A Novel Lipidomic Tool for Monitoring ER-stress and its role in diet induced cellular dysfunction.

The endoplasmic reticulum (ER) is a sub-cellular organelle with a central importance in the processing of newly synthesized proteins. A variety of pathophysiologies have been linked to ER dysfunction including obesity, neurodegeneration, ageing and drug toxicity. During ER dysfunction unfolded proteins collect in the ER and this is referred to as ER-stress. This has been observed in both in vitro and in vivo models. Prolonged ER-stress results in cell death by apoptosis. As part of a previous PhD studentship in the Griffin group we developed a combined lipidomic and proteomic approach for mapping changes in sub-cellular organelles. This approach involves the ultracentrifugation of cells or tissues across a density gradient to separate out organelles according to their differential densities. We have extended the original proteomic method to include a parallel lipidomic workflow, allowing us to monitor changes in organelle composition for both proteins and lipids, terming this new approach localisation of lipids (LOL). While originally developed to follow peroxisome proliferation following chemical/toxicant exposure this novel poly-omic tool can be used to follow changes in other sub-cellular organelles.

This PhD proposal is to develop this lipidomic tool further to investigate the area of diet induced ER-stress. With increased consumption of Western diets, high in saturated fats, one of the consequences is an increase in ER-stress which is thought to subsequently lead to diseases such as type 2 diabetes and cardiovascular disease. However, we do not know whether the ER stress arises from either the fat inducing protein mis-folding directly or whether the cell membrane fluidity is altered and this results in the stress. This project fits with the BBSRC recent research call for mechanistic understanding of nutritional interventions. We will initially investigate ER-stress at the cellular level in yeast and human hepatocytes using known chemical inducers of the process to develop a combined proteomic and lipidomic map of the cellular changes induced. The Griffin group regularly cultures these cell lines and hence this should provide a robust system to optimize the workflow. We will perform lipidomics in the Griffin laboratory using UPLC high resolution mass spectrometry to profile a range of lipid species including phospholipids, triglycerides, diglycerides, cholesterol esters and sphingolipids. Proteomics will be performed with Kathryn Lilley who heads the Cambridge Centre for proteomics, Biochemistry and the Cambridge Systems Biology Centre. This will largely be completed in year 1. We will then compare this with simulated diet induced changes by supplementing cell culture media with either saturated or unsaturated fats to see how dietary fats influence ER stress. This system will be developed in year 2 and applied in year 3. In year 4 the student will focus on writing up her/his results for publication and completion of their thesis with the intention to submit at ~3.5 years.