BIOCHEMICAL AND MOLECULAR BASIS OF ETF-QO DEFICIENCY

戊二酸血症II型(GA2)是一种由于电子转移黄素蛋白(ETF)或电子转移黄素蛋白-泛醌氧化还原酶(ETF-QO)缺乏而引起的遗传性代谢性疾病。由于ETF是(至少)9种黄素蛋白脱氢酶的电子受体，这两种蛋白中的任何一种的缺失都会损害线粒体的氧化途径。这项提议关注的是ETF-QO，以及它对ETF的反应。利用最近建立的ETF-QO基因的结构来扩增单个外显子，将鉴定GA2患者的ETF-QO突变，这些患者患有免疫和酶促ETF-GO缺乏症。突变型和野生型ETF与ETF-QO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使用了定点突变体，根据对几个ETF(与Kim博士合作确定的)的晶体结构的比较，预测它们将参与复杂的形成。要研究定点突变的基本原理也是基于之前的实验，这些实验表明，在ETF与ETF-QO的相互作用中，含有特定赖氨酸的β-ETF亚单位区域。ETF-QO的三维结构将通过X射线结晶学确定，以更好地了解突变的影响和野生型蛋白质的功能。最后，我们将建立表达系统，以研究选定的致病突变对该完整膜蛋白的黄素和铁硫氧化还原中心的影响。

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.