We propose investigating basic principles of eukaryotic translation initiation using molecular biology and nuclear magnetic resonance techniques. Regulation of translation is a crucial aspect of benign and malignant cell growth. In this proposal we plan to study proteins that are key factors of translation initiation, their interaction with mRNA and principles of the regulation of translation initiation as mediated by protein phosphorylation. The research will be focused on the eukaryotic translation initiation factor eIF4E, its interaction with the mRNA m7GpppX cap structure and with other proteins. The protein eIF-4E is a central factor of translation initiation; its importance for translation is comparable to that of the TATA binding protein (TBP) for transcription. It shows no homology to any protein with known 3D structure. Phosphorylation of initiation factors plays an important role in the regulation of translation initiation. For example, insulin-stimulated cell growth is related to phosphorylation (apparently via the MAP kinase pathway) of an elF-4E binding protein that will be studied in this project as well (4E- BP2). In the first phase of the proposed research the solution structure of elF4E will be solved, complexed with the minimum-size cap structures, m7GpppG and m7Gpp. The effect of phosphorylating Ser-53 on the structure and function of eIF-4E will also be studied. A family of recently identified eIF-4E binding proteins (4E-BP1, 4E-BP2 and PHAS-I) has been shown to inhibit translation initiation by binding to elF4E. Expression systems for these proteins of ca. 118 to 120 residues are available to us. The solution structures of one or several of these proteins will be pursued. Subsequently, we will study the binding of these proteins to eIF- 4E to investigate the mode of translation initiation. Phosphorylation of 4E-BP on Ser63 disables 4E-BP to bind elF-4E. Thus, the effect of phosphorylation on the conformation of 4E-BP will be investigated. On a broader scale, this research is aimed at elucidating the role of elF-4E for cell growth and cell transformation, and at understanding the mechanism of translation initiation in general. We expect that this research will be relevant to the understanding of tumorigenic uncontrolled cell growth.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA068262-04
Application #
2882428
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Mietz, Judy
Project Start
1996-05-08
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Obayashi, Eiji; Luna, Rafael E; Nagata, Takashi et al. (2017) Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5. Cell Rep 18:2651-2663
Salvi, Nicola; Papadopoulos, Evangelos; Blackledge, Martin et al. (2016) The Role of Dynamics and Allostery in the Inhibition of the eIF4E/eIF4G Translation Initiation Factor Complex. Angew Chem Int Ed Engl 55:7176-9
Edmonds, Katherine A; Wagner, Gerhard (2015) (1)H, (13)C, and (15)N backbone and sidechain chemical shift assignments for the HEAT2 domain of human eIF4GI. Biomol NMR Assign 9:157-60
Sekiyama, Naotaka; Arthanari, Haribabu; Papadopoulos, Evangelos et al. (2015) Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1. Proc Natl Acad Sci U S A 112:E4036-45
Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak et al. (2015) eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference. Nat Commun 6:7194
Leigh, Kendra E; Sharma, Mayuri; Mansueto, My Sam et al. (2015) Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication. Proc Natl Acad Sci U S A 112:9010-5
Yi, Tingfang; Kabha, Eihab; Papadopoulos, Evangelos et al. (2014) 4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance, proliferation and metastasis. Oncotarget 5:6028-37
Mahalingam, Poornachandran; Takrouri, Khuloud; Chen, Ting et al. (2014) Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in vitro characterization as inhibitors of protein-protein interaction. J Med Chem 57:5094-111
Yefidoff-Freedman, Revital; Chen, Ting; Sahoo, Rupam et al. (2014) 3-substituted indazoles as configurationally locked 4EGI-1 mimetics and inhibitors of the eIF4E/eIF4G interaction. Chembiochem 15:595-611
Luna, Rafael E; Akabayov, Sabine R; Ziarek, Joshua J et al. (2014) Examining weak protein-protein interactions in start codon recognition via NMR spectroscopy. FEBS J 281:1965-73

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