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: 6336230
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336230
• 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缺乏的重要性的一个困难是，能量/氧化代谢受损也发生在其他神经退行性疾病中，包括 CAGn/Qn 疾病。该项目将测试多种可能损害线粒体的机制，包括 CAGn/Qn 疾病中的 KGDHC。我们已经证明，组织转谷氨酰胺酶（tTGase；来自豚鼠肝脏）可以将 Qn 扩展共价连接到 KGDHC 和 3-磷酸甘油醛脱氢酶上，从而使它们失活。组蛋白的 tTGase 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）转谷氨酰胺酶，导致恶性循环，尽管线粒体中没有发现转谷氨酰胺酶和在 CAGn/Qn 紊乱中积累的聚集体。将进行六个系列的实验。 (I) 人脑转谷氨酰胺酶似乎与广泛使用的豚鼠肝酶不同，将被纯化、表征、克隆和测序。 (ii)由于转谷氨酰胺酶活性在人成纤维细胞中几乎不存在，因此其活性将通过提高胞质Ca2+、通过地塞米松诱导以及可能通过转染来增加。细胞中异常的存在和时间将根据转谷氨酰胺酶在诱导的时间和数量方面的增加来确定。这些研究将阐明该模型中是否发生线粒体损伤（包括 KGDHC），以及它是否先于或随后出现其他细胞损伤证据。 (iii) 将在转基因 CAGn/Qn 小鼠（项目 5）的大脑中检查作为体外转谷氨酰胺酶底物的蛋白质（包括 KGDHC）。这些研究将确定这些模型中能量代谢成分（包括 KGDHC）是否发生变化，以及它们是在聚集体积累之前还是之后发生。 (iv) 合理设计的转谷氨酰胺酶抑制剂将用纯化的蛋白质、培养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细胞模型中测试ε-(γ-谷氨酰)赖氨酸作为CAGn/Qn疾病标记物的效用。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物积累的结果，那么未来的研究将集中于线粒体（包括 KGDHC）对 AD 以及其他神经退行性疾病中积累的聚集物的影响。