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Study of the eIF5A impact on cellular proteomic profile using Saccharomyces cerevisiae as model

Grant number: 15/23203-0
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): August 01, 2016
Effective date (End): September 30, 2019
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal researcher:Cleslei Fernando Zanelli
Grantee:Natália Moreira Barbosa
Home Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
Associated scholarship(s):17/23993-6 - Impact of the role of eIF5A in the translation of proteins imported into the Endoplasmic Reticulum and mitochondria, BE.EP.DR

Abstract

The translation factor 5A (eIF5A) is highly conserved in Archaea and eukaryotes and is essential for cell viability. This is the only protein known to contain the amino acid residue hypusine, essential for eIF5A function, generated by a post-translational modification. The functional homolog of eIF5A in bacteria is the EF- P factor, which also undergoes a modification of lysyl or arginyl- lysylation rhamnosylation in different species. The N and C terminal domains of eIF5A are superimposable on domains I and II of EF- P and the position of modification loop are also conserved. The crystal structure of EF-P bound to 70S ribosome shows that this factor is located between the P and E sites and the modification loop near the peptidyl transferase center of the ribosome. Using data from tRNA and rRNA hydroxyl radical cleavage it was modeled the eIF5A location on 80S similarly as reported for EF-P structure. These structural data suggest an EF-P/eIF5A function in peptide bond formation if the E site is not occupied by deacylated tRNA. Besides eIF5A and EF- P have already been associated with the translation initiation, more recent studies have established a role in the translation elongation, more specifically on the elongation of consecutive proline-containing sequences (poly- P), which are able to induce a ribosome stalling due to low reactivity of proline for peptide bond formation. However, different proteomic cellular profile studies concluded that not only polypeptides containing poly-P have its translation dependent on EF- P and the absence of this factor does not affect all poly-P polipeptides. An analysis integrating data from different studies led to the conclusion that residues immediately before and immediately after consecutive proline residues inducing ribossmoal stalling and the translation initiation rate and poly-P sequence position also affect the dependence of EF- P for protein translation. Still, there are few studies with proteomic profile in the absence of eIF5A function and the proteins which syntheses are dependent on eIF5A are not well known. Furthermore, eIF5A is associated with cell proliferation at different organisms, more specifically in the G1/S transition, and different types of human tumors, which reinforces the role of eIF5A in translation of proteins related to these functions. Thus, the present study aims to search for the proteins which syntheses are dependent on eIF5A by proteomic profile comparison between wild-type strains and eIF5A mutants in Saccharomyces cerevisiae. We propose two strategies: 1. define the proteomic profile by mass spectrometry (LC-MS/MS) using Stable Isotope Labeling by amino acids in cell culture (SILAC) for quantitative differential correlation between samples of wild and eIF5A mutant; and 2- setting proteomic profile for GFP fluorescence using a 4156 collection of strains, each one containing a different ORF fused to the C-terminal GFP and a protein RFP constitutively produced as normalizing, both in the wild and eIF5A mutant background. Both methodologies will be conducted in collaboration with researchers at the University of Toronto (Dr. William W. Navarre and Dra. Brenda Andrews) and the University of British Columbia (Dr. Leonard J. Foster), experts in these matters .In this way, it will contribute significantly to understand which proteins have their translation dependent on eIF5A function and what are the determinants of this dependence.

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Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
BARBOSA, Natália Moreira. Estudo do impacto da função do Fator de Início de Tradução de Eucariotos (eIF5A) no perfil proteômico celular utilizando o modelo de Saccharomyces cerevisiae. 2019. 98 f. Doctoral Thesis - Universidade Estadual Paulista "Júlio de Mesquita Filho" Faculdade de Ciências Farmacêuticas..

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