BIOCHEMICAL AND MOLECULAR BASIS OF ETF-QO DEFICIENCY

ETF-QO 缺陷的生化和分子基础

• 批准号: 6581866
• 负责人: FRANK E FRERMAN
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581866
• 关键词: Escherichia coli; NAD(P)H dehydrogenase; X ray crystallography; electron transport; enzyme deficiency; flavins; flavoproteins; gene expression; gene mutation; genetic mapping; human genetic material tag; inborn aminoacid metabolism disorder; iron sulfur protein; mental retardation; molecular pathology; protein protein interaction; protein structure function; site directed mutagenesis; tissue /cell culture; ubiquinone

Glutaric acidemia type II (GA2) is an inherited metabolic disease resulting from deficiency of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Because ETF is the electron acceptor for (at least) nine flavoprotein dehydrogenases, deficiency of either of these proteins impairs mitochondrial oxidation pathway. This proposal focuses on ETF-QO, and on its reaction with ETF. Mutations in ETF-QO from GA2 patients with immunologically and enzymatically established ETF-GO deficiency will be identified, using the recently established structure of the ETF-QO gene to amplify individual exons. The interactions between mutant and wild- type ETFs and ETF-QO will be investigated in kinetic experiments. These experiments employ site-directed mutants in two regions that, based on comparisons of the crystal structures of several ETFs (determined in collaboration with Dr. Kim), are predicted to participate in complex formation. The rationale for the site-directed mutations to be studied is also based on previous experiments that implicate specific lysine-containing regions of the beta-ETF subunit in the interaction of ETF with ETF-QO. The three-dimensional structure of ETF-QO will be determined by x-ray crystallography, to better understand the effects of mutations and the functioning of the wild-type protein. Finally, we will establish expression systems to investigate the effects of selected disease- causing mutations on the flavin and iron sulfur redox centers of this integral membrane protein.

谷氨酸II型（GA2）是一种遗传代谢性疾病，是由于电子转移黄蛋蛋白（ETF）或电子转移黄素 - 泛蛋白 - 泛素氧化还原酶（ETF-QO）引起的。由于ETF是（至少）九种黄素蛋白脱氢酶的电子受体，因此这些蛋白质中的任何一个的缺陷都会损害线粒体氧化途径。该提案侧重于ETF-QO及其与ETF的反应。将使用ETF-QO基因的最近建立的结构来放大各个外部EXON，从而鉴定出具有免疫学和酶促建立的ETF-GO缺乏的GA2患者ETF-QO的突变。突变体和野生型ETF与ETF-QO之间的相互作用将在动力学实验中进行研究。这些实验在两个区域采用了定向的突变体，这些突变体基于对几个ETF的晶体结构的比较（与Kim博士合作确定），预计将参与复杂的形成。要研究的位置定向突变的基本原理也基于先前的实验，这些实验暗示了β-ETF亚基的特定含赖氨酸的特定区域与ETF与ETF-QO的相互作用。 ETF-QO的三维结构将由X射线晶体学确定，以更好地理解突变的作用和野生型蛋白的功能。最后，我们将建立表达系统来研究所选疾病的影响 - 导致突变对这种整体膜蛋白的黄素和铁硫氧化还原中心。