INBORN ERRORS OF PYRUVATE METABOLISM

丙酮酸脱氢酶复合物（PDC）在细胞内的代谢中起着关键作用。 用于能量生产和生物合成的丙酮酸代谢 流程.人类PDC的遗传缺陷与先天性 乳酸性酸中毒，各种神经功能障碍，经常过早死亡。 这个项目的长期目标是研究分子基础 的遗传缺陷的PDC在受影响的患者，并加强我们的 理解结构-功能关系和调节 PDC的丙酮酸脱氢酶（E1）组分，两者均在 蛋白质和DNA。在过去的四年里，我们和其他人已经确定了 人E1 α基因编码区的特异性突变 位于X染色体上然而，对功能知之甚少。 这些突变在蛋白质水平上的意义。而且我们的 对E1缺乏症的临床异质性的理解仍然是 支离破碎E1组分，α 2 β 2四聚体，催化 复合物中的限速步骤，并受到共价键的调节 修改;我们对其活性位点性质的理解以及 这两个分股发挥的作用非常有限。虽然E1（和 因此PDC）受到短期监管，其长期监管 激素在转录水平上的作用还知之甚少。初始 人E1 α和E1 β基因启动子区的分析 表明这些启动子可能代表了独特的变异， “管家基因”的转录调控。“五个具体目标 本续期申请中提出的建议是（i）调查以下人员的作用 活性位点中的特定氨基酸残基，（ii）研究 这两个亚基的结构-功能关系以及 三个磷酸化位点调节催化活性，（iii） 分析E1 α启动子区的转录调控， (iv)分析人类E1 β启动子的转录调控- 调控区，和（v）表征PDC缺陷型中的遗传缺陷。 患者我们将过表达定点突变的人E1蛋白， 研究E1的结构-功能关系的基础。动力学 突变蛋白的参数、光谱和结合特性将被 测定野生型和突变型启动子的转录活性 将使用DNA酶I足迹法、凝胶迁移率变化和 报告基因在转染细胞中的表达。基因缺陷将 使用酶测定、Western和北方分析进行分析，以及 患者特异性cDNA的测序。选定的突变将被重新创建 使用定点诱变和突变蛋白的过表达 功能分析。 我们多方面的实验方法是 旨在加深我们对 E1在催化、转录调控中的关系 基因，以及E1基因缺陷的分子基础。

The pyruvate dehydrogenase complex (PDC) plays a pivotal role in the metabolism of pyruvate for energy production and for biosynthetic processes. Genetic defects of the human PDC are associated with congenital lactic acidosis, variable neurological disability, and often early death. The long term goals of this project are to investigate the molecular basis of genetic defects of PDC in affected patients and to enhance our understanding of the structure-function relationships and regulation of the pyruvate dehydrogenase (E1) component of PDC, both at the level of protein and DNA. During the past four years, we and others have identified specific mutations in the coding region of the human E1alpha gene localized on chromosome X. However, little is known about the functional significance of these mutations at the protein level. Furthermore, our understanding of the clinical heterogeneity of E1 deficiency remains fragmentary. The E1 component, an alpha2beta2 tetramer, catalyzes the rate-limiting step in the complex and is subject to regulation by covalent modification; our understanding of the nature of its active site and the roles played by these two subunits is very limited. Although the E1 (and hence PDC) is subject to short-term regulation, its long-term regulation at the transcriptional level by hormones is poorly understood. Initial analyses of the promoter regions of the human E1alpha and E1beta genes indicate that these promoters may represent unique variations to transcriptional regulation of "housekeeping genes." Five specific aims proposed in this renewal application are to (i) investigate the roles of specific amino acid residues in the active site, (ii) investigate the structure-function relationships of the two subunits and the roles of the three phosphorylation sites in regulation of catalytic activity, (iii) analyze the transcriptional regulation of the E1alpha promoter region, (iv) analyze the transcriptional regulation of the human E1beta promoter- regulatory region, and (v) characterize genetic defects in PDC-deficient patients. We will overexpress site-directed mutant human E1 proteins to investigate the basis for structure-function relationships of E1. Kinetic parameters, spectral and binding properties of the mutant proteins will be determined. Transcriptional activity of the wild-type and mutant promoters will be analyzed using DNase I footprinting, gel mobility shift and expression of a reporter gene in transfected cells. Genetic defects will be analyzed using enzyme assays, Western and Northern analyses, and sequencing of patient-specific cDNAs. Selected mutations will be recreated using site-directed mutagenesis and overexpression of the mutant proteins for functional analysis. Our multifaceted experimental approach is designed to enhance our understanding of the structure-function relationships of E1 in catalysis, transcriptional regulation of the E1 genes, and the molecular basis of genetic defects of E1.