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

一种用于监测内质网应激及其在饮食引起的细胞功能障碍中的作用的新型脂质组学工具。

基本信息

批准号：
1944308
负责人：
金额：
--
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1944308
关键词：
Novel Lipidomic Tool Monitoring ER

项目摘要

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.

内质网（ER）是一种亚细胞细胞器，在新合成蛋白质的加工过程中具有核心重要性。多种病理生理学与ER功能障碍有关，包括肥胖、神经变性、衰老和药物毒性。在ER功能障碍期间，未折叠的蛋白质聚集在ER中，这被称为ER应激。这在体外和体内模型中都已观察到。延长的ER-应激导致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质组学和蛋白质组学的方法来绘制亚细胞器的变化。这种方法涉及细胞或组织在密度梯度上的超离心，以根据它们的差异密度分离出细胞器。我们已经扩展了原来的蛋白质组学方法，包括一个平行的lipidomic工作流程，使我们能够监测蛋白质和脂质的细胞器组成的变化，这种新的方法定位脂质（LOL）。虽然最初开发用于在化学/毒物暴露后跟踪过氧化物酶体增殖，但这种新型多组学工具可用于跟踪其他亚细胞器的变化。这个博士建议是进一步开发这种脂质组学工具，以研究饮食诱导的ER应激领域。随着西方饮食消费的增加，饱和脂肪含量高，后果之一是ER压力增加，这被认为会导致2型糖尿病和心血管疾病等疾病。然而，我们不知道ER应激是否直接来自脂肪诱导蛋白的错误折叠，或者是否细胞膜流动性改变而导致应激。该项目符合BBSRC最近的研究呼吁对营养干预措施的机械理解。我们将首先在酵母和人肝细胞中使用已知的化学诱导剂在细胞水平上研究ER应激，以开发诱导的细胞变化的蛋白质组学和脂质组学组合图谱。Griffin小组定期培养这些细胞系，因此这应该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高分辨率质谱法进行脂质组学分析，以分析一系列脂质物质，包括磷脂、甘油三酯、甘油二酯、胆固醇酯和鞘脂。蛋白质组学将与凯瑟琳Lilley谁负责剑桥中心蛋白质组学，生物化学和剑桥系统生物学中心。这项工作将在第一年基本完成。然后，我们将比较这与模拟饮食诱导的变化，通过补充细胞培养基与饱和或不饱和脂肪，看看饮食脂肪如何影响ER应激。该系统将在第二年开发，并在第三年应用。在第4年，学生将专注于撰写她/他的成果，以出版和完成他们的论文，并打算在约3.5年提交。