A Novel Lipidomic Tool for Monitoring ER-stress and its role in diet induced cellular dysfunction.
This studentship represents a exciting collaboration between the Griffin group (metabolomics/lipidomics) in the Department of Biochemistry and the Cambridge Systems Biology Centre and Safety and Environmental Sciences (SEAC) at Unilever as part of an BBSRC DTP iCASE award.
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. However, we still don’t understand the underlying mechanisms that induce ER-stress, although it is known that increased consumption of saturated fats, as occurs when eating a westernised diet, can induce ER-stress and may contribute to both type 2 diabetes risk and the process of ageing.
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 project aims 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. We also have evidence that novel lipid signalling pathways may be involved. 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 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 this cell line 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. 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.
Applicants should have a strong background in analytical chemistry, with those with experience in LC-MS being particularly welcome.
This studentship is funded by a combination of the University of Cambridge BBSRC DTP scheme and Unilever as part of an iCASE award.
Roberts LD, McCombie G, Titman CM, Griffin JL. A matter of fat: an introduction to lipidomic profiling methods. J Chromatogr B Analyt Technol Biomed Life Sci.; 871(2):174-81 (2008).
Roberts LD, Hassall DG, Winegar DA, Haselden JN, Nicholls AW, Griffin JL. Increased hepatic oxidative metabolism distinguishes the action of Peroxisome proliferator-activated receptor delta from Peroxisome proliferator-activated receptor gamma in the ob/ob mouse. Genome Med. 1(12):115 (2009).
Dunn WB, Broadhurst DI, Atherton HJ, Goodacre R, Griffin JL. Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Chem Soc Rev.; 40(1):387-426 (2011).
Heather LC, Wang X, West JA, Griffin JL. A practical guide to metabolomic profiling as a discovery tool for human heart disease. J Mol Cell Cardiol. 2013 Feb;55:2-11.
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