Structural studies of eukaryotic protein synthesis factor complexes eIF2B and eIF2/eIF2B, critical for translational control in eukaryotic cells

All organisms are composed of cells. Cell growth and cell division are coordinated and controlled by a wide range of signals that ensure they occur at the correct places and times. Hence developing embryos require periods of rapid growth while adults require much slower growth to replace damaged or dying cells. When there is a loss of growth control, diseases such as cancer can develop, while a failure to promote growth when required can cause a failure to repair damaged cells or cause tissue wasting. We have been studying how cells control the conversion of nutrients into the new proteins that are required for life. Proteins perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make 3-dimensional structures that are important for each to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of the genes in the genome. This is termed 'protein synthesis' and it is the final step in the pathway called 'gene expression' which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within 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 adapter molecules called transfer RNAs (tRNAs) that bring the necessary amino acids together.

This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act that are called protein synthesis initiation factors. They direct the ribosome and a specialised tRNA called initiator 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. This 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. This proposal concerns factors designated eIF2B and eIF2, two factors critical for regulating ribosome binding of Met-tRNAi. eIF2B is known as a factor that 'switches on' its partner eIF2 so that eIF2 can bind to Met-tRNAi and recruit it to ribosomes
In this proposal we describe preliminary experiments, which show that the factor eIF2B is twice as big as was previously thought and also provide clues to its overall structure in three dimensions. Because proteins cannot be seen with the naked eye or light microscopes, we propose here to use the technique called cryo electron microscopy to rapidly freeze highly purified protein samples in liquid nitrogen and then magnify them so that we can build a three-dimensional model of eIF2B and study how it binds to its partner protein called eIF2.
By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help understand control of cell growth and provide further insight into vanishing white matter disease, 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 for commercial products or those that grow cells by fermentation because it will allow an improved understanding of protein synthesis mechanism. Detailed structural information will help unravel the precise controls of protein synthesis will assist in the design of optimized commercial protein expression or fermentation systems.

Unravelling the nature and control of molecular interactions drives much molecular biological research. For example the large G protein family function as molecular switches, controlled by interacting ligands and regulatory partners. In protein synthesis the G protein eIF2 functions to bind initiator tRNA to ribosomes in all eukaryotic cells. Its GTP/GDP status is controlled by two other translation factors called eIF2B and eIF5. Several observations are important for this proposal. eIF2 and eIF2B are both multisubunit protein complexes of 3 and 5 different subunits.
We: 1) find that eIF2B is a dimer of two pentamers with a combined size of ~600 kDa, large enough for visualization via single particle electron microscopy; 2) have optimized conditions to purify eIF2 and eIF2B complexes for structural biology and 3) provide preliminary electron microscopy using negative-stained samples that reveal that eIF2B is amenable to cryo electron microscopy.
In the case for support we propose a series of experiments to use highly purified reagents available in the Pavitt laboratory for studies to determine (i) the overall structure of the eIF2B complex and (ii) identify subunit positions within this structure (iii) define how it interacts with its partner eIF2 to promote nucleotide exchange and (iv) following phosphorylation of eIF2, where it forms an inhibitory complex critical for translational control. In addition we propose supporting molecular studies to define the stoichiometry of eIF2:eIF2B for guanine nucleotide exchange and whether this is altered by phosphorylation of eIF2, an event critical for translational control in all eukaryotic cells. Together these studies will define structurally steps in the global protein synthesis pathway critical for translational control.

This work will examine the mechanisms of regulation 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. To promote understanding of research findings and communication, GP joined the management committee of an EU-funded COST (Co-operation in Science and Technology) Action called MYELINET (Myelin Orphan Diseases in Health), to coordinate the study, training and treatment for the many orphan diseases affecting the CNS myelin. The European Leukodystrophy Association (ELA) represents patient groups and organise patient-scientist meetings to facilitate face-to-face contact between researchers and to communicate their research findings directly to patients/ parents. GP spoke at a meeting in 2009. GP has also presented our work to clinical geneticists at Manchester children's hospital (Feb 2012). GP will continue to pursue/engage with these clinical/ patient groups to promote greater awareness and understanding of our research to promote its impact.
The project may have industrial interest. Bulk growth of yeast is used to produce a variety of organic molecules, beverages/food stuffs and biopharmaceuticals. Applications include where there is a requirement to highly express specific proteins, for example therapeutic or industrially relevant proteins. 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. The Centre of Excellence for Biopharmaceuticals (COEBP) within FLS at Manchester actively engages with potential industrial partners. GP and other academics have met with several company representatives with the aim of developing commercially relevant projects. Further meetings are planned for 2013/14. 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 has 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. Similarly AR has contributed to displays and EM demonstrations on the theme of Biological Electron Microscopy. In addition AR organised an EM image display for a local Science Fair and hosted visits from school parties, 6th form colleges, and technical colleges to view our EM. We will run similar activities at future events and expect the employed RA to contribute. We have also hosted secondary school work experience students to gain hands on more in-depth experience of lab-life and intend offering similar future places.