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氏病（AD）的一致特征。活性氧介导的损伤和线粒体功能障碍可能是因果关系的。线粒体α-酮戊二酸脱氢酶复合物（KGDHC）在AD中始终缺乏缺陷，并且似乎特别脆弱于氧化应激相关的损伤。自上次提交以来，遗传研究似乎已经证实，在四个不同的研究中，总共1400多名受试者，AD和DLST基因的多态性之间的关联编码了KGDHC的E2K亚基。遗传不平衡已在40个瑞典AD家族以及“零星”高加索人（附录1）和日本受试者中显示出来。这些发现导致了以下假设：1）KGDHC在AD中的缺乏通常是由于KGDHC对氧化应激的敏感性。 2）DLST的遗传异常可能是患者亚组中AD的危险因素。生化研究（AIM 1）将确定脆弱的亚基，以及负责KGDHC在AD中脆弱性的关键氨基酸或假肢群体。该方法需要定量三个kgdhc和重组人KGDHC亚基。我们将测量三个KGDHC亚基的酶活性和蛋白质免疫化学水平，以鉴定亚基脆弱性，并使用质谱和氨基辅助测序来帮助识别至关重要的氨基酸或假肢。 DLST中致病性突变的遗传搜索（目标2）将主要从瑞典AD家族中测试家族病例，并且还从高加索犹太人队列中选择了“散发性”案例，该病例具有“处于DLST基因型”的``处于危险''的风险。如果发现了合理的DLST突变（AIM 2），那么KGDHC蛋白对各种代谢损伤的反应以及在培养的细胞茎中的反应（AIM 2），则使用重组E2K和使用AIM 1中开发的测试参数来测试该突变的结果。