Inborn Errors of Metabolism in Cell Culture

描述（由申请人提供）： 该项目的长期目标是了解由大分子催化机器的先天错误引起的遗传代谢疾病的分子基础。所研究的模型系统是一个高度保守的人α-酮酸脱氢酶复合物家族，其大小从400万至1000万道尔顿，包括支链α-酮酸脱氢酶复合物（BCKDC）、丙酮酸脱氢酶复合物（PDC）和α-酮戊二酸脱氢酶复合物（KGDC）。这些线粒体大分子结构在支链氨基酸和葡萄糖降解途径中催化α-酮酸的氧化脱羧。催化组分的组织类似于与E3结合蛋白偶联的24-梅里克体转酰酶或60-梅里克体转乙酰酶（在人PDC的情况下）形成结构核心，脱羧酶/脱氢酶（E1）、二氢硫辛酰胺脱氢酶（E3）的多个拷贝通过非共价相互作用连接到该结构核心。BCKDC在枫糖性血尿病（MSUD）中缺乏，表现为通常致命的α-酮症酸中毒、脑病和智力迟钝。人类PDC的缺乏导致葡萄糖利用受损和新生儿乳酸过多，并与2型糖尿病相关。在这个竞争性的更新申请中，我们将继续剖析BCKDC的结构和功能以及MSUD的生化和结构基础。然而，研究范围将扩展至包括人PDC和人KGDC的同源组分。具体目标是：1）证实了一种新的分子开关，该开关协调两个顺序的半反应（2）剖析人二氢硫辛酰胺脱氢酶（E3）缺乏的结构和生化基础，即E3缺乏型MSUD，并描述人BCKDC和KGDC中E3结合的结构决定因素; 3）确定多聚体人PDC支架的三维结构，并评估E3结合蛋白在人PDC核心组装中的作用。这些研究旨在增加我们对这个大型催化机器家族如何在生物化学和结构水平上工作的理解，以及保守的催化机制和大分子组装如何被致病的人类突变破坏。从这项研究中获得的知识将为开发更有效的MSUD和新生儿乳酸脱氢酶治疗提供一个框架。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of inherited metabolic diseases caused by inborn errors of macromolecular catalytic machines. The model system under study is a family of highly conserved human alpha-ketoacid dehydrogenase complexes ranging from 4- to 10-million daltons in size, which comprises the branched-chain alpha-ketoacid dehydrogenase complex (BCKDC), the pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex (KGDC). These mitochondrial macromolecular structures catalyze the oxidative decarboxylation of alpha-ketoacids in the branched-chain amino acid and glucose degradative pathways. The organization of catalytic components is similar in that the 24- meric transacylase or the 60-meric transacetylase coupled with the E3-binding protein (in the case of human PDC) forms the structural core, to which multiple copies of a decarboxylase/dehydrogenase (E1), a dihydrolipoamide dehydrogenase (E3) are attached by non-covalent interactions. BCKDC is deficient in Maple Syrup Urine Disease (MSUD) manifested by often-fatal alpha-ketoacidosis, encephalopathies and mental retardation. Deficiency in human PDC results in impaired glucose utilization and neonatal lactic acidemias, and is associated with type 2 diabetes. In this competing renewal application, we will continue to dissect the structure and function of BCKDC as well as the biochemical and structural basis for MSUD. However, the scope of the investigation will be extended to include the cognate components of human PDC and human KGDC. Specific Aims are: 1) To corroborate a novel molecular switch that coordinates the two sequential half-reactions (decarboxylation and reductive acylation) catalyzed by the E1b component of human BCKDC; 2) To dissect the structural and biochemical basis for human dihydrolipoamide dehydrogenase (E3) deficiency, i.e. the E3-deficient form of MSUD, and to delineate structural determinants for E3 binding in human BCKDC and KGDC; and 3) To determine three-dimensional structure of the multimeric human PDC scaffold and to assess the role of the E3-binding protein in the core assembly of human PDC. These studies are aimed to increase our understanding of how this family of large catalytic machines works at the biochemical and structural level, and how the conserved catalytic mechanisms and macromolecular assemblies are disrupted by disease-causing human mutations. The knowledge obtained from this investigation will provide a framework for developing more effective therapies for MSUD and neonatal lactic acidemias.