GTP-binding to eIF2B as a novel mechanism for G protein activation in protein synthesis initiation

We study how cells control the conversion of nutrients into the new proteins that are required for life-the growth and division of cells. Proteins are biological machines that perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make specific 3-dimensional structures that are important for each protein to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of our genes in the genome. Termed 'protein synthesis' this is the final step in the gene expression pathway, which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within relatively large and complex molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers' called protein synthesis factors and adapters called transfer RNAs (tRNAs). In concert they bring the necessary amino acids together with the instructions to ensure the correct proteins are made at the right time.
This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act termed protein synthesis initiation factors. They direct the ribosomes and a specialised tRNA that starts proteins with the amino acid methionine (designated Met-tRNAi) to the correct start place on each mRNA. This is critical to make the right proteins in every cell and must be done both accurately and rapidly. Initiation is the most complex phase of protein synthesis and the least well understood at the molecular level. Key to this proposal are factors designated eIF2B, eIF2 and eIF5. These three factors are critical for regulating ribosome binding of Met-tRNAi by controlling the energy charge on the eIF2 factor. The energy status is determined by binding nucleotides called GTP and GDP. When bound to GTP eIF2 is switched on and can bind to Met-tRNAi and when bound to GDP it cannot bind to Met-tRNAi and so is switched off. We have been studying this switching mechanism, as it is central to the control of protein synthesis. eIF5 switches eIF2 off, while eIF2B switches it on. We have recently found that the eIF2B protein also binds GTP. This unexpected finding has implications for how eIF2B works. eIF2B is complex factor and we have identified approximately which part of the protein GTP binds to - called the gamma subunit. We know that the gamma subunit is critical for both eIF2B known functions, first its GDP/GTP exchange function and also a second role we recently found, a function that displaces eIF5 from eIF2 so that eIF2 is free for nucleotide exchange. The experiments we describe in this proposal are aimed at understanding (i) why eIF2B binds GTP, (ii) where exactly it does bind and (iii) how important it is for each of the eIF2B roles in cells. Our idea is that GTP is directly handed from eIF2B to eIF2, which would be a novel way for this type of protein to function.
By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help scientists understand cell growth better and provide further insight into disease mechanisms, for example vanishing white matter disease, a genetic disorder which is caused by mutations in eIF2B. The work may also be of interest to industries eg those that produce specific proteins as drug therapeutics or commercial products or those that grow cells by fermentation, because improved understanding of protein synthesis mechanisms will assist in the design of optimized commercial protein expression or fermentation systems.

Unravelling the control of molecular interactions drives much molecular biological research. For example the G protein family function as molecular switches, controlled by interacting ligands and regulatory partners. In protein synthesis initiation the G protein eIF2 binds initiator tRNA to ribosomes in all eukaryotic cells in a GTP-dependent manner. Its GTP/GDP status is controlled by both eIF5 (GAP/GDI) and eIF2B (GDF/GEF).
Our novel recent observations important for this proposal are that:
1) eIF2B has a new second function required to displace eIF5 from eIF2-GDP/eIF5 complexes (termed GDF activity) to free eIF2 from its inhibitory complex prior to activation.
2) The eIF2Bgamma subunit (eIF2Bg) is critical for both GDF and GEF activities.
3) Surprisingly eIF2Bg binds GTP.
Our data suggest that GTP-binding to eIF2B is critical for its functions. GTP binding is highly unusual for GEFs. We will exploit our knowledge and available yeast genetic tools to test the hypothesis that GTP-binding by eIF2Bg is important for both eIF2B GDF and GEF functions and that GTP is transferred directly from eIF2B (GEF) to eIF2 (G protein) to promote protein synthesis. This would demonstrate a novel GEF mechanism.
Specifically we will:
1. Identify the GTP-binding site on eIF2Bg by molecular and genetic approaches.
2. Determine the impact of eIF2Bg mutations (made in 1) affecting GTP-binding and/or causing disease on eIF2B activities in vivo.
3. Develop new kinetic assays to monitor eIF2, eIF5 and eIF2B interactions critical for the GDI GDF and GEF activities of eIF5 and eIF2B using fluorescently labelled GDP/GTP and proteins with stopped flow fluorescence methods.
4. Use the new methods to assess the importance and contribution of GTP binding to eIF2B for its GDF and GEF activities in vitro.
Together these studies will kinetically define steps in the global protein synthesis pathway critical for all eukaryotic cells.

This work will examine the mechanisms of translation in eukaryotes by the protein synthesis factor eIF2B. Yeast has served as a paradigm for the study of translational regulation across all eukaryotes. Much of the current understanding in the translation control field has stemmed from this work in yeast. Outside of the immediate academic fields the research may be of interest to clinical scientists, medical practitioners, patient groups, industrial scientists and the wider public. Scientists/clinicians are seeking to control protein synthesis therapeutically. For example, developing specific inhibitors for use in cancer treatments. In addition mutations in eIF2B cause a fatal human disorder-VWM. This has raised interest in translational control and in my laboratory's research studies. The disease-causing mutations affect the ability of glial cells to correctly form myelin sheaths around neuronal axons, and manifests as ataxia. This is a chronic-progressive disorder; patients suffer seizures, comas and deteriorate leading to a premature death. Our results may impact on the understanding of VWM pathology. To promote understanding of research findings and communicate with clinicians and patient groups, GP joined the management committee of an EU-funded COST Action (Co-operation in Science and Technology) called MYELINET (Myelin Orphan Diseases in Health). This formed to promote understanding and to coordinate the study, training and treatment for the many orphan diseases affecting the CNS myelin. The European Leukodystrophy Association (ELA) represented patient groups and organises patient-scientist meetings to facilitate face-to-face contact between researchers and to communicate their research findings directly to patients/parents. GP spoke to patients and their families at a meeting in 2009. Myelinet was superseded by an EU-FP7 LeukoTreat to which GP contributed. GP has also presented work to clinical geneticists at Manchester children's hospital (Feb 2012). GP will continue to pursue opportunities to engage with appropriate clinical/ patient groups to promote greater awareness and understanding of our research.
Industrial interests. Yeast is used to produce a variety of organic molecules, beverages/food stuffs and biopharmaceuticals. Translational control is clearly linked to both bulk cellular growth and recombinant protein expression, therefore a greater understanding of these processes can only benefit industries using such methods. BBSRC and EPSRC have developed Networks in Industrial Biotechnology and Bioenergy (NIBBs). GP has joined BioProNET set up to enhance processes for production of biologics. BioProNET is led by A. Dickson at Manchester and M. Smales at Kent. GP has met with company representatives with the aim of developing commercially relevant projects. Novozymes at Nottingham is one partner who we are actively engaging with. GP will endeavour to make contact with additional industrial partners to promote our science. Should specific commercial exploitation opportunities arise, we will make full use of the University of Manchester Intellectual Property company (UMIP) to maximize the commercial impact. UMIP has resources and experience to facilitate potential commercial application through contracts, patents and has funding available to pump-prime projects requiring development.
Public engagement is also part of our impact plan. FLS holds an annual Community Open Day. GP contributed in 2012 offering the public insight into our science through interactive table-top activities and poster information to engage children and adults alike to promote scientific understanding and enthusiasm. We will run similar activities at future events and expect the employed RA to contribute. GP has also hosted secondary school work experience students to gain hands on more in-depth experience of lab-life and intend offering similar future places.