Gabrielle Dias Salton1, Claudia Cilene Fernandes Correia Laurino2, Nicolás Oliveira Mega3, Andrés Delgado-Cañedo4, Niclas Setterblad5, Maryvonnick Carmagnat6, Ricardo Machado Xavier7, Elizabeth Cirne-Lima8, Guido Lenz9, João Antonio Pêgas Henriques10, Jomar Pereira Laurino11. 1. a Post-Graduation Program in Cellular and Molecular Biology, Molecular Radiobiology Laboratory, Biotechnology Center , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil , Cryobiology Unit and Umbilical Cord Blood Bank, Hemotherapy Service , Hospital de Clínicas de Porto Alegre , Porto Alegre (RS) , Brazil. 2. b Molecular Biology for Auto-immune and Infectious Diseases Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil . Embriology and Cellular Differentiation Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre; Faculdade de Veterinária , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil . Faculdade Nossa Senhora de Fátima , Caxias do Sul (RS) , Brazil . Instituto Brasileiro de Saúde , Porto Alegre (RS) , Brazil. 3. c Animal Biology Post-Graduation Program , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil. 4. d Biotechnology Research Center for Interdisciplinary Research , Universidade Federal do Pampa , São Gabriel (RS) , Brazil. 5. e Imaging, Cell Selection and Genomics Platform , Institut Universitaire d'Hématologie, Hôpital Saint-Louis , Paris , France. 6. f Immunology and Histocompatibility Laboratory AP-HP , INSERM UMRS 940, Institut Universitaire d'Hématologie , Paris , France. 7. g Molecular Biology for Auto-immune and Infectious Diseases Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil. 8. h Embriology and Cellular Differentiation Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre; Faculdade de Veterinária , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil. 9. i Cell Signaling Laboratory, Biophysics Department, Biotechnology Center and Post-Graduation Program in Cellular and Molecular Biology , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) , Brazil. 10. j Molecular Radiobiology Laboratory, Biotechnology Center and Post-Graduation Program in Cellular and Molecular Biology , Universidade Federal do Rio Grande do Sul , Porto Alegre (RS) ; Biotechnology Institute , Universidade de Caxias do Sul , Caxias do Sul (RS) , Brazil. 11. k Biotechnology Institute , Universidade de Caxias do Sul, Caxias do Sul (RS) and Instituto Brasileiro de Saúde , Porto Alegre (RS) , Brazil.
Abstract
BACKGROUND: Eukaryote initiation factor 2 subunit β (eIF2β) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2β contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2β was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2βΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS: eIF2βΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2β translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2βΔ3K did not make this translocation. DISCUSSION: eIF2β is involved in the protein synthesis process and should act in nuclear processes as well. eIF2βΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2βΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2β polylysine stretch domains are promising targets for this.
BACKGROUND: Eukaryote initiation factor 2 subunit β (eIF2β) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2β contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus. METHODS: The gene eIF2β was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2βΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence. RESULTS:eIF2βΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2β translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2βΔ3K did not make this translocation. DISCUSSION: eIF2β is involved in the protein synthesis process and should act in nuclear processes as well. eIF2βΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2βΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2β polylysine stretch domains are promising targets for this.
Entities:
Keywords:
Cell Proliferation; Cell viability; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factors; Nucleolar organizer region associated proteins; Protein Biosynthesis; Translational Medical Research
Authors: Maria A Ferraiuolo; Chung-Sheng Lee; Lian Wee Ler; Jeanne L Hsu; Mauro Costa-Mattioli; Ming-Juan Luo; Robin Reed; Nahum Sonenberg Journal: Proc Natl Acad Sci U S A Date: 2004-03-15 Impact factor: 11.205
Authors: I B Rosenwald; R Kaspar; D Rousseau; L Gehrke; P Leboulch; J J Chen; E V Schmidt; N Sonenberg; I M London Journal: J Biol Chem Date: 1995-09-08 Impact factor: 5.157
Authors: Rita Spilka; Christina Ernst; Helmut Bergler; Johannes Rainer; Susanne Flechsig; Alexander Vogetseder; Eva Lederer; Martin Benesch; Andrea Brunner; Stephan Geley; Andreas Eger; Felix Bachmann; Wolfgang Doppler; Peter Obrist; Johannes Haybaeck Journal: Cell Oncol (Dordr) Date: 2014-07-29 Impact factor: 6.730