eIF2B (eukaryotic initiation factor 2B): regulation of its activity and expression, and its roles in translation initiation

The production of proteins is a key process in all living cells, allowing them to use the genetic information held in the genes in their DNA. Most genes contain the information for making proteins, which are the cellular components that carry out almost all the functions of the cell.

To make the protein coded for by a given gene, the letters in the DNA are first copied to a second similar molecule, known as mRNA, in a process called transcription. This mRNA molecule is then 'read' by molecular machines called ribosomes in order to produce the protein, a process known as translation. The proteins expressed within a cell determine its properties, e.g., how it will function and respond to its environment.

The process of protein synthesis consumes a large proportion of a cell's energy and nutrients, and so it must be very tightly controlled. Furthermore, this process can be altered to favour the production of different proteins by allowing the machinery to preferentially 'read' certain mRNA molecules, which therefore allows the cell to respond quickly to a stressful environment caused, for example, by a reduction in available nutrients or energy. This kind of mechanism can also control the expression of different proteins as a cell goes through its stages of growth and division, the cell cycle.

A key part of the control of protein synthesis involves proteins termed 'initiation factors', which are the focus of this research. These initiation factors control the attachment of the mRNA molecules to ribosomes and are vital to allow protein synthesis to start. They are also a key point of regulation under a wide variety of conditions because they control which mRNA molecules to recruit and even exactly where to start reading their code. The main focus of this research is a factor termed eIF2B, which is composed of 5 separate protein 'subunits'. We will also study two proteins that work with eIF2B in the process of protein synthesis, eIF2 and ABC50. The applicant's laboratory has many years' experience in investigating all these proteins.

Disruption of the normal function of eIF2B can lead to disease. Slight spelling mistakes in the genes that encode the 5 subunits of eIF2B lead to an often-serious brain disease, known as 'vanishing white matter'. It also responds to insulin, and so is mis-regulated in type I (insulin-dependent) diabetes. This implies that the proper functioning of eIF2B is necessary to maintain a healthy cell. Furthermore, eIF2B, eIF2 and ABC50 limit the capacity of cells to make proteins that are used as drugs or in disease diagnosis, and which are of great commercial value.

We aim to study four main aspects of eIF2B:
1. How it is controlled, based on new discoveries made by the applicant's laboratory or others, to learn more about how this important protein can be controlled under different conditions in the cell;

2. How cells can control how much of this protein they contain, and how this is related to the control of the amounts of eIF2 and ABC50: this is important as changes in the amounts of all these proteins will affect both how fast proteins can be made and how protein production is controlled;

3. How changes in the amounts or the cellular activity of eIF2B (and ABC50) affect the production of specific proteins. This is important since, as described above, changes in protein synthesis have a major affect on how much of different proteins are present in cells, and thus on the function and 'health' of the cell;

4. Its structure, using state-of-the-art techniques to investigate the way in which its five subunits are arranged and how they interact with its partner protein, eIF2.

Protein synthesis (mRNA translation) and its control play key roles in regulating gene expression, and thus the 'proteome' and consequently impacts on many cellular functions including cell growth and proliferation. mRNA translation is regulated by multiple mechanisms and this impacts both on general protein synthesis and on the production of specific proteins or protein isoforms. Eukaryotic initiation factor (eIF)2B plays a key role in these aspects of translational control. Composed of five different subunits, eIF2B acts as the guanine nucleotide-exchange factor (GEF) which promotes production of active GTP-bound eIF2. eIF2 brings the initiator methionyl-tRNA to the ribosome. Its function is stimulated by the ATP-binding cassette protein ABC50.

Building on the extensive previous work and expertise of the PIs' research team, together with appropriate research collaborations, we will:

1. extend our studies on the control of eIF2B by post-translational modifications to investigate novel phosphorylation events catalysed by kinases involved, e.g., in cell cycle control and to acetylation, which is emerging as a major mechanism for regulating cytoplasmic proteins;

2. initiate studies into the unexplored area of the control of the levels of eIF2B subunits and related proteins. This includes work on their transcriptional control by mitogenic or stress signalling and the translational control of eIF2B expression through the mammalian target of rapamycin and also cellular stress pathways;

3. use reporter constructs and new technologies (deep sequencing) to examine the impact of eIF2B, and its control, and of ABC50 on the initiation of the translation of specific mRNAs, including the use of different start codons;

4. use advanced biophysical methods (cryo-electron microscopy) to gain insight into the structural organisation of the pentameric eIF2B complex and its regulated interaction with eIF2.

Protein synthesis is a key process in all cells and helps to determine the amounts of different proteins that are made. Correct control of protein synthesis is crucial for healthy cells and organisms, and protein synthesis plays a key role in ageing/longevity. Understanding it is thus of central importance for a wide range of scientists working in biochemistry, cell biology, biotechnology and biomedical sciences.

Our findings will be disseminated through the specialist peer-reviewed literature; presentations at suitable scientific conferences, through the PI's links with industry; and the media, as appropriate. We anticipate publishing at least 4-5 research papers in high-impact international journals, and presenting the data at national ('Translation UK'), European (2013 Heidelberg Translation meeting) or North American (2012/4 Cold Spring Harbor Translational Control meetings), and other relevant (e.g., biotechnology, signalling) conferences.

The impact of the proposed research falls logically into the following areas, for the indicated beneficiaries:

1. understanding the control of eIF2B activity by post-translational modifications will provide key new insights into the control of protein synthesis, e.g., its links to second messengers and cell cycle control. This will benefit investigators studying the control of gene expression, cell physiology and metabolism, as well as those studying translational control mechanisms and the links between the dysregulation of protein synthesis and human disease/ageing.

2. investigating the control of the levels of expression of eIF2B, and of eIF2 and ABC50, will pave the way to a fuller understanding of the ways by which the cellular capacity for protein synthesis (translation factor levels) is controlled, via transcriptional and posttranscriptional mechanisms. The latter are likely to reveal new autoregulatory control of eIF2B levels. This is important for understanding the regulation of anabolic processes and cell growth/proliferation. This directly benefits UK/EU biotechnologists and companies working to enhance production of recombinant proteins ('biologic' drugs) which are of rapidly increasing interest and value. More cost-effective production of such drugs will benefit the health-care sector and its users, i.e., the general public, by making them more readily available at affordable prices.

3. understanding the roles played by eIF2B, eIF2 and ABC50 regulating the translation of specific mRNAs, and selection of different start codons, is fundamentally important for knowledge of the regulation of the expression of specific proteins or protein isoforms, i.e., the control of the cellular proteome.

4. understanding the structural organisation of the eIF2B complex and its interactions with its substrate eIF2 is an outstanding issue of fundamental importance for knowledge of protein synthesis and its control.

The project will improve understanding of the mechanisms governing the activity and cellular levels of key components of the protein synthetic machinery, thus aiding the growing number of systems/computational biologists who are modelling key processes such as protein synthesis.

The PI has >20 years experience of managing a large research group. The PI will meet every other week meetings with the PDRAs and other group members working on related research, to coordinate their work, and exchange data, ideas and reagents. Through weekly lab meetings, the PI encourages continuous close links between all lab members working on aspects of protein synthesis and its control.

The ability to achieve the objectives of this study is very high given that (i) the PI has extensive experience in studying eIF2B and ABC50, including numerous discoveries related to their function and control; (ii) both named PDRAs already have several years expertise in working with eIF2B, and (iii) appropriate collaborations are already in place (Richter) or agreed (Coldwell, Passmore).