Molecular Genetics and Biochemistry of KGDHC in Alzheimer's Disease

阿尔茨海默病 KGDHC 的分子遗传学和生物化学

• 批准号: 6287804
• 负责人: KWAN-FU REX SHEU
• 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/6287804
• 关键词: Alzheimer's disease; Jewish; Scandinavian country; biopsy; clinical research; enzyme activity; enzyme complex; family genetics; genetic polymorphism; genetic susceptibility; human subject; immunochemistry; mass spectrometry; mitochondria; molecular genetics; neural degeneration; neurochemistry; neurogenetics; oxidative stress; oxoglutarate dehydrogenase; postmortem; protein sequence

Mitochondrial dysfunction and oxidative stress are consistent features of Alzheimer's disease (AD). The reactive oxygen species-mediated damage and mitochondrial dysfunction may be causally related. The mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) is consistently deficiently in AD, and appears to be particularly vulnerabl4e to oxidative stress-related insults. Genetic studies since the previous submission appear to have confirmed, in four different studies with a total of over 1400 subjects, an association between AD and a polymorphism of the DLST gene that encodes the E2K subunit of KGDHC. The genetic disequilibrium has been shown in members from 40 Swedish AD families, and in "sporadic" Caucasian (Appendix 1) and Japanese subjects. These findings have led to the following hypotheses: 1) The deficiency of KGDHC in AD is usually due to the susceptibility of KGDHC to oxidative stress. 2) A genetic anomaly of DLST may be a risk factor for AD in a subgroup of patients. Biochemical studies (Aim 1) will identify the vulnerable subunit, and the crucial amino acids or prosthetic groups responsible for the vulnerability of KGDHC in AD. The approach requires quantitation of each of the three KGDHC and recombinant human KGDHC subunits. We will measure the enzyme activities and protein immunochemical levels of the three KGDHC subunits to identify the subunit vulnerability, and use mass spectroscopy and amino aid sequencing to help identify the crucial amino acids or prosthetic groups. Genetic search (Aim 2) for pathogenic mutations in DLST will test primarily familial cases from the Swedish AD families, and also selected "sporadic" cases from the Caucasian Jewish cohort who possess the 'at risk" DLST genotype. A candidate, if found in familial AD cases, will be tested for genetic linkage in affected Swedish families. The biochemical tests (Aim 1) will also be used in Project 3 to define the responses of KGDHC proteins to various metabolic insults and under KGDHC deficiency conditions in cultured cell stems. If a plausible DLST mutation is found (Aim 2), then the consequence of that mutation will be tested using recombinant mutant E2k and using test parameters developed in Aim 1.

线粒体功能障碍和氧化应激是阿尔茨海默病（AD）的一致特征。活性氧介导的损伤和线粒体功能障碍可能存在因果关系。线粒体 α-酮戊二酸脱氢酶复合物 (KGDHC) 在 AD 中始终存在缺陷，并且似乎特别容易受到氧化应激相关损伤的影响。自上次提交以来的基因研究似乎已经在四项不同的研究中证实了 AD 与编码 KGDHC E2K 亚基的 DLST 基因多态性之间的关联，该研究涉及总共 1400 多名受试者。 40 个瑞典 AD 家族的成员以及“零星”白种人（附录 1）和日本受试者中都显示出遗传不平衡。这些发现得出以下假设：1）AD中KGDHC的缺乏通常是由于KGDHC对氧化应激的敏感性。 2) DLST 遗传异常可能是部分患者发生 AD 的危险因素。生化研究（目标 1）将确定 AD 中 KGDHC 的脆弱亚基以及关键氨基酸或辅基。该方法需要对三个 KGDHC 和重组人 KGDHC 亚基中的每一个进行定量。我们将测量三个 KGDHC 亚基的酶活性和蛋白质免疫化学水平，以识别亚基的脆弱性，并使用质谱和氨基酸测序来帮助识别关键的氨基酸或辅基。针对 DLST 致病性突变的基因搜索（目标 2）将主要测试来自瑞典 AD 家族的家族病例，并且还从具有“危险”DLST 基因型的高加索犹太群体中选择“散发”病例。项目 3 还将使用生化测试（目标 1）来确定 KGDHC 蛋白对各种代谢损伤和代谢损伤的反应。在培养细胞干中的 KGDHC 缺乏条件下，如果发现合理的 DLST 突变（目标 2），则将使用重组突变体 E2k 并使用目标 1 中开发的测试参数来测试该突变的结果。