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

内质网(ER)是一种亚细胞器，在新合成蛋白质的加工过程中起着核心作用。多种病理生理学因素与内质网功能障碍有关，包括肥胖、神经退行性变、衰老和药物毒性。在内质网功能障碍期间，未折叠的蛋白质聚集在内质网中，这被称为内质网应激。在体外和体内模型中都观察到了这一点。长时间的内质网应激可通过细胞凋亡导致细胞死亡。作为Griffin小组以前的博士研究生的一部分，我们开发了一种结合脂体学和蛋白质组学的方法来绘制亚细胞细胞器的变化图。这种方法涉及跨越密度梯度的细胞或组织的超速离心，以根据细胞器的不同密度分离出细胞器。我们已经扩展了原来的蛋白质组学方法，包括并行的脂肪组学工作流，使我们能够监测蛋白质和脂类细胞器组成的变化，将这种新方法称为脂类定位(LOL)。虽然最初是为了跟踪化学/毒物暴露后的过氧化物体增殖而开发的，但这种新的多组体工具也可以用于跟踪其他亚细胞细胞器的变化。这项博士建议是为了进一步开发这种脂体学工具来研究饮食诱导的内质网应激的区域。随着富含饱和脂肪的西方饮食的增加，其后果之一是内质网应激增加，这被认为随后会导致2型糖尿病和心血管疾病等疾病。然而，我们不知道内质网应激是由脂肪诱导蛋白直接错误折叠引起的，还是细胞膜流动性改变而导致的应激。这个项目符合BBSRC最近的研究，呼吁从机制上理解营养干预。我们将首先在酵母和人类肝细胞的细胞水平上研究内质网应激，使用已知的这一过程的化学诱导剂来开发诱导细胞变化的蛋白质组和脂组学联合图谱。格里芬团队定期培养这些细胞系，因此这将提供一个强大的系统来优化工作流程。我们将在Griffin实验室使用UPLC高分辨率质谱仪进行脂类组学，以分析一系列脂类，包括磷脂、甘油三酯、二甘油酯、胆固醇酯和鞘磷脂。蛋白质组学将与剑桥蛋白质组学、生物化学中心和剑桥系统生物学中心的负责人凯瑟琳·利利一起进行。这将在第一年基本完成。然后我们将通过向细胞培养液中添加饱和或不饱和脂肪来比较模拟饮食诱导的变化，看看饮食脂肪如何影响内质网应激。这个系统将在二年级开发，并在三年级应用。在四年级，学生将专注于撰写她/他的成果，以便发表和完成他们的论文，并打算在~3.5年内提交。

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.