Eukaryotic initiation factor 5 guanine-nucleotide dissociation inhibitor activity and control of translation initiation

Interactions between proteins modulate essential functions, ensuring that organisms and their cells can grow and perform tasks at the correct time and place. Many proteins act together within complexes either with other proteins or also with other cell components (lipids, DNA or small molecules for example) to coordinate a common function. One group of proteins relevant to this proposal is required make (or synthesize) all new proteins in each cell in every organism. These are called 'protein synthesis factors'. Understanding how protein synthesis factors function and interact with each other is, therefore, fundamental to understanding how proteins are made in all cells, in all organisms. By improving our understanding how processes such as protein synthesis work in normal cells it can help scientists understand diseases in which this process is altered. From previous work done we know that defects in protein synthesis factors can be associated with a wide range of diseases in mammals such as forms of diabetes, obesity, or how infectious agents such as viruses are able to cause infections. Control of protein synthesis is also critical for organism responses to nutrient deprivation, stress, as a well as during embryo development including differentiation of tissues, for nervous system function including memory, and during the aging process. In work leading up to this proposal we identified a novel interaction between two protein synthesis factors known as eIF2 and eIF5 that is critical for protein synthesis regulation and describes a new function for the eIF5 protein. This work has defined a new step in the chain of events necessary for starting (or initiating) new rounds of protein synthesis. By discovering this new step, it has implications for how scientists understand the mechanisms of protein synthesis control. We now have a leading advantage, as we are the first researchers to uncover this. We propose here a set of detailed molecular experiments to characterise its role. Rather than using animals for this work, we will use purified systems in test tubes and perform some experiments in yeast cells which are an excellent cellular model system to study universal translational controls common to all forms of life. In addition to furthering our basic science knowledge, our findings may be directly relevant to several industries. For example, several new advanced medicines used to treat serious life-threatening diseases, or produce vaccines are proteins made specifically from different cell systems. Knowledge of our work could help maximize the production of each product, thereby helping to reduce costs of production.

CONFIDENTIAL Translational control in eukaryotic cells is critical for gene regulation during nutrient deprivation and stress, development, differentiation, nervous system function including memory, aging, and disease. One protein synthesis factor that is critical for many of these controls and will be studied in this proposal is eukaryotic initiation factor (eIF) 2: a general eukaryotic protein synthesis initiation factor that functions to deliver initiator-tRNA to 40S ribosome/mRNA complexes. eIF2 is a G protein that switches between GDP and GTP-bound forms and this is regulated by a GTPase activating protein (GAP) eIF5, a guanine nucleotide exchange factor (GEF) eIF2B and protein kinases that phosphorylate eIF2 to control both general and gene specific translation. In work leading to this proposal, we describe a new activity for eIF5. It possesses GDP dissociation inhibitor (GDI) activity. This separate eIF5 function defines a new step in the initiation pathway. Hence eIF5 GAP activity switches eIF2 to the inactive eIF2-GDP complex and its GDI function prevents premature GDP dissociation. We show using yeast genetic methods that mutants affecting GDI function have a dramatic effect on translational control via eIF2 phosphorylation: highlighting that GDI function is critical for normal translational controls common to all eukaryotes. We are now proposing here a work program designed to address important related questions that will define GDI activity further and investigate how the eIF2/eIF5 GDI complex is disrupted by a GDI dissociation factor (GDF). We propose that GDF function is a novel activity of eIF2B, in addition to its known GEF function. eIF2B GEF activity is controlled by eIF2 phosphorylation. We will also investigate whether eIF2B GDF activity is similarly controlled by the same signalling event. As eIF2B interaction with phospho-eIF2 regulates translation in diverse and important contexts, this work will be of wide interest and high impact.

The program of work described in this application will examine the mechanism and regulation of translation in eukaryotes. To date, the use of yeast biochemical and genetic techniques has served as a paradigm for the study of translational regulation across all eukaryotes and much of the current understanding in the translation control field has stemmed from this work. Therefore, the project will impact primarily on researchers in the immediate and wider scientific community. As we are studying control of a GTP-binding G-protein our finding would also be of interest to cell biologists and other scientists interested in G protein signaling and control mechanisms. The findings from this research will be disseminated through publications in the scientific literature, presentations at scientific conferences by both applicant and the employed researcher, and if appropriate via press releases coordinated through the University press office. Materials such as yeast strains, bacterial strains, and DNA reagents that will be produced during the research will be stored in the laboratory in catalogued archives and will be made available to other researchers in the UK and overseas upon request. Outside of the immediate academic fields, the research will be of interest to clinical scientists, medical practitioners, patient groups and the wider public. This stems from the fact that that mutations in the translation initiation factor eIF2B cause a fatal human genetically inherited disorder. This has created wider interest in translational control and in my laboratory's research studies in particular. The mutations affect the ability of glial cells to correctly form myelin sheaths wrapped around the axons of neurons, which initially manifests as ataxia. This is a chronic-progressive disorder; patients suffer seizures, comas and deteriorate over time, with loss of quality of life and a premature death. To promote understanding of research findings and communication with relevant groups, I have joined the management committee of an EU-wide COST (Co-operation in Science and Technology) Action called MYELINET (Myelin Orphan Diseases in Health) which was formed to promote better understanding and to coordinate the study, training and treatment for the many orphan diseases affecting the CNS nerve-insulating myelin. This network includes representatives from 20 EU countries. Patient groups are represented including The European Leukodystrophy Association (ELA) who organise patient-scientist meetings to facilitate face-to-face contact between researchers and patients/ parents to enable scientists to communicate their research findings directly. I will continue to engage with these groups during the lifetime of this award to promote greater awareness and understanding of our research. In addition, the Faculty of Life Sciences employs a full-time public engagement coordinator (Helen Jopling) who promotes public engagement activities at all levels. We will liaise with her to ensure that our science can be promoted more widely. The project may have significant industrial implications as bulk growth of yeast and human cell lines are used to produce a whole variety organic molecules (including biofuels) and beverages/food stuffs and biopharmaceuticals. Applications include where there is a requirement to highly express specific proteins, for example therapeutic monoclonal antibodies. 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. If 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.