GLUTARIC ACIDEMIA TYPE II -- MUTANT ELECTRON TRANSFER FLAVOPROTEIN

拟议研究的目标是继续研究戊二酸。 II型酸血症，一种人类先天的氨基酸和脂肪酸氧化错误， 在ETF：泛醌氧化还原酶(ETF：QO)上，该蛋白是 在一些疾病患者中缺乏，通常会催化 电子转移黄素蛋白(ETF)与细胞间的电子转移 线粒体呼吸链。我们对这个问题的调查已经 已经持续了十多年，现在已经定位为 大大提高了我们对电子转移方式的理解 ETF的黄素中心和铁硫氧化还原中心之间的影响：QO和 线粒体内膜中的辅酶Q(泛醌)，以及如何 该蛋白的突变扰乱了其功能，并导致戊二酸血症 II型。了解这种蛋白质的功能是至关重要的，如果治疗 通过基因转移的方法，什么时候和如果他们是 开发，是成功的，关于基因和它的 对于理解ETF：QO不足的原因，监管可能很重要 常伴有严重的先天性肾囊性疾病。我们现在 手头有五个克隆的95%以上的猪肉ETF：QO cDNA BlueScrip，并在两个克隆中克隆了大约85%的人类ETF：QO基因。 本资助期的具体目标是：(A)完成对猪肉的分析 和人类ETF：QO cDNA，(B)使用人类探针来定义 ETF：QO缺陷的GA2患者，以及(C)开发结构-功能图 QO，标识与ETF、Flavin和 铁-硫氧化还原中心和泛苯二酚。使用站点定向的实验 将对脱氮副球藻ETF-QO进行诱变以 解决不同结构域的氨基酸序列如何变化的问题 改变酶的功能。

The objective of the proposed research is to pursue studies on glutaric acidemia type II, a human inborn error of amino and fatty acid oxidation, and on ETF:ubiquinone oxidoreductase (ETF:QO), the protein which is deficient in some patients with the disease and which normally catalyzes electron transfer between electron transfer flavoprotein (ETF) and the mitochondrial respiratory chain. Our investigations on this subject have been ongoing for more than ten years, and are now positioned to substantially improve our understanding of how electron transfer is effected between the flavin and iron-sulfur redox centers of ETF:QO and coenzyme Q (ubiquinone) in the inner mitochondrial membrane, and how mutations of the protein perturb its function and lead to glutaric acidemia type II. Knowledge of how this protein functions is crucial if treatment for the disease by gene transfer approaches, when and if they are developed, is to be successful, information about the gene and its regulation may be important in understanding why ETF:QO deficiency is so frequently associated with severe congenital renal cystic disease. We now have on hand over 95 percent of pork ETF:QO cDNA in five clones in BlueScript, and about 85 percent of the human ETF:QO cDNA in two clones. Specific aims for this funding period are to (a) complete analysis of pork and human ETF:QO cDNA, (b) to use the human probe to define mutations in ETF:QO-deficient GA2 patients, and (c) to develop a structure-function map of ETF:QO, identifying domains which interact with ETF, the flavin and iron-sulfur redox centers, and ubiquinone. Experiments using site-directed mutagenesis of the ETF-QO of Paracoccus denitrificans will be performed to address issues of how changes in amino acid sequence in various domains alter enzyme function.