Effects of CAG/Qn Expansions on KGDHC and Other Enzymes

CAG/Qn 扩展对 KGDHC 和其他酶的影响

• 批准号: 6287807
• 负责人: JOHN P BLASS
• 金额: $ 18.12万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-02 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6287807
• 关键词: Alzheimer's disease; Huntington's disease; calcium metabolism; enzyme activity; enzyme inhibitors; genetically modified animals; human tissue; laboratory mouse; metabolism; mitochondria; molecular cloning; neural degeneration; nucleic acid repetitive sequence; oxidation; oxidative phosphorylation; oxoglutarate dehydrogenase; protein glutamine gamma glutamyltransferase; protein purification

A difficulty in interpreting the significance of the well-documented deficiency of the alpha-ketoglutarate dehydrogenase complex in Alzheimer's Disease (AD) is that impaired energy/oxidative metabolism also occurs in other neurodegenerative disorders including the CAGn/Qn disorders. This project will test a number of mechanisms that might damage mitochondria, including KGDHC, in the CAGn/Qn disorders. We have shown that the enzyme tissue transglutaminase (tTGase; from guinea pig liver) can covalently attach Qn expansions to KGDHC and to glyceraldehyde-3-phosphate dehydrogenase, inactivating them. tTGase action of histone aggregates them. A variety of other proteins were not substrates. Mitochondrial damage might impair calcium regulation and thereby activate (calcium-dependent) transglutaminase, leading to a vicious spiral, even though transglutaminase and the aggregates which accumulate in the CAGn/Qn disorder are not found in mitochondria. Six series of experiments will be done. (I) The human brain transglutaminase(s), which appear to differ from the widely used guinea pig liver enzyme, will be purified, characterized, cloned, and sequenced. (ii) Since transglutaminase activity is almost absent from human fibroblasts, its activity will be increased by raising cytosolic Ca2+, by dexamethasone induction, and possibly by transfection. The presence and timing of abnormalities in the cells will be determined in relation to the increases in transglutaminase in terms of both time and quantity of enzyme induced. These studies will clarify whether damage to mitochondria (including to KGDHC) occurs in this model and whether it precedes or follows other evidence of cell damage. (iii) Proteins which are transglutaminase substrates in vitro (including KGDHC) will be examined in the brains of transgenic CAGn/Qn mice (with Project 5). These studies will determine whether changes in constituents of energy metabolism (including KGDHC) occur in these models and whether they precede or follow accumulation of aggregates. (iv) Rationally designed transglutaminase inhibitors will be tested with purified proteins, cultured cells, and in transgenic animals. (v) The utility of epsilon-(gamma- glutamyl)lysine as a marker for CAGn/Qn disorders will be tested, in human tissues as well as in animal and cultured cell models. If these studies indicate that mitochondrial damage in the CAGn/Qn disorders is a consequence of the accumulation of intracellular aggregates, future studies will focus on the effects of mitochondria (including KGDHC) of the aggregates that accumulate in AD as well as those in other neurodegeneration.

解释阿尔茨海默病（AD）中α-酮戊二酸脱氢酶复合物的充分记录的缺乏的重要性的困难在于受损的能量/氧化代谢也发生在包括CAGn/Qn病症的其他神经退行性病症中。该项目将测试一些可能损害线粒体的机制，包括CAGn/Qn疾病中的KGDHC。我们已经表明，酶组织转氨酶（tTGase;从豚鼠肝脏）可以共价连接Qn扩展KGDHC和甘油醛-3-磷酸脱氢酶，使它们失活。组蛋白的TGase作用使它们聚集。许多其他蛋白质不是底物。线粒体损伤可能会损害钙调节，从而激活（钙依赖性）转氨酶，导致恶性螺旋，即使在CAGn/Qn疾病中积累的转氨酶和聚集体在线粒体中没有发现。将进行六个系列的实验。(I)人脑转氨酶似乎与广泛使用的豚鼠肝酶不同，将对其进行纯化、表征、克隆和测序。(ii)由于人成纤维细胞中几乎不存在转氨酶活性，其活性将通过提高胞质Ca 2+、地塞米松诱导以及可能通过转染而增加。将根据诱导的酶的时间和量确定细胞中异常的存在和时间与转氨酶升高的关系。这些研究将阐明在该模型中是否发生线粒体（包括KGDHC）损伤，以及它是在细胞损伤的其他证据之前还是之后。(iii)将在转基因CAGn/Qn小鼠脑中检查体外转氨酶底物蛋白（包括KGDHC）（项目5）。这些研究将确定在这些模型中是否发生能量代谢成分（包括KGDHC）的变化，以及它们是在聚集体积累之前还是之后。(iv)合理设计的转氨酶抑制剂将用纯化的蛋白质、培养的细胞和转基因动物进行测试。(v)将在人体组织以及动物和培养的细胞模型中测试β-（γ-谷氨酰）赖氨酸作为CAGn/Qn病症的标志物的效用。如果这些研究表明CAGn/Qn疾病中的线粒体损伤是细胞内聚集体积累的结果，那么未来的研究将集中在AD中聚集体的线粒体（包括KGDHC）的作用以及其他神经变性中的线粒体作用。