Ligand modulation of the Integrated stress response

Proteins perform nearly all functions in cells needed for life. Each protein is made from amino acids linked in chains and which folded into unique structures that enable each protein to fulfil individual roles in the body. The instructions required to make each protein correctly are determined by the DNA sequences of our genes in the genome. Called protein synthesis, it is critical that the instructions are decoded accurately at the right place and the right time. This enables organ and cell-specific proteins to only be made in those tissues and cells where they are required. It is also important that cells can both control and rapidly change which proteins they make at any one time, so that organisms can respond rapidly to changes around them. Examples include: 1) changes in protein synthesis in brain cells help to form memories; 2) during early pregnancy to ensure that embryos develop the right tissues in the right places; 3) when people are infected with viruses; and 4) when people suffer from diseases such as obesity or cancer.
Protein synthesis occurs within relatively large and complex molecular machines called ribosomes that decode instructions relayed from the genome. This is made possible by the action of 'helpers' called protein synthesis factors and adapters called transfer RNAs (tRNAs). Together they bring the necessary amino acids together with gene instructions to ensure the correct proteins are made at the right time and place.
This proposal concerns how protein synthesis is regulated by stress in a process widely called the integrated stress response (ISR). The protein synthesis factor called eIF2 brings the starting tRNA (tRNAi) to the ribosome to begin making every protein. eIF2 is known to be controlled by the ISR. When this control is out of balance it can contribute to common diseases such as cancers, diabetes, heart disease and a range of neurodegenerative conditions. We know the central element of the ISR is a reaction by which eIF2 can be modified response to stress by protein enzymes called protein kinases and that the modified version is switched off. A second enzyme called a phosphatase can switch eIF2 back on again to reset the control switch. So while it appears we understand this regulatory circuit, we now know that eIF2 interacts with many other protein synthesis factors rather than being found free in cells. How the regulatory kinases and phosphatases can access eIF2 when it is also interacting with these different other proteins is not known. Understanding this will inform how rapidly eIF2 can be switched between 'on' and 'off' states and identify which forms of eIF2 are resistant to change. Current models of how this works assume only free eIF2 can be modified. Our preliminary data questions this assumption and provides a strong basis to evaluate which forms of eIF2 can be regulated. We propose here a series of biochemical experiments to address which eIF2-containing complexes can be switched on and off in the ISR. The knowledge gained in understanding these reactions and interactions could help explain different responses to stress in different tissues and may help inform the design of better therapeutics for a wide range of conditions.

Unravelling the mechanisms controlling gene expression is critical to our understanding of the normal life course and how organisms respond to environmental challenges. Eukaryotic protein synthesis is one such fundamental cellular process that requires multiple controlled steps via complex molecular and modular interactions. In all eukaryotic cells in response to diverse stresses reversible phosphorylation of eIF2 on ser51 [eIF2(P)] down-regulates bulk protein synthesis and stimulates translation of specific mRNAs. Following stress resolution dephosphorylation restores proteostasis. This is termed the integrated stress response-ISR. Importantly, an aberrant ISR contributes to multiple diseases, so its proper functioning is critical for a healthy life course. During the translation cycle eIF2 interacts with multiple ligands. It is not known how eIF2 ligands impact upon kinase and phosphatase actions. Current models of kinase/ phosphatase action assume only free eIF2 can be modified. Our preliminary data questions this assumption and provides a strong basis for us to evaluate which eIF2 complexes are modifiable. We propose here to build upon our existing strengths and develop a biochemical approach using highly-purified translation factors, tRNAi and ribosomes to form a range of stable pre-initiation factor complexes and to systematically evaluate how these complexes alter the ability of eIF2 kinases and phosphatases to modify eIF2 at its regulatory phosphorylation site. This work will allow both the development of new models of the ISR as well as inform studies of ISR modulating compounds and disease mechanisms.

Academic Impact
This work will examine a new level of control of protein synthesis that operates during the integrated stress response (ISR) in eukaryotic cells. The work will be primarily of interest to those studying the mechanism and control of protein synthesis. However as the ISR operates in all eukaryotic organisms, the research will provide broad new insight into a mechanism of translational control covered in general molecular biology textbooks. This means that there is potentially broad impact of this research in an academic setting for both university level teaching and to stimulate future research.
Industrial and Economic Impact
The integrated stress response pathway is of interest for potential therapeutic intervention. As such pharmaceutical companies are interested in modulating the ISR. We have initiated dialog with one major company (GSK, letter attached) to ensure that our findings can be rapidly translated and extended towards therapeutic avenues.
More widely cell-based expression systems a used to produce a variety of biopharmaceuticals, and because Translational control and the ISR is clearly linked to both bulk cellular growth and recombinant protein expression, a greater understanding of these processes can only benefit industries using such methods. GP joined BioProNET a BBSRC Network in Industrial Biotechnology and Bioenergy (NIBB) set up to enhance processes for production of biologics. Should specific commercial exploitation opportunities arise, we will make 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 sector and Societal Impact
Mutations in translation factors cause genetic disorders. Our results may impact on the understanding of disease pathology. To promote understanding of research findings and communicate with clinicians and patient groups, GP has made contact with clinical geneticists in Manchester and has international links with clinical paediatricians interested in rare brain disorders including those affected by translation factor mutations. The European Leukodystrophy Association (ELA) organises patient-scientist interaction meetings to facilitate face-to-face contact between researchers who communicate their research findings directly to patients/parents. GP spoke to patients and their families at meetings in 2009 and 2015. GP will continue to pursue suitable opportunities to engage with appropriate clinical/ patient groups. This will promote greater awareness and understanding of our research among these parties. More locally UoM and FBMH organises community open days to engage local public in research. PI and RA will engage in these events. We will also host school children in our lab. They will gain experience of academic research environments and be enthused to study STEM subjects at a higher level.

Training of skilled researchers
One postdoctoral researcher and one technician will be trained as part of this project. They will gain extensive experience in state of the art RNA biology and protein analysis techniques. In addition by collaborating with other world-leading experts in academia and industry they will build connections for their future career development. They will therefore become equipped to undertake challenges relevant to careers in either academic research or industry.