AML等肿瘤频发的IDH2突变体的结构、功能与稳定性研究

发布时间：2018-04-27 04:03

本文选题：IDH2 + 野生型　；参考：《浙江理工大学》2017年硕士论文

【摘要】：异柠檬酸脱氢酶(Isocitrate dehydrogenase,IDH)是一类在三羧酸循环中发挥关键作用的酶家族。人类基因组有5种IDH基因,可编码生成三种异柠檬酸脱氢酶(IDH1、IDH2及IDH3),其中IDH2位于线粒体,催化异柠檬酸氧化脱羧生成α-酮戊二酸(α-KG)和NADPH。已有研究表明在不同肿瘤中存在各种IDH突变体,包括急性髓细胞白血病(AML)、脑肿瘤、神经胶质瘤等,其最为常见的突变碱基位于R132(IDH1)、R140与R172(IDH2)。IDH突变体将α-KG转化生成肿瘤代谢物2-羟戊二酸(2-HG),从而紊乱了一系列α-KG依赖性双加氧酶反应。具体表现为:促进组蛋白赖氨酸甲基化;抑制10-11易位酶家族(TET)羟甲基化,进而激活CpG岛的DNA甲基化;稳定缺氧诱导因子(HIF-1α)的表达,上调血管内皮生长因子(VEGF)。目前与癌症发生和增殖密切相关的IDH2突变体的结构、功能及稳定性的研究鲜有报道,本课题通过分析不同突变体的IDH2结构、功能与稳定性,将对深入理解IDH2突变导致肿瘤发生、增殖的分子机制及其诊疗提供一些线索和参考。本研究选用哺乳细胞表达载体pCMV6-AC-Myc-His,通过基因克隆、重叠PCR、双酶切鉴定与测序成功构建11个IDH2重组表达载体:WT、R140G、R140Q、R140W、R172S、R172K、R172M、R172W、R172G、R172C、R172P。在此基础上,在293T与小鼠小胶质细胞BV2中瞬时转染过表达IDH2野生型与不同突变体,检测蛋白表达与相关信号通路(Western)、细胞代谢(检测酶活力、葡萄糖及乳酸、ROS变化)、蛋白降解(Western)、蛋白质与热激蛋白90相互作用(免疫共沉淀)等实验,通过生物信息学同源模型构建,分析突变对IDH2结构的影响,并与药物导致蛋白降解的结果进行相互验证与比对,探究IDH2野生型和突变体之间结构、功能与稳定性的差异。结果表明:1)野生型与突变体R140/R172在293T和BV2细胞中表达水平具有明显差异;2)突变体IDH2酶活力显著低于野生型;3)在293T细胞中,与野生型IDH2相比较,R172S、R172K、R172M、R172W、R172G、R172C、R172P突变体过表达降低细胞乳酸含量,R172S、R172K、R172W、R172G突变体过表达导致葡萄糖含量升高,R140G、R140Q、R140W、R172S、R172K、R172M、R172W、R172G、R172C突变体ROS含量均显著降低;不同突变体的过表达对细胞PI3K/AKT/mTOR信号通路影响不同,其中多个突变体过表达抑制pAKT和pS6的表达,并导致Cyclin D1表达下调;这些结果表明突变体IDH2过表达减缓了细胞代谢与生长。而在BV2细胞中,过表达R140Q、R140W、R172S、R172K、R172M、R172W、R172G、R172C、R172P突变体增加了TNF-α表达;其中R172S、R172M、R172W、R172G突变体过表达诱导IL-6表达,推测这些突变体的过表达可能与炎症反应的发生有一定的关联;4)泛素化蛋白连接酶抑制剂Bortezomib对野生型IDH2表达无影响,而对突变体R140G、R140Q、R140W、R172S、R172K表达敏感,促使其蛋白降解;对过表达的突变体R172M、R172W、R172G、R172C、R172P具有耐药性,药物处理过后的表达水平并没有发生明显变化。同源模建结构分析表明相应的点突变影响了蛋白质的结构。HSP90抑制剂17-AAG对突变体IDH2过表达并无影响,免疫共沉淀结果表明HSP90与IDH2无相互作用。以上研究将可能为IDH2作为标靶提供一些重要的信息与理论依据。
[Abstract]:Isocitrate dehydrogenase (IDH) is a family of enzymes that play a key role in the three carboxylic acid cycle. There are 5 IDH genes in the human genome, which can encode three ISO citrate dehydrogenases (IDH1, IDH2 and IDH3), in which IDH2 is located in the mitochondria and catalyzes the oxidation of ISO decarboxylic acid to produce alpha ketopurate (alpha -KG) and NADPH.. A variety of IDH mutants have been found in different tumors, including acute myelocytic leukemia (AML), brain tumor and glioma. The most common mutation base is located in R132 (IDH1), and R140 and R172 (IDH2).IDH mutants transform alpha -KG into tumor metabolite 2- hydroxyl diacid (2-HG), thereby disorder a series of alpha -KG dependence. The reaction of dioxygenase is specific: promoting the methylation of protein lysine, inhibiting the methylation of the 10-11 translocation enzyme family (TET), activating the DNA methylation of CpG Island, stabilizing the expression of HIF-1 alpha (HIF-1 alpha) and up regulating the vascular endothelial growth factor (VEGF). The structure of the IDH2 mutant, which is closely related to the occurrence and proliferation of cancer, is present. The study of energy and stability is rarely reported. By analyzing the IDH2 structure, function and stability of different mutants, this topic will provide some clues and references for the understanding of the molecular mechanism and diagnosis and treatment of IDH2 mutation leading to tumor occurrence and proliferation. This study selects mammalian cell expression vector pCMV6-AC-Myc-His, and reduplication of PCR through gene cloning. 