Regulation and structural organisation of a key regulatory translation factor eIF2B

The production of proteins (protein synthesis) is an essential process for living cells. It is needed for them to grow, divide and survive. Protein synthesis is stimulated by hormones, like insulin, and decreased when nutrients are scarce. The availability of amino acids, the building blocks for proteins, is especially important in controlling this process. The applicant's laboratory studies how protein synthesis is normally controlled and why errors in these control systems lead to human disease. One of the key cellular components that control protein synthesis is a protein termed 'eukaryotic initiation factor 2B' (eIF2B). The applicant has studied eIF2B and its control for a more than 15 years. The activity of eIF2B is stimulated by amino acids and by hormones. It is inhibited under stressful conditions where protein synthesis slows down. Several years ago, the applicant's laboratory identified a key link between insulin and the control of eIF2B, which helped to pave the way to a better understanding of the actions of insulin. This laboratory has now identified a new way in which amino acids can control eIF2B. eIF2B is made up of five components, called subunits, one of which carries out the cellular function of eIF2B. This is the one that is known to be important for the control of eIF2B, which operates through the addition or removal of phosphate groups (called 'phosphorylation-dephosphorylation', a very common way of regulating proteins). The other four subunits have ancillary functions in the control of eIF2B or in the assembly of the eIF2B 'complex'. Recent work has shown that changes (mutations) in the genes for the subunits of eIF2B are responsible for an inherited severe brain disease called 'vanishing white matter' (VWM for short) which mainly affects children. This underlines the fact that the proper actions and control of eIF2B are crucial. It is important to understand how these mutations affect eIF2B and why this leads to a brain disease. The principal aims of this project are: 1. to find out how amino acids control the activity of eIF2B: in particular we wish to identify the links (so-called 'signalling components') that relay information about the availability of amino acids to control eIF2B's activity. This goal is important for understanding how protein synthesis is controlled. It will also provide valuable new information about the ways that nutrients are detected by animal cells and affect their functions. 2. to discover other ways in which the function of eIF2B can be controlled. By applying start-of-the-art 'proteomic' techniques, we will learn more about the control of eIF2B by phosphorylation-dephosphorylation. We will apply this technology to learn more about the ways in which eIF2B is controlled under different conditions that affect protein synthesis. This aim is important for achieving a more complete understanding of the control of eIF2B, a key regulatory molecule in animal cells. 3. to investigate how the five different subunits of eIF2B work together to create a properly functioning eIF2B protein, although our initial data have already provided insights into this. We will build on this information to establish how this protein works. We will use both biochemical methods and new biophysical techniques. This aspect is also very relevant for understanding how mutations that cause the disease VWM affect the function of eIF2B. 4. to apply our established methods to explore how VWM mutations affect the function, assembly and control of eIF2B. This work will be valuable in understanding both the disease processes that lead to VWM and the properties of eIF2B itself. 5. eIF2B is known to control the production of specific proteins. We will use information gained in this project to extend our understanding of how it does this, and of the implications for the expression of the genetic information of mammalian cells.

Eukaryotic initiation factor (eIF) 2B is a guanine nucleotide-exchange factor (GEF) that is needed to generate the active GTP-bound form of eIF2, the protein that brings the initiator methionyl-tRNA to the ribosome for every initiation event. eIF2B is a heteropentamer (subunits alpha-epsilon) and thus more complex than other GEFs. eIF2B plays a key role in mRNA translation and in its control. Mutations in any subunit of eIF2B lead to a severe inherited human neurodegenerative disorder, 'vanishing white matter' (VWM). We have recently shown that amino acids positively regulate eIF2B by inhibiting the phosphorylation of an inhibitory site in eIF2Bepsilon (Ser525), the catalytic subunit. eIF2B is also activated by hormones and growth factors. Here, we will (A) explore the control of eIF2B by amino acids, including identifying and characterizing the Ser525 kinase, which may be linked to a new nutrient-sensing mechanism. Our pilot data show that there are additional phosphorylation sites in eIF2B. We will (B) use proteomic methods to identify them and then study their regulation and roles in controlling eIF2B activity. We will also study (A,B) the interplay between Ser525, Ser540 (an inhibitory site in eIF2Bepsilon that is regulated by insulin) and the other sites in the control of eIF2B activity, to gain clearer insights into the different inputs into the control of this key translation factor. We will (C,D) build upon our recent work to study the intersubunit interactions involved in assembling eIF2B holocomplexes, using complementary biochemical and biophysical approaches. The findings will help our sixth objective (E), characterizing the effects of VWM mutations in each eIF2B subunit on the assembly, activity and function of eIF2B complexes. Lastly, (F) we will explore the role of changes in eIF2B activity on start-codon selection. This is important for understanding the cellular consequences of changes in eIF2B activity and VWM mutation