Role of translation elongation factor 1B (eEF1B) in regulating protein synthesis in response to oxidative stress in yeast

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

All organisms must respond to changes in their external environment. With the availability of genome sequences much attention has focused on analyzing the changes in gene expression/transcription profiles during these adaptive responses. The translation of mRNA into protein is a fundamental component of the gene expression pathway. However, relatively little is known regarding the role of translational control mechanisms in the response to stress conditions, which is the focus of this proposed study. This application is based on our preliminary findings showing that oxidative stress causes a rapid inhibition of protein synthesis. Such a global inhibition of protein synthesis is widely recognised as a response of biological systems to stress conditions. Preventing protein synthesis during stress conditions may allow time for organisms to direct gene expression towards the production of new molecules required to protect against or detoxify the stress. Our data show that oxidative stress inhibits protein synthesis at multiple levels. The goal of this comprehensive research programme is to understand the molecular details of these regulatory mechanisms. This study will focus on oxidative stress which is a major problem for most biological systems. Reactive oxygen species and free radicals are produced as toxic by-products of normal metabolism and through exposure to environmental factors including sunlight. All organisms, including humans, contain effective antioxidants such as vitamins A and C and enzymes, such as catalase and superoxide dismutase, that can detoxify these harmful molecules. However, under extreme conditions reactive oxygen species can overwhelm the antioxidant defences resulting in a so-called 'oxidative stress'. It is important to understand how cells respond to an oxidative stress because it is implicated in many diseases including cancer, neurodegenerative and cardiovascular diseases. In addition, oxidative damage to cells and tissues can contribute to the decline in physiological function that occurs in ageing cells. This research will make use of the yeast Saccharomyces cerevisiae as a model organism. Yeast offers an ideal model system to study these types of processes since it is genetically tractable and has served as the organism of choice for most post-genomic studies. There is also a high degree of conservation between the stress-protective systems in yeast and human cells making it an ideal organism for this study.

Technical Summary

Inhibition of protein synthesis is a common response to cellular stress. We have found that a complex pattern of translational regulation occurs in response to oxidative stress, with many mRNAs differentially regulated. This regulation involves the well-known Gcn2-mediated inhibition of translation initiation. Additionally, our data indicate that translation is regulated at the post-initiation phase by elongation factor 1B (eEF1B). Elongation begins with the insertion of an aminoacyl-tRNA into the empty A-site of an elongating ribosome catalysed by elongation factor 1 (eEF1). eEF1 comprises a G protein (eEF1A) and a guanine nucleotide exchange factor (eEF1B). eEF1B contains two subunits (alpha and gamma). TEF5 encodes the catalytic subunit (eEF1B alpha), which is essential for viability. Two non-essential genes, TEF3 and TEF4, encode gamma isoforms. Our preliminary data indicate that a tef3 mutant shows less inhibition of protein synthesis and mutants lacking TEF3 are resistant to H2O2 implicating eEF1B in the response to oxidants. This project will identify the role of eEF1B in the oxidative stress response. We will determine how eEF1B activity is modulated in response to oxidants by examining the composition and activity of the eEF1B complex during different growth and stress conditions. The role eEF1B in the response to oxidants will be determined by examining the rate and fidelity of translation elongation. We will investigate the overlapping roles of translation initiation and elongation in the response to oxidative stress by comparing the roles of eEF1B and Gcn2 in mRNA-specific translational regulation. Post-initiation regulation of translation has not been widely considered as a regulated step in protein synthesis and this work programme will provide a better understanding of how gene expression is modulated by ROS to control protein production.

Publications

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Grant CM (2011) Regulation of translation by hydrogen peroxide. in Antioxidants & redox signaling

 
Description We have analyzed the regulation of protein synthesis in response to oxidative stress conditions induced by exposure to H2O2 in the yeast Saccharomyces cerevisiae. This is because our previous data have shown that H2O2 causes an inhibition of translation initiation that is dependent on the Gcn2 protein kinase which phosphorylates the a-subunit of translation initiation factor eIF2. In this current project we have shown that translation is also regulated at the elongation stage via the activity of the eEF1B elongation factor.

1) We have examined the role of Tef3 and Tef4 in the regulation of translation elongation during oxidative stress conditions. Tef3 and Tef4 are two isoforms of yeast eEF1B??and our data indicate that they play different roles in the oxidative stress response. Tef3, but not Tef4, is required to inhibit protein synthesis in response to hydrogen peroxide exposure. Interestingly mutants lacking TEF3 do not show an inhibition of growth during oxidative stress presumably because they can maintain protein synthesis under these stress conditions. To elucidate the role of Tef3 and Tef4 in the stress response we have carried out detailed biochemical analysis. Our data suggest that Tef3 regulates the catalytic activity of eEF1B? in response to oxidative stress conditions. We are currently working up the methodology to examine the guanine nucleotide exchange activity of Tef5. These assays will be performed in the presence and absence of Tef3 and Tef4 in cells exposed to oxidative stress. A manuscript is in preparation pending these final experiments. This is a particularly interesting finding since there is increasing evidence from diverse organisms suggesting that that eEF1B plays important roles in the control of gene expression, particularly during altered growth conditions.

2) We have measured average mRNA ribosomal transit times to directly determine whether Tef3 and Tef4 influence the rate of elongation. We have previously shown that ribosomal transit times are increased by approximately 50% in response to adaptive doses of hydrogen peroxide. Interestingly, there is no such increase in transit time in a tef3 mutant confirming that Tef3 is required to inhibit protein synthesis in response to oxidative stress conditions. Inhibiting protein synthesis may prevent continued gene expression during potentially error-prone conditions as well as allow for the turnover of existing mRNAs and proteins whilst gene expression is reprogrammed to deal with the stress. We have examined the fidelity of protein synthesis and found that misreading is elevated in eEF1B mutants.

3) We originally proposed to determine whether the regulation of translation initiation and elongation is required for mRNA-specific translational control. We have identified several mRNAs which show increased polysome-association in response to hydrogen peroxide stress. We chose to focus on Hsp30 and found that the levels of a tagged protein (Hsp30::TAP) are significantly elevated in response to oxidative stress. Interestingly, this increase appeared to depend on Gcn2 and eEF1B. Unfortunately however, these differences were found to depend on the nature of the tag used and no increased in Hsp30 was detected using an Hsp30::HA construct. We also attempted to raise antibodies against Hsp30 to enable us to examine a non-tagged protein but were unsuccessful. Given the tag-dependent differences in Hsp30 expression we stopped working on this aspect of the project.

The major impacts of this research will be on (a) the bioscience research community with specific interests in translational regulation, (b) on medical biosciences, since alterations in protein synthesis and folding are implicated in numerous disease processes and (c) on Industrial research aimed at maximising protein production from biological systems for biotechnological and biopharmaceutical applications. In the longer term the research will impact the UK economy through the National Health Service and patients via improved diagnosis and the potential for therapeutic intervention and management of diseases, and the industrial sector, by providing genetic tools for improvement of existing strains, or generation of strains with novel characteristics. This may be through new drugs to tackle the ever increasing problem of amyloidosis in an ageing population.
Exploitation Route To rpovide a framework for understanding the regulation of translation during stress conditions in eukaryotes
Sectors Pharmaceuticals and Medical Biotechnology