim JR, Saini P, Buskirk AR, Dever TE. eIF5A promotes translation of polyproline motifs. Mol Cell 2013; 51:35-45.
  5. Sathe L, Bolinger C, Mannan MA, Dever TE, Dey M. Evidence that base-pairing interaction between intron and mRNA leader sequences inhibits initiation of HAC1 mRNA translation in yeast. J Biol Chem 2015; 290:21821-21832.

Collaborators

  • Lina Basel-Vanigaite, MD, Tel Aviv University, Tel Aviv, Israel
  • Guntram Borck, MD, PhD, Universität Ulm, Ulm, Germany
  • Madhusudan Dey, PhD, University of Wisconsin-Milwaukee, Milwaukee, WI
  • Alan Hinnebusch, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
  • Susumu Katsuma, PhD, University of Tokyo, Tokyo, Japan
  • Frank Sicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada

Contact

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

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