11 IDH2 recombinant vectors were successfully constructed by double enzyme digestion and sequencing: WT, R140G, R140Q, R140W, R172S, R172K, R172M, R172W, R172G, R172C, and transiently transfected with mouse microglia to express wild and different mutant bodies, detect protein expression and related signaling pathways, and cell metabolism Enzyme activity, glucose and lactic acid, ROS change), protein degradation (Western), protein and heat shock protein 90 interaction (immunoprecipitation) and other experiments, constructed by bioinformatics homologous model, analyze the effect of mutation on the structure of IDH2, and verify and compare with the results of drug induced protein degradation and explore the wild type and process of IDH2. The difference between the structure, function and stability of the variant. The results showed that: 1) the expression level of the wild type and the mutant R140/R172 in the 293T and BV2 cells was significantly different; 2) the activity of the mutant IDH2 enzyme was significantly lower than that of the wild type; 3) in 293T cells, the R172S, R172K, R172M, R172W, R172G, R172C, mutants were overexpressed in comparison with the wild type IDH2. The over expression of low cell lactic acid, R172S, R172K, R172W, R172G led to the increase of glucose content, and the content of R140G, R140Q, R140W, R172S, R172K, R172M, R172W, R172G, and mutant were significantly reduced, and the over expression of the mutants had different effects on the signaling pathway. The results showed that the expression of Cyclin D1 was down regulated, and the results showed that the overexpression of the mutant IDH2 slowed down the cell metabolism and growth. In BV2 cells, the overexpression of R140Q, R140W, R172S, R172K, R172M, R172W, R172G, R172C, mutants induced the expression of alpha. The overexpression of some mutants may be associated with the occurrence of inflammatory reactions; 4) ubiquitin protein ligase inhibitor Bortezomib has no effect on the expression of wild type IDH2, but is sensitive to the expression of mutant R140G, R140Q, R140W, R172S, R172K, and promotes its protein degradation; the mutant R172M, R172W, R172G, R172C, R172P has resistance, The expression level after treatment did not change significantly. Homologous model construction analysis showed that the corresponding point mutation affected the protein structure.HSP90 inhibitor 17-AAG on the overexpression of the mutant IDH2, and the immunoprecipitation results showed that there was no interaction between HSP90 and IDH2. The above study would provide a target for IDH2 as a target. Some important information and theoretical basis.

【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2

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