Investigation of the metabolic changes caused by isocitrate dehydrogenase mutations using mass spectrometry

使用质谱法研究异柠檬酸脱氢酶突变引起的代谢变化

• 批准号: 1809885
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1809885
• 关键词: Investigation; metabolic; changes; caused; isocitrate

Isocitrate dehydrogenase mutations have previously been found to be associated with tumour cell growth, especially the mutation of arginine 132 to histidine (R132H) although other mutations have been observed. These mutations are found in the majority of grade 2 and grade 3 gliomas in humans, especially impacting those between the ages of 20 and 40 and currently have one of the lowest 5-year survival rates of all cancer types making them a prime clinical target. The R132H mutation disrupts the normal citric acid cycle conversion of isocitrate to 2-oxoglutarate and catalyses the production of 2-hydroxyglutarate from both species, possibly due to competitive binding to the isocitrate dehydrogenase active site which has been shown to be moderated by R132. The physiological purpose for the upregulation of 2-hydroxyglutarate is not currently known and requires further investigation as several competing mechanisms of action have been postulated. These include the inhibition of 2-oxoglutarate dependent oxygenases, which alters the expression of the genes involved in cell differentiation and posttranslational modification of proteins. The mutation has however, been observed in early glioma development suggesting a pro-oncogenic role. Loss of function mutations have also been observed in other cancer types which results in the accumulation of fumarate and succinate which are also citric acid cycle metabolites of prime interest.Mass spectrometry remains the primary analytical method for metabolomics due to its high sensitivity and resolution which allow the detection of metabolite ions at femtomolar concentrations. This allows the identification of small molecules that are the products of cellular processes. Recent advances in metabolomics, especially ion-chromatography coupled with mass spectrometry, allow small polar and physiologically ionic metabolites, which are not observed using normal chromatographic techniques, to be identified in complex mixtures.This work aims to improve the methods of quantitative and qualitative analysis of metabolites by electrospray ionisation mass spectrometry of a range of biological systems. The characterisation of metabolites from wild-type and mutant cell lines and various tissues. This will include the optimisation of ion chromatography and other chromatographic techniques to allow the separation and identification of metabolites with database comparison and quantification to measure cellular concentrations. Tissue culture of various cell lines with isocitrate dehydrogenase mutations will be required and the use of stable isotope labelled media will be used to follow glycolytic and citric acid cycle pathways by carbon flux analysis with the eventual aim of the identification of targets for pharmaceutical intervention to disrupt cancer cell growth.This project falls within the EPSRC Physical Sciences - Analytical Science research area.

此前已发现异柠檬酸脱氢酶突变与肿瘤细胞生长有关，特别是精氨酸132突变为组氨酸(R132H)，尽管也观察到了其他突变。这些突变在人类大多数2级和3级胶质瘤中被发现，特别是影响那些年龄在20岁到40岁之间的人，目前这些突变是所有癌症类型中五年存活率最低的之一，使他们成为主要的临床目标。R132H突变扰乱了异柠檬酸到2-氧戊二酸的正常柠檬酸循环，并催化了两个物种2-羟基戊二酸的产生，这可能是由于R132竞争结合到异柠檬酸脱氢酶活性部位。2-羟基戊二酸上调的生理目的目前尚不清楚，需要进一步研究，因为已经假设了几种相互竞争的作用机制。其中包括抑制依赖2-氧戊二酸的加氧酶，该加氧酶改变与细胞分化和蛋白质翻译后修饰有关的基因的表达。然而，在胶质瘤的早期发展中观察到了这种突变，这表明它具有促肿瘤作用。在其他类型的癌症中也观察到了功能突变的丧失，导致富马酸和琥珀酸积累，这也是柠檬酸循环中最感兴趣的代谢物。由于其高灵敏度和分辨率，使得能够检测到毫微摩尔浓度的代谢物离子，质谱仪仍然是代谢组学的主要分析方法。这使得能够识别作为细胞过程产物的小分子。代谢组学的最新进展，特别是离子色谱与质谱联用技术的进展，使人们能够在复杂的混合物中鉴定出用常规色谱技术无法观察到的小的极性和生理离子代谢物。这项工作旨在改进一系列生物体系的电喷雾电离质谱法对代谢物进行定性和定量分析的方法。来自野生型和突变型细胞系和各种组织的代谢物的特征。这将包括优化离子层析和其他层析技术，以便通过数据库比较和量化来分离和鉴定代谢物，以测量细胞浓度。将需要对具有异柠檬酸脱氢酶突变的各种细胞系进行组织培养，并将使用稳定同位素标记的介质通过碳通量分析来跟踪糖酵解和柠檬酸循环途径，最终目的是确定药物干预的目标，以干扰癌细胞的生长。该项目属于EPSRC物理科学-分析科学研究领域。