学位論文 A novel role of elF5A in HIF-1α-mediated hypoxic response

TARIQ MOHAMMAD

内容記述
主指導教員 : 吉田稔
Chapter 1: Introduction1.1 Eukaryotic initiation factor 5A (eIF5A)……………………………11.2 Post-translational modifications of eIF5A……………………………11.3 Role of eIF5A in translation…………………………………………21.4 Role of eIF5A in cancer………………………………………………3Chapter 2: Regulation of eIF5A acetylation2.1 Introduction…………………………………………………………………072.2 Materials and methods……………………………………………………092.2.1 Compounds and cell culture2.2.2 Antibodies and immunoblotting2.2.3 Hypoxic conditions2.3 Results and discussion……………………………………………………102.3.1 Increasing glucose concentration induced acetylation of eIF5A2.3.2 acetylation of eIF5A is induced by hypoxia2.3.3 Effect of HDAC inhibitors and hypoxia on eIF5A acetylationSummary…………………………………………………………………………11Chapter 3: eIF5A is essential for HIF-1α expression in hypoxia3.1 Introduction…………………………………………………………………143.1.1 Hypoxia inducible factor 1 alpha (HIF-1α)3.1.2 Regulation of HIF-1α3.1.3 HIF-1α and cancer progression3.2 Materials and methods……………………………………………………183.2.1 Compounds and cell culture3.2.2 Antibodies and immunoblotting3.2.3 RNAi and hypoxia3.2.4 GC7 treatment and hypoxia3.3 Results and discussion……………………………………………………193.3.1 Time course of eIF5A acetylation and HIF-1α expression is different3.3.2 eIF5A is essential for HIF-1α expression in hypoxia3.3.3 Functional eIF5A is important for HIF-1α expression in hypoxia3.3.4 GC7 treatment or eIF5A knockdown-mediated suppression of HIF-1α is independent of prolyl hydroxylase (PHD)3.3.5 Mechanism of HIF-1α suppression by GC7 treatment or eIF5A knockdownSummary……………………………………………………………………22Chapter 4: eIF5A is required HIF-1α-mediated transcriptional activation in hypoxia4.1 Introduction………………………………………………………………294.2 Materials and methods…………………………………………………304.2.1 Compounds and cell culture4.2.2 Antibodies and immunoblotting4.2.3 RNA interference, GC7 treatment and hypoxia4.2.4 RNA extraction and qPCR4.3 Results and discussion…………………………………………………32Summary……………………………………………………………………33Chapter 5: eIF5A is important for growth of tumor spheroid5.1 Introduction………………………………………………………………375.2 Materials and methods…………………………………………………385.2.1 Compounds and cell culture5.2.2 Antibodies and immunoblotting5.2.3 RNA interference and GC7 treatment5.3 Results and discussion………………………………………………395.3.1 HIF-1α knockdown leads to reduction in the size of spheroid5.3.2 eIF5A knockdown has negative effect on spheroid growth5.3.3 Functional eIF5A is essential for spheroid growthSummary……………………………………………………………………40Chapter 6: Conclusions…………………………………… 44ReferencesReferences…………………………………………………………45
Eukaryotic translation initiation factor 5A (eIF5A) is an important cellular protein and is highly conserved from bacteria to humans. eIF5A is essential for cell growth and survival. Initially eIF5A was identified to be a translation initiation factor. However recent findings suggested that eIF5A is not involved in translation initiation but has a role in translation elongation and other cellular processes such as mRNA transport. eIF5A is quite unique as it is the only cellular protein containing an unusual, polyamine-derived amino acid, hypusine [Nε- (4-amino 2-hydroxybutyl) lysine]. A conserved lysine residue of eIF5A i.e., lysine 50 of human eIF5A, is modified to hypusine. eIF5A undergoes acetylation, at lysine 47. Our lab previously reported that acetylation regulates subcellular distribution of eIF5A and that acetylated eIF5A localizes to the nucleus. Regulation of eIF5A acetylation by cellular environment is unknown. In the current study I focused to reveal a novel function of eIF5A, under different cellular environments.In order to unravel the role of eIF5A, I studied physiological conditions promoting eIF5A acetylation. I found glucose and hypoxia as the cellular cues of eIF5A acetylation induction. eIF5A was found to be an important factor for cell growth and survival in hypoxia. Furthermore, I found that hypusinated eIF5A is essential for the expression of HIF-1α in hypoxia. In the absence of eIF5A HIF-1α is degraded by the proteasome. My study revealed the importance of eIF5A in tumor growth and survival. On the basis of my observations, it is suggested that hypusination of eIF5A may be a potential target for therapeutic intervention.
